EXPRESSION OF LUTEINIZING HORMONE RECEPTOR (LHR) IN THE

LEYDIG CELL CULTURE OF SPRAGUE DAWLEY RAT THAT INDUCED BY

ADVANCED GLYCATION END-PRODUCT 200g/mL AND GAMMA MANGOSTEEN

5M

GRADUATING PAPER

Submitted to the Board of Examiners as a Partial

Requirement for the attainment of Bachelor of Medicine Degree
in Universitas Gadjah Mada,
Yogyakarta, Indonesia

Written by :

Muhammad
Herdhana Ash
Shidiqi
12/328510/KU/
14897

FACULTY OF MEDICINE
Universitas GADJAH MADA
YOGYAKARTA

2015

AUTHENTIC STATEMENT

I, the undersigned

1
 Name : Muhammad Herdhana Ash Shidiqi

Student Number : 12/Ku/328510/148097

Faculty : Medicine

Hereby declare that i am the sole writer of this thesis.

I am responsible for the work submitted in this thesis

and it does not contain any material written by other

universities, except as specified in acknowladgement or

in footnotes.

Yogyakarta, january 25th 2015

The writers

Muhammad Herdhana Ash Shidiqi

APPROVAL PAGE

i
EXPRESSION OF LUTEINIZING HORMONE RECEPTOR (LHR) IN THE LEYDIG
CELL CULTURE OF SPRAGUE DAWLEY RAT THAT INDUCED BY ADVANCED
GLYCATION END-PRODUCT 200G/ML AND GAMMA MANGOSTEEN 5M
By :

Muhammad Herdhana Ash Shidiqi

12/328510/KU/14897

2
 Tested and approved on -

Team of Graduating Paper Examiner

Material Advisor Methodology Advisor

Dr dr. Dicky Moch. Rizal, dr. Rustamaji,M.Kes

Sp. And., AIFM, M.Kes

NIP 196910081996011001 NIP 11201100094

Expert Advisor

Dr. dr Rul Afiyah Syarif M.kes

NIP 197007091999032001

ACKNOWLEDGEMENT

By Gods grace finally i am done with my research report.

First and foremost i would like to thank to my belove parents,
my father, M.Subchi Yusuf and my mother, Umu Salamah, for
their endless love and support for making me who am i today
and being able finish this research report. I also would like
to thank Allah swt, for without him nothing is posible.
I would like to convey my indebtedness to my content
Dr.dr.Dicky moch Rizal Sp. And., AIFM, M.Kes and dr.rustamaji
M.Kes for their consultation and guidance in this whole
publication paper.

Not forgetting a special thanks to Dr.dr.Rul Afiyah

Syarif M.kes who was willing to be examiner for the research.

3
 At the same time to my other friends, seniors and
secretaries at the international program, your help is deeply
appriciated for lending me a hand in completing my thesis.
Finally, i really hope that this thesis will be
beneficial for the readers and for the advanvement or medical
theory.

Thank you

Yogyakarta,25 januari 2016

4
Contents

AUTHENTIC STATEMENT....................................................................................................II
APPROVAL PAGE....................................................................................................................II
ACKNOWLEDGEMENT.......................................................................................................III
LIST OF FIGURES....................................................................................................................3
TABLE LIST..............................................................................................................................4
LIST OF ATTACHMENT..........................................................................................................5
ABBREVIATION LIST.............................................................................................................6
ABSTRACT...............................................................................................................................7
CHAPTER I. INTRODUCTION...............................................................................................9
I.1. Background....................................................................................................................10
I.2.Problem Formulation......................................................................................................13
I.3. Objective........................................................................................................................13
I.4. Research Authenticity....................................................................................................14
I.5. Research Benefit............................................................................................................14

CHAPTER II. LITERATURE REVIEW.................................................................................15

II. 1. Literature Review........................................................................................................15
II. 1. 1. Luteinizing Hormone and Luteinizing Hormone Receptor..................15
II.1.2. The Destruction of Luteinizing Hormone Receptors and Effects of It.. .17
II.1.3. Advanced Glycation End-product.........................................................21
III.1.4. Correlation AGE and The Destruction of Cell......................................25
III.1.5. Correlation Between AGE and The Diseases.......................................27
II.1.6. Gamma Mangosteen........................................................................... 29
II.2. Basic Theory.................................................................................................................32
II.3. Teoritical Framework....................................................................................................35
II.4. Conceptual Framework................................................................................................37
II.5. Hypothesis....................................................................................................................38

CHAPTER III. RESEARCH METHOD..................................................................................39

III.1. Study Design...............................................................................................................39
III.2. Time and Study Setting...............................................................................................39
III.3. Research Population and Subject................................................................................39
III.4. Study Variables............................................................................................................40
 III.5. Study Material and Tools............................................................................................40
III.6. Operational Definition of Variable..............................................................................41
III.7. Conduct of The Research............................................................................................42
III. 8. Data Analysis.............................................................................................................45

CHAPTER IV. RESULT AND DISCUSSION........................................................................46

IV. 1. Result.........................................................................................................................46
IV. 1.1. Result of Normality Statistical Data...................................................46
IV. 1. 2. The Expression of LHR in The Leydig Cell Culture.............................46
IV. 2. Discussion...................................................................................................................48

CHAPTER V. CONCLUSION AND SUGGESTION.............................................................56

V. 1. Conclusion...................................................................................................................56
V. 2.Suggestion.....................................................................................................................56

CHAPTER VI. REFERENCES...............................................................................................57

CHAPTER VII. ATTACHMENT............................................................................................62

LIST OF FIGURES

Figure 1. The seven transmembrane -helix structure of

a G protein-coupled receptor such as LH 16

Figure 2. Glucose and AGE formation pathway 23

 Figure 3. AGE-RAGE interaction and NF-kB leading to

oxidant stress and generation of pro-

inflammatory cytokines 25

Figure 4. Gross appearance of manogsteen and chemical

structure of Gamma Mangostin 30

TABLE LIST

Table 1. The Mean Value of Research Subject

Characteristic 47
 Table 2. The Mean Value of Post-Hoc Least Significant

Difference(LSD) analysis.

47

LIST OF ATTACHMENT
 Attachment1. Detail of Data
Statisti 61
Normality of Data Distribution
Attachment2.
Tes
61
Attachment3. One way ANOVA Test and Test of
Homogeneity of
Variances 62
Attachment4. post hoc Statistical
Test 63

Attachment5. Grafic of Regression Standardized

Residual Dependent
Variable 64

ABBREVIATION LIST

3-DG :3-deoxyglucosone

AGE :Advanced Glycation End-product

COX-2 : Cyclooxygenase-2 Enzyme

CML :Carboxymethyl-lysine

DHT :Dihidrotestosterone

ELISA :Enzyme-linked Immunosorbent Assay

LH :Luteinizing Hormone

LHR :Luteinizing Hormone Receptor

NF-kB :Nuclear Factor kappa-B

PGE2 :Prostaglandin E2

RAGE :Receptor for Advanced Glycation End Products

ROS :Reactive Oxygen Species

ABSTRACT

EXPRESSION OF LUTEINIZING HORMONE RECEPTOR (LHR) IN THE LEYDIG

CELL CULTURE OF SPRAGUE DAWLEY RAT THAT INDUCED BY ADVANCED
GLYCATION END-PRODUCT 200g/ml AND GAMMA MANGOSTEEN 5M

Dicky Moch Rizal 1, Muhammad Herdhana Ash Shidiqi 2, Rustamaji3

1 Medicine Study Program, Faculty of Medicine UGM
2 Department of Physiology, Faculty of Medicine UGM
3 Department of Pharmacology & Therapy, Faculty of Medicine UGM

Background: Advanced Glycation End Products (AGE) are

protein or lipids that to be the result of a series of
chemical reactions after an initial glycation reaction. AGE
will cause cell destruction,which increasing Oxydative
stress and inflamation. Gamma mangostin is a major bioactive
compounds in mangosteen extract and one of the major
subtances that classified in xanthone derivates. It is
contains antioxidant and can decrease inflammatory response
that caused by AGE

Objectives: To look the expression of Luiteinizing Hormone

receptor (LHR) in the Leydig Cell Cultures of Sprague Dawley
rat that induced by AGE and given Gamma Mangosteen. Then
comparing with Leydig cell culture of Sprawgue Dewley that
induced by AGE only.
Methods: Used an experimental laborarory study on expresion of
Luteinizing Hormone Receptor (LHR) level data of Leydig cell
culture Sprague Dawley rat that induced by Advanced glycation
End-products and given gamma mangostin compared with leydig
cell culture Sprague Dawley rat that induced by AGE only. Data
processing by using SPSS aplication Statistics 22.0 by one way
ANOVA statistical test to know significance of on expresion of
Luteinizing Hormone Receptor (LHR) level on every Leydig cell
culture group.
Result: No significant difference on expresion of Luteinizing
Hormone Receptor (LHR) 5level of Leydig cell culture that only
given AGE (7,79 pg/ 10 /24h) 5
with group that given AGE dan
gamma mangosteen (8,06 pg/ 10 /24h) with p value p=0,10.
Conclusion: The Expression of Luiteinizing Hormone Receptor
(LHR) level is higher on the leydig cell culture which given
Advanced Glycation End-products compere with control (no
treatment). After giving gamma-mangostin, the expression of
Luiteinizing Hormone Receptor (LHR) level is higher then
leydig cell culture which only given Advanced Glycation End-
product. The mean of LHR levels5
in the all group, include in
the Expected levels.(3.9 pg/ 10 /24h).

Keywords: Expresion of Luteinizing Hormone Receptor (LHR)

level, Leydig cell culture, Sprague Dawley rat, Advanced
glycation end products, gamma mangostin.
 EXPRESI RESEPTOR LUTEINIZING HORMONE (LHR) PADA KUTUR SEL
LEYDIG TIKUS SPRAGUE DAWLEY RAT YANG DIINDUKSI ADVANCED
GLYCATION END-PRODUCT (AGE)200g/ml AND GAMMA MANGOSTEEN 5M

Dicky Moch Rizal 1, Muhammad Herdhana Ash Shidiqi 2, Rustamaji3

1 Program Studi Pendidikan Dokter FK UGM
2 Departemen Fisiologi FK UGM
3 Departemen Farmakologi & Terapi FK UGM

INTISARI
Latar Belakang: Advanced Glycation End Products (AGE) adalah
protein atau lipid yang menjadi hasil dari serangkaian
reaksi kimia setelah awalan reaksi glikasi. AGE akan
menyebabkan seuatu kerusakan pada sel dengan peningkatan
stres oksidatif dan inflamasi. Gamma mangosteen merupakan
senyawa bioaktif utama pada extrak manggis dan merupakan
salah satu zat utama pada klasifikasi turunan xanthone.
Gamma mangostin mengandung antioksidan dan dapat menurunkan
respon inflamasi yang di sebabkan AGE.

Tujuan Penelitian: Mengamati ekspresi dari reseptor hormon

Luiteinizing (LHR) pada kultur sel Leydig tikus Sprague
Dawley yang diinduksi dengan AGE dan diberi Gamma Mangosteen.
Lalu dibandingkan dengan kultur sel Leydig tikus Sprague
Dawley yang hanya diberi AGE saja.
Metode: Analisis experimental laboratory data Ekspresi
receptor LH pada kultur sel Leydig tikus Sprague Dawley yang
diinduksi Advanced glycation end products dan diberi gamma
mangosteen dibandingkan dengan yang tidak diberi gamma
mangosteen. Pengolahan data dilakukan dengan menggunakan
software SPSS Statistics 22.0 dengan uji statistik one way
ANOVA untuk mengetahui kebermaknaan kadar testosteron pada
tiap kelompok kultur sel Leydig
Hasil: Tidak terdapat perbedaan bermakna pada kadar ekspresi
5
reseptor LH yang hanya diberi AGE saja (7,79 pg/ 10 /24h)
dengan kelompok yang diberi AGE dan gamma mangosteen (8,06 pg/
5
10 /24h), dengan nilai p=0,10.
Kesimpulan: Ekspresi reseptor LH lebih tinggi pada kultur sel
Leydig yang diberi AGE saja dibandingkan dengan kelompok
kontrol (tidak diberi perlakuan). Setelah pemberian gamma
mangosteen, kadar ekspresi reseptor LH lebih tinggi
dibandingkan dengan kltur sel Leydigyang di beri AGE saja.
Rerata kadar ekspresi reseptor Lh pada semua kelompok termasuk
5
dalam rentang kadar yang diharapkan (3.9 pg/ 10 /24h).
Kata kunci: kadar ekspresi reseptor LH, kultur sel Leydig,
tikus Sprague Dawley, Advanced glycation end products, gamma
mangosteen.

CHAPTER I. INTRODUCTION

I.1. Background

Infertility affects in average 15%20% couples,

therefore, a major health problem. Incidence of male

infertility is rising due to various genetic, infectious, and

environmental factors. The most difficult problems for

patients to prevent and treat are those presenting with

azoospermia (no spermatozoa in the ejaculate). Where the sperm

production is influenced by the level of testosterone

adequate. Testosterone levels are maintained by Leydig cells

in order to be sufficiently differentiated and stimulated by

Luiteinizing Hormone (LH)(Goluza et al., 2014).

Especially sexual development for male is regulated by

Chronin gonadotropin and LH. Luiteinizing Hormone is a hormone

secreted by anterior pituitary in response to Gonadotropin-

Releasing Hormone (GnRH) released by the hypothalamus. This

hormone will give an effect on the leydig cells surface in

the testis after binding with their receptors (Jabbour, 2014).

Luteinizing Hormone Receptor (LHR) is a special receptor for

LH. Interaction of this binding will initiates a sequence of

membrane and intracellulare events. The activation leads to

increase testosterone production and male sexual development.

Testosterone is secreted by Leydig cells effect: (1)

Reproductive system before birth,(2) Sex-specific tissues

after birth, (3) developing the sex drive at puberty, (4)

Secondary sexual characteristic. (5) Exert a protein anabolic

effect, promote growth factors (Sherwood, 2011).

Distruction of the Leydig cells automatically induce

deactivation of LHR or the distruction of LHR itself. There

are many factors that induce damaging of Leydig cells, such as

tumor or cancer, trauma, inflamation process in the testis,and

loss of mutation functions of the gene for the Luteinizing

Hormone Receptor in male caused by pseudohermaphroditism

associated with Leydig-cell hypoplasia (Liu et al., 2015),

etc. The inflamation factor which damages Leydig cells mostly

caused by Advanced Glication End-product (AGE) effects on

intracellular function. A receptor for AGE will produce

expression after getting stimulation of AGE binding. The

result of expression is NF-kB sites control cellular

expression that linking Receptor AGE to the inflamatory

response ( Li et al., 1997).

In the ageing process will increase the product of

glycation process. The product is Advanced Glycation End-

products (AGE). It is caused by defense mechanisms of

cellular antioxidant depletion and the generation of oxigen

free radicals. For this case also could be found in the people

with Diabetes Mellitus. Advanced Glycation End-products be

able to induce destruction of cell then automatically will

destroy any tissues organ by process of oxidative stress

(Bierhaus et al.1997).

Therefore, if the Leydig cells are destruction or cells

can not be stimulated by LH, automatically the production of

testosterone will decrease or none at all. Deficiency of

testosterone will make problems in male such as loss the

libido, erectile disfunction, depression, derease cognitive

ability, lethargy, osteoporosis, and lost of muscle strength.

This contellation of symptoms is known collectively as

andropause, or ADAM (Andropause Deficiency of the Aging Male)

(Rajfer J., 2013).

Recently, have been done many researches about benefits of

mangosteen fruit. Gamma Mangostin is a major bioactive

compounds in mangosteen. Gamma Mangostin is one of the major

subtances that classified in xanthone derivates. According to

some researches, Gamma Mangostin has advantages for medical

treatment as anti-inflamatory and hight antioxydant. The

result of research in Taiwan about Gamma Mangostin could be

the medicine as anti-cancer and preventing for cancer(Hui-

Fang Chang et al, 2012). Mangosteen is one of the fast-growing

tropical fruits in South Eastern Asia. So, it is easily to be

found in Indonesia.

In the research about Gamma Mangostin. It exhibited

enhancement of Natural Killer (NK) cell activity in a mouse

model, and decreasing the level of prostaglandin E2 (PGE2)

through inhibition of cyclooxygenase (COX-2) activity and

Nitric Oxyde Synthase (NOS), and production cardiovascular

protective effects as well as strong antioxidant. Gamma

Mangostin also strongly inhibited human cell proliferation

(Hui-Fang Chang et al., 2012). The benefit of Gamma Mangostin

is about anti-inflamation also has explained and proofed of

other research. Gamma Mangosteen is effective in inhibition of

activated of substance that induce inflamatory genes,

including TNF, IL-1, IL-6, IL-8, and MCP-1(Bumrungpert et

al., 2009).

I.2.Problem Formulation

From the introduction, the problem described can be

formulated as below:
Is The expression of Luiteinizing Hormone Receptor (LHR)

higher on the leydig cell culture which given Advanced

Glycation End-products 200g/ml and Gamma Mangosteen 5M.

compared with the leydig cell culture which given Advanced

Glycation End-products 200g/ml only?

I.3. Objective

This study aims to look the expression of Luiteinizing

Hormone receptor (LHR) in the Leydig Cell Cultures of Sprague

Dawley rat that induced by AGE and given Gamma Mangosteen.

Then comparing with Leydig cell culture of Sprawgue Dewley

that induced by AGE only.

I.4. Research Authenticity

There are some researches about the effect of Advanced

Glycation End-product on the body and the advantage of Gamma

Mangosteen in health field. As for as nothing research found

the relation of variable on this research or about the

Expression of Luteinizing Hormone Receptor (LHR) in the Leydig

Cell Culture of Sprague Dawley Rat that induced by Advanced

glycation End-product and Gamma Mangosteen.

I.5. Research Benefit

This research is expected to prove the benefits of Gamma

Mangosteen as prevention of inflamation process or destrution

Leuitizing Hormone Reseptors and also preventing decrease of

testosterone levels that resulted infertility. In this case

Advanced Glycation End Products effect (AGE) as the factor of

destruction. The developing knowledge of health professional

about the benefits of mangosteen, especially for the Gamma

Mangosteen. Furthermore, it can be the basic for studies aimed

to prevent the destruction by AGE on Leydig cells. People also

can be protected by the adverse effects of AGE on the health

of the body organs, especially against the Leydig cells in the

testis.

CHAPTER II. LITERATURE REVIEW

II. 1. Literature Review

II. 1. 1. Luteinizing hormone and luteinizing hormone receptor.

Gonadotropins is Glycoprotein hormone. It regulates

gonadal function, and has two type of hormones. There are

Luteinizing hormone (LH) and follicle-stimulating hormone

(FSH). It composed of a common -subunit that is non-

covalently associated with a hormone-specific -subunit.

Gonadotropins exert their action through gonadotropin

receptors, the Luteinizing Hormone Receptor and the FSH

receptor (FSHRA) are primarily expressed in reproductive

organs and act coordinately to control steroidogenesis,

folliculogenesis, and ovulation (Ogiwara et al., 2013). The

luteinizing hormone receptor (LHR) is a member of the G

protein-coupled receptor family. Although this receptor is a

monomeric glycoprotein. The LHR consist of 674 amino acids and

has molecular mass of about 85-95 KDA based on the extent of

glycosylation. The LHCG receptor possess seven membrane-

spanning domains or transmembrane helices. Binding of LH and

hCG to this receptore are biologically functional and thus

capable of activating cellular pathways leading to secretion

of a steroid hormone product (Roes et al., 2000).

Figure 1. The seven transmembrane -helix structure of a G

protein-coupled receptor such as LHR. (Iriana et al., 2009)

Start from production of GnRH by the hypothalamus, and

subsequently GnRH stimulates the production of LH by the

anterior pituitary. and LH binds to the LH receptor in Leydig

cells. and there was activation of testosterone production.

Leydig cells require cholesterol, because it is the essential

precursor for all steroid hormones. in other words,

cholesterol is the basic ingredient of testosterone.

Cholesterol can either be incorporated by the cell through

receptor-mediated endocytosis from Low Density Lipoprotein. It

can be synthesized de novo within the leydig cell starting

from acetyl-coenzime A. In addition testosteron signaling

regulates lipid homeostasis in leydig cell. Briefly

testosterone production begins from pregnenolone, which is

converted to progesterone 3-beta hydroxysteroid dehydrogenase.

Then in the metabolism into androstenedione. Then subsequently

androstenedione is converted to testosterone by 17-beta

hydroxysteroid dehydrogenase(Nieschlag et al., 2010).

II.1.2. The Destruction of luteinizing hormone receptors and effects

of it.

In the testis, there is a significant population of

macrophages located in the interstitial compartment. They have

Fc and complement receptors, express macrophage-specific

markers, produce interleukins, phagocytose and kill pathogenic

organisms. It means there are inflamation process in this area

which will give the cellular destruction on the leydig cell.

The level of testosterone depending on the nature of Leydig

cells change within the gonad. If Leydig cells are poorly

differentiated from their mesenchymal precursors. It has not

capable for maintaining the normal testosterone production.

However, many cases testosterone levels are maintained by

Leydig cells which sufficiently different and stimulated by

LH(Goluza et al., 2014). The conclusion is in inflamation

process could damage cell organs, which is LHR on Leydig cell.

So, it is possible to decrease testosterone production.

Aging has an association with testosterone level in the

serum. The main problem of it lies on the binding site of LH

with their receptor (LH Receptor). The research that proof

such correlation has been made by using leydig cell of Brown

Norway rats, with various ranges of age, which were isolated

and long-term cultured with LH. In young rat specimens,

experimental suppression of serum LH levels resulted in the

decreases of Leydig cell volume and T production. In contrast,

in the old specimens, Leydig cells volume and T production is

reduced. Leydig cells from both old control rats and young LH-

suppressed rats have reduced numbers of LH binding sites.

However, whereas the cells from young LH-suppressed rats

produced cAMP at the high levels of young control cells, the

old cells produced far less cAMP. cAMP is the classical second

messenger involved in LH-induced leydig cell testosterone

production, and it is generation solely depends on LH and its

receptor interaction and effector activation. The old cells

failed to respond to LH due to the changes in LH receptor

number and affinity, and/or the ability of the cells to

produce 3,5-cAMP in response to LH. So, the reduced ability of

old Leydig cells to produce cAMP results from a deficiency in

LH binding. Thats why, administered LH therapy failed to

increase testosterone production by old Leydig cells (Chen et

al., 2002).

Radiation therapy is also one of the most important cases

that affected the destruction of LHR. This case has already

proven by previous researches, which used cultured human

leydigicell in prostatic carcinoma patient that exposed to

different dose of fractioned gamma radiation. The treatments

were divided into 2 kinds, which are low dose (2 Gy and 4 Gy)

and high dose (6,7,8,9,10, Gy) of radiation therapy. The study

shows us that radiation therapy with various dose give adverse

effects on the leydig cell function include on LHR, for

example in the radiation therapy of Acute Lymphblastic

Leukemia, Testicular Seminoma, Unilateral germ cell cancer,

carcinoma in situ of the testis, Pelvic radiotherapy for

prostatic carcinoma, total body irradiation for bone marrow

transplantation, Radioiodine therapy for thyroid carcinoma,

and also in the case of healthy adult men, a single dose

of 600 rad of radiation directed. Because, testis is one of

the most important radio sensitive tissues , which even very

low doses of radiation deteroriatite testicular function

(Sivakumar et al., 2006).

The result of this study also proves that Leydig cell

surface LH/hCG receptor concentration signicantly decreased,

following higher doses of radiation exposure (6 Gy and above).

However, lower doses (2 and 4 Gy) did not signicantly affect

the receptor concentration. Another result is that Gamma

radiation was found to have an inhibitory effect on both basal

and LH-stimulated cAMP production in a dosedependent manner.

While the lower doses (2 and 4 Gy) did not show any

appreciable change in the generation of cAMP, higher doses (6

Gy and above) brought down the cAMP generation in both basal

and LH-stimulated conditions. The depressed LH-stimulated cAMP

production in higher doses treated cells in such ways, may be

due to subnormal Leydig cell surface LH receptors and

associated impairment in downstream events such as

transmembrane signaling and activation of effector system.

Since the binding of LH to its receptor and the activation of

effector system are the important steps involved in the

activation of steroidogenesis, the observed decrease's in LH/

hCG receptors, cAMP production, and steroidogenic enzymes are

the factors that account for the subnormal testosterone

recorded in the clinical cases after radiation exposures

(Sivakumar et al., 2006).

Leydig cells reside in the interstitium of the testes and

contribute about 75% of the total testosterone produced by

normal adult male to support spermatogenesis and to maintain

masculinity (Sivakumar et al., 2006). The destruction on LHR

causes many clinical manifestation. One of this condition is

hypogonadism or decreasing of testosterone level. It is caused

by decreasing or lossing the response of LHR binding and LH,

because of this the testosterone levels significantly

decreasing.

Actually, the causing of hypogonadism can be of central

(hypothalamic or pituitary) or testicular origin, or a

combination of both. In men, the major gonadal steroid hormone

is testosterone. For example during puberty, this hormone

important for the development of male secondary sexual

characteristics, stimulation of sexual behavior and function,

and initiation of sperm production. Than In adult males this

hormone also important, testosterone is involved in

maintaining muscle mass and strength, fat distribution, bone

mass, red blood cell production, male hair pattern, libido and

potency, and spermatogenesis. Another that many effects which

caused lack of testosterone levels, such as loss of libido,

erectile dysfunction, diminished intellectual capacity,

depression, lethargy, osteoporosis, loss of muscle mass and

strength, and some regression of secondary sexual

characteristics. (Carnegie., 2004).

II.1.3. Advanced Glycation End-product

Advanced Glycation End Products (AGE) are protein or

lipids that become non enzymatically glycated and oxidized

after contact with aldose sugars. It can be said to be the

result of a series of chemical reactions after an initial

glycation reaction (Schmidt et al.,1994; Singh et al., 2001).

In the early glycation and oxidation processes result in the

formation of Schiff bases and Amadori products, Further

glycation of proteins and lipids causes molecular

rearrangements that lead to the generation of AGEs. AGEs it

self may fluoresce, produce reactive oxygen species (ROS),

bind to specific cell surface receptors, and form cross-links.

(Schmidt et al.,1994 ; Brownlee et al., 1985). In the begining

step of Maillard reaction or well known as glycation is

schiff-base formation. It is formed by glucose reaction with

Ammino protein group (NH2). Then this Schiff-base will convert

becoming cethoamine as amadori product which more stable

(e.g.HbA1c). Free reactive carbonil group from amadori product

which responsible to some glycation biologic consequense and

Amadory product could be degragated become many carbonil

subtances. One of very reactive such as 3-deoxy-glucosone,

which react again with free amino group for creating

intermediate glycation product. It contributes formation of

Advanced Glycation End-product, include dicarbonyl

intermediates, such as 3-deoxy-glucosone, glyoxal and methyl-

glyoxal. Glyoxal and methyl-glyoxal could be formed by glucose

autooxidation and glikolipid product. The begining glycation

products and intermediate could be slowly becomes getting some

re-forming chemistry complex, to create AGE stable subtances

and irreversible. It induces ROS and interact the surface

structure of some cells. AGEs consist of some chemistry

structure, include 2-(2-furoyl)-4(5)-furanyl- 1H-imidazole

(FFI), 1-alkyl-2-formyl-3,4-diglycosyl pyrroles (AFGPs), N-q-

carboxy-methyl-lysine (CML), pyrraline dan pentosidine. N-q-

carboxy-methyl-lysine (CML) is main AGEs that accumulated in

vivo (Basta et al.,2004; Huebschmann et al., 2006). Another

formation AGEs mechanism includes carbonyl stress pathway,

which this oxydation from glucose and lipid. It will create

dicarbonyl intermediate sunstance which uses reactive

carbonile group to bind with ammino acid and form AGEs.

Another mechanism is by aldose reductasemediated polyol

pathway (Huebschmann et al., 2006).

 Figure 2. Glucose and AGE formation pathway (Singh et al.,
2001).

Advanced Glycation End(AGE) product may modify the

extracellular matrix; modify the action of hormones, cytokines

and impact function of intracellular protein. The important

factors of AGEs formation include the rate of turnover of

protein for glycoxidation, hyperglycemia level, and

environment that extent of oxidant stress. (Schmidt et al.,

2001; Brownlee et al., 1995; Schmidt et al., 1999; Fu et al.,

1994).
 Advanced glication End-product has a receptor for creating

reaction on target cells. Receptors for AGE will initiates the

intracellular signaling that disrupts cellular function

through its recognition and binding of AGEs. Advanced

Glication End Receptor (RAGE) is a member of the

immunoglobulin superfamily of receptors. (Schmidt AM et al.,

1992 ; Sugaya K. et al., 1994). The location of this receptor

in chromosome 6 in the major histocompatibility complex

between genes for class II and III (Sugaya et al., 1994). AGEs

binding on RAGE induces to form intracellular reactive oxygen

some expressions of cytocine will increase,also tumour

necrosis factors (TNF- dan TNF-), interleukins (IL) 1, 6,

8,18 and interferon- (Wright, 2006). In several part of the

RAGE promoter has Nuclear factor (NF)-kB sites, an interferon-

response element, and an NF-interleukin-6 (IL-6) DNA binding

motif (Li et al., 1997). RAGE will produce expression after

had stimulation, one of them is NF-kB sites control cellular

expression that linking RAGE to the inflamatory response.( Li

et al., 1997). RAGE has a 332-amino acid extracellular

component consisting of 2 C-type domains preceded by 1

V-type immunoglobulin-like domain (Yan et al., 2003). RAGE

also has a singgle transmembrane domain followed by a highly

changed 43 amino acid cytosolic tail andthe cytosolic tail is

critical for RAGE-induced intracellular signaling. (schmidt et

al., 2001).
 Figure 3. AGE-RAGE interaction and NF-kB leading to
oxidant stress and generation of pro-inflammatory cytokines
(Singh et al., 2001).

III.1.4. Correlation AGE and the destruction of cell.

In the destruction of organ Advanced glication End-product

(AGE) can destruct directly on DNA or binding result of AGE

and the receptor, which producing free radical (Reactive

Oxygen Species). A free radical is defined as any chemical

species that contains unpaired electron. This unpaired

electron usually produces a highly reactive free radical.

Oxygen is the most common source of free radical in biological

system. Oxidative stress and nitrosative stress are term of

harmfull effects and biological damage caused by Reactive

Oxygen Species (ROS) and Reactive Nitrogen Species(RNS)(Valko

et al., 2007). When there is an imbalance between over

production of free radical(ROS/RNS) and decrease of

antioxidant molecules will cause side effect in the body. Over

production of ROS or RNS can damage and inhibit the normal

functions of lipids, protein and DNA. This effect is due to

intracellular reduction of oxygen into ROS, which is toxic to

cells and tissues.(Wickens et al., 2001).

The most susceptible sites of damaging areas that caused

by ROS is cell membrane. Cell membrane fatty acids and form

lipid peroxides can react with free radicals. Accumulation of

lipid peroxides can lead to production of carcinogenesis

agents like malondialdehyde. Cell membrane damage via lipid

peroxidation can permanently impair fluidity and elasticity of

the membrane that can lead to the cell rupture. (Lipman et

al., 1983 ; Ran et al., 2006).

Another main targets for free radicals attact are

Proteins. Overproduced radicals can react with protein

aminoacids to oxidize and cross-link them. This reaction can

impaire the function of important celullar and extracellular

protein like enzymes and connective tissue protein

permanently. Other targets for free radical attach is DNA. DNA

can break its strands that caused by interaction with free

radical.(Stadtman et al., 1995 ; Ames et al., 1993; Linn et

al., 1995).

III.1.5. Correlation between AGE and the diseases

High level of AGE in body could cause many clinical disease

manifestation. According to the explanation above, AGE induces

Free-radical production as well as depleting nitric oxide

concentration. Than leading to oxidative stress. As nitric

oxide is vasodilatory and has anti ploriferative effects on

vascular smooth mucle. AGE accumulation could therefore result

in vascular thickening with loss elasticity, hypertension and

endothelial dysfunction. This condition also to affect tissue

remodelling resulting in activation of smooth muscle

proliferation that automatically will increasing vascular

permeabillity. This changes are important part in the

pathophysiological feature associated with diabetes (Boel et

al., 1995; Ceriello ., 1999).

In the diabetic condition shows that AGE concentration

will increse in a few weeks and in the systemic, kidney, skin

and vascular tissue will be higher (Baynes JW, Thorpe SR .,

1999). the accumulation of serum AGEs in diabetic nephropathy

may be mainly due to decreased removal in the kidney rather

than increased production by high glucose levels or oxidative

stress. The increasing of AGE level often found on the people

who have Diabetes Melitus type 2. On this condition of the

people, will happen formation and desposition of AGE molecule,

which the result of protein glycation and glucose or lipid.

these are caused by the increasing of blood glucose. The role

of AGE has been reported that Advanced Glycosylation End-

products (AGEs) play an important role in the development of

diabetic complications, include the following: (1) formation

of cross-links between key molecules in the basement membrane

of the Extra Celullar Membrane, permanently altering cellular

structure; and (2) interaction of AGEs with RAGE on cell

surfaces, altering cellular function (Goldin et al., 2015).

AGE may contribute to eventual neuronal dysfunction and

death as an important factor in the progression of various

neurodegenerative diseases, including Alzheimer's Disease

(AD). The investigation the role of AGE modification in AD and

other neurodegenerative disorders, in the reaseach performed

immunohistochemical studies using antibodies for AGEs, A,

tau, ubiquitin, and apolipoprotein E (ApoE). They research

data demonstrated that AGE modification was involved in

pathological changes observed in both AD and other

neurodegenerative disorders, implying that AGEs is important

factor in the progression of various neurodegenerative

disorders. The binding of AGEs to specific receptors can also

generate oxidative stress. As well as the production of pro-

inflammatory cytokines. In various neurodegenerative diseases

other than AD, AGEs were present in some neuropathological

structures, including senile plaques, NFTs, Pick bodies, and

granulovacuolar degeneration. Under these circumstances,

oxidative stress may be induced by AGE generation and

receptor-mediated reactions (Sasaki et al., 1998)

II.1.6. Gamma Mangosteen

Gamma Mangosteen is a substance of the mangosteen fruit,

especially in the skin of it. Mangosteen is a tropical fruit.

There are many places for mangosteen fruit in the South

Eastern Asia, especially Indonesia. The taste of mangosteen

flesh is sweet and the skin has many benefits and one of it is

high anti-oxidant. The major bioactive compounds found in

mangosteen are alpha and gamma mangosteen. The chemocal name

of Gamma-mangostin is; 31271-07-5; 1,3,6,7-tetrahydroxy-2,8-

bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one which has

molecular formula; C23H24O6 and 396.43306 g/mol of Molecular

weight. The benefit as anti-oxidant has been proved that the

Gamma Mangosteen is more pothent than vitamin C as an

antioxidant (Chin et al., 2008). there are another benefits of

Gamma Mangosteen which proved by researcher. And that another

benefit is anti-inflammatory which blocking the release of

prostaglandin (PGE2) and inhibiting the expression of

cyclooxigenase-2(COX-2) in protein and RNA levels (Nakatani et

al., 2002; Nakatani et al., 2004). Gamma Mangosteen is also

known effective for the treatment and prevention of non

insulin-dependent diabetes by exhibited inhibitory activity

against alpha-amylase (Loo AEK et al., 2007).

 A C

Figure 4. Gross appearance of manogsteen (A and B

(Shibata et al., 2013)and chemical structure of Gamma
Mangostin (C) (Chang et al., 2012).

The study that was conducted by Bumrungpert et al (2009)

has proven that Mangosteen (that contained Alpha Mangosteen

and Gamma Mangosteen) acts as an anti inflamatory

agent. First, in dose 3 mmol/L mangosteen most effectively

decreased the mRNA levels of several candidate inammatory

genes exposed to lipopolysaccharide (LPS) which inammation

induced by 10 mg/L LPS in primary cultures of human

adipocytes. In other words, mangosteen is effectively

decreases LPS-induced Inflammatory gene expression. Decreased

the induction by LPS of inammatory genes, including tumor

necrosis factor-a, interleukin (IL)-1b, IL-6, IL-8, monocyte

chemoattractant protein-1, and Toll-like receptor-2 In this

case, they demonstrated that Gamma Mangosteen was more

effective than alpha mangosteen.

Secondly, they examined the effects of alpha and gamma

mangosteen on mitogen-activated protein kinases (MAPK)

phosphorylation. The MAPK acts as the activator of

transcription factors that induce inflammatory gene

expression. The data demonstrated that alpha and gamma

mangosteen attenuate the activation by LPS of MAPK in primary

cultures of human adipocytes. This case also demonstrated that

gamma mangosteen was more effective than alpha mangosteen on

an equimolas basis. Thirdly, Alpha and Gamma Mangosteen are

effective to decrease LPS-mediated NF-kB and AP-1 activation.

The Activation of NF-kB and AP-1 play important roles in the

transcriptional activation of inflammatory genes. In this

result also demonstrate that Gamma Mangosteen is more

effective than alpha mangosteen in blocking LPS-mediated NF-kB

and AP-1 activity in primary cultures of human adipocytes.

Other result also shows that mangosteen prevents LPS-

induced insulin resistance, especially in gamma attenuated the

suppression by LPS of the mRNA levels of PPARg and

adiponectin. These genes are needed for insulin stimulated

glucose uptake and utilization. This prevention activity

becomes possible by inhibiting inflammation and the

suppression of PPARg or its target genes, because inflammation

in white adipose tissue (WAT) is intimately linked to insulin

resistance.
 II.2. Basic Theory

Luteinizing Hormone (LH) is Gonadotropin Hormone that

composed of a common -subunit which is non-covalently

associated by a hormone-specific -subunit. Luteinizing

Hormone has many functions to target organ which purposed, one

of the example is leydig cell in the Testis. The process of it

is the binding of LH and LHR in the leydig cell which create

the activation of Testosteron production. The function of

Testosterone is very importance inside of our body,such as for

the development of male secondary sexual characteristics,

stimulation of sexual behavior and function, and initiation of

sperm production, maintaining muscle mass and strength, fat

distribution, bone mass, red blood cell production, male hair

pattern, libido and potency, and spermatogenesis. If there is

a destruction on Leydig cell in the Testis will create

destruction on Luteinizing Hormone Receptor (LHR). Because of

the reason, it will create many clinical manifestations, such

as loss of libido, erectile dysfunction, diminished

intellectual capacity, depression, lethargy, osteoporosis,

loss of muscle mass and strength, and some regression of

secondary sexual characteristics.

The destruction on Leydig cell or one of cell organ

(Luteinizing Hormone Receptor), as known many factors that

influenced. One of them is caused by stress oxydative which

induced by Advanced Glycation End-product (AGE). Advanced

Glycation End Products (AGE) is a protein or lipid becoming as

non enzymatically glycated and oxydized after reacting with

Aldose Sugars. Advanced Glycation End-product itself needs a

receptor for giving a reaction on the target. The Rcseptors

for AGE will initiate the intracellular signaling that

disrupts cellular function through its recognition and binding

of AGEs. Advanced Glication End Receptor (RAGE) is a part of

the immunoglobulin superfamily of receptors. The response of

AGE creates a formation for reactive oxygen species (ROS) in

the intracellular, which activating NF-B. So, there are many

expressions of cytokine will increase, such as tumour necrosis

factors (TNF- and TNF-), interleukins (IL) 1, 6, 8 and 18,

and interferon-. The most susceptible sites of damaging areas

that caused by ROS is cell membrane. Because of that could

caused cell rupture and LHR in the membrane cell getting

damage. The increasing of Advanced Glycation End-product often

found on Diabetes mellitus type 2 patients. On Diabetses

Mellitus type 2 patients has formation and deposition

advanced glycation end products (AGEs) molecule which the

result of protein glycation and lipid or glucose cause of the

increasing of blood glucose in the body.

Gamma Mangostion is a substance derived from the

mangosteen fruit which found in Indonesia. Gamma Mangosteen is

already known has benefits as anti oxidant and inhibit the

process of inflammation with blocked prostaglandin (PGE2)

release and inhibited the expression of COX-2 in protein and

RNA levels. So, it is approximatelly could prevent the

destruction of leydig cell which caused by AGE. It is

developed formany kind of treatments for disease.

 II.3. Teoritical Framework

Explanation :
: getting of
: binding
--- : preventing decrease
: caused
: decrease
 Shortly, Advanced Glycation End-product could be found in

environment, aging process, hypoglycemia in the Diabetic

patient. After binding AGE with AGE receptor in leydig cell

will increase the ROS and some inflammatory factors, such as

interleukin (1, 6, 8, 18), Interferon- , NF-KB, and Tumor

Necrosis Factor (TNF- and TNF-). The increasing of ROS and

inflammatory process on leydig cell will create the

destruction for leydig cell itself. On the ROS it self could

destroy protein structure, DNA, and lipid Membrane on the cell

itself which will induce cell destruction. The destruction of

this cell will create destruction for LH receptor on Leydig

cell. Another case ROS has effect for lipid membrane will

destroy the LH receptor directly. The decrease of LH receptor

will create many clinical manifestation. One of them is the

decrease of testosterone level, such as depression, erectile

dysfunction, hypogonadism, decrease cognitif ability,

osteoporosis and, loss of muscle strength.

With additional of Gamma Mangosteen expected could

prevent and decrease inflammatory process. Because of Gamma

Mangosteen has a function as antioxydant.

 II.4. Conceptual Framework

Advaced Glycation End-product

e leydigcell culture of sprawgue dewley rats Cell damage

The expression of Huteinizing Hormone R

Gamma Mangosteen

Explanation;

: Become (encounter)

: affects

: applied

shortly, The leydig cell culture of sprawgue dewley rats

will give Andvanced Glication End which cause the destruction

of cell. So it has effect on the decreasing of Huteinizing

Hormone Receptor (LHR) expresions. After the additional of

Huteinizing Hormone Receptor.

 II.5. Hypothesis

There is the increase of Luteinizing Hormone Receptor

(LHR) expressions on the Leydig Cell Culture of Sprague Dawley

Rat that induced by Advanced Glycation End-product and Gamma

Mangosteen compared to the leydig cell culture which only

given Advanced Glycation End-product.

CHAPTER III. RESEARCH METHOD

III.1. Study Design

This research use secondary data with experimental

laboratory study.

III.2. Time and Study Setting

This research is conducted in the laboratory of

Endocrinology, Physiology Department of the Faculty of

Medicine Uiversitas Gadjah Mada on april 2014 until april

2015.

III.3. Research Population and Subject

1. Population : the population research is using by the

result data of research by dr.Dicky Moch Rizal.

2. Subject : the subject of research based on inclusion and

exclusion criteria.

1)Inclusion criteria : the value of Luteinizing Hormone

Receptor (LHR)expresion on the Leydig cell culture in Sprague

Dawley rat which induced by Glycation End-product

(AGE)comparing with Leydig cell culture in Sprague Dawley rat

which induced by AGE and given Gamma Mangosteen. As the result

data of research by Dr.dr. Dicky Moch Rizal Sp.And.,

AIFM,M.Kes. Leydig cell cutures in this research devided into

3 groups, there are ;

1. group 1 : Leydig cell culture (control, without treatment)

2. group 2 : Leydig cell culture + AGE- BSA 200g/ml.

3. group 3 : Leydig cell culture + AGE- BSA 200g/ml + Gamma

Mangostin 5M.

2)exclussion criteria : the result data of research by

Dr.dr.Dicky Moch Rizal Sp.And., AIFM,M.Kes which not include

the expression of Luteinizing Hormone Receptor (LHR).

III.4. Study Variables

Variables of this study are:

a. Dependent variable: Expression of Luteinizing Hormone

Receptor (LHR)in the leydig cell culture.

b. Independent variables: solution of AGE-BSA and Gamma

Mangosteen.

III.5. Study Material and Tools

This research used secondary research result from

Dr.dr.Dicky Moch Rizal Sp. And., AIFM, M.Kes. below is

material and tools used by Dr.dr.Dicky Moch Rizal Sp. And.,

AIFM, M.Kes to get primarry result:

1 Leydig cell culture production : laminar airflow

(Labconco), pH meter electric, volumetrik pipe,

eppdendorf pipe, cold Sentrifuge, Autoclaft (Hirayama),

CO2incubator, vortex, magnetic stirer, medium bottle,

culture plate 24 well, milipore diameter pore 0,22 um.

2 Leydig cell culture media: Ham medium 12:DMEM, Percoll,

Fetal bovine serum (FBS), Penstrep, gentamicin,

fungizone, amphotericin, Phosphat buffer saline (PBS),

 kolagenase, triphan blue, 3HsD, AGE-BSA dari Biovision,

Gamma Mangosteen of sigma Aldrich, ELISA kit

3 Observation of LH receptor expressions
ELISA kit. Reagents, Assay plate, Standard, Antibody,

HRP-conjugate, Wash Buffer, Substrate A, Substrate B,

Stop Solution, Adhesive Strip, Instruction manual.

III.6. Operational Definition of Variable

1. leydig cell culture is leydig cell culture taken of

Sprague Dawley rat testis organ which 90 days alive. And

the culture raised after getting confluent mostly for 7

days on special culture plate which content 24 wells. The

amount of leydig cell every a well is 1x105.

2. Gamma Mangosteen is derivate of xanthone. It is from the

skin of mangosteen fruit which extracted as powder. The

value of Gamma Mangosteen is 10M.

3. AGE-BSA solution as Advanced Glycation End Products-

Bovine Serum Albumin asextraction product of BSA which

incubated and added by glucose. The value is 200g/ml

4. The expression of LH receptor is amount of LH receptor

expression level on Leydig cell culture which measurable

by ELISA methode and has sensitivity 3.9 pg/dL.

III.7. Conduct of The Research

1. data collection

The collection of data researched on Expression of

Luteinizing Hormone Receptor (LHR) in the Leydig Cell

 Culture of Sprague Dawley Rat that induced by Advanced

glycation End-product and Gamma Mangosteen. It is from

the result research by Dr.dr.Dicky Moch Rizal, Sp.And.,

AIFM, M.Kes. Below is the explanation of research

procedure by Rizal (2014, 46-64) to get primary data.

a Removal of testis
90 days aged Sprague Dawley mices testicle with the
weight around 200 gram will be fasted for about 10 hours
before the taking of testis. The mice is anestesized with
0,3ml/100grBB HCL cetamine i.m. After that, the four leg
of the mice fixed with rope in the surgery table. Fur in
the area abdomen then drabbled with with wet cotton and
shaved until the skin in the surgery area is revealed.
The area then sterilized with alcohol and incisied for
about 2 cm long along the stomach center line with
scalpel. Following that, the specimen then peritoneum
incisied for about 1,5-2 cm long. Using a pair of curving
tweezers and small scissors, a cutlet for about 1 cm long
and 0,5 inch anterior towards the genital area was made
in the center line along the lower abdomen. The mices
skin was so loose that it can be opened to the right and
left direction to take every testis from just one
incision. Both vas deferens will then revealed from one
side of the testis. The left vas deferens then gently
gripped with pliers and lifted partially so that the
cutlet can be observed more clearly.
Vas deferense then ligated and cut in a way that has
explained above. After the testis had lifted, the cut
area then cleaned and observed if there is any
hemorrhage. After that, he peritoneum and skin were sewed
back with thread that can be absorbed by the body. The
scar then applied with iodine until dried. Lastly,
termination was done by applying an extended dose of
anesthesia.
b Leydig Isolation and Cell Culture
1 Prepare testicle and wash with PBS twice. After
that, mince it manually.
2 Prepare 0,25mg/ml Collagenase; 7ml per 1 mice.
Drop a few in the minced testicle and mince it
chemically.
 3 The result from previous action then will be put
in 37C incubation tube (2 mices in a 50 conical
tube, and then divided into 2)
4 Prepare 28ml DMEM. The result from the incubation
process then will be homogenized. After 2 minutes,
put supernatant
5 Supernatan will then be 1250rpm centrifuged for 7-
10 minutes, which will be resulted in pellets.
6 Throw away the supernatant and add 2ml DMEM in the
pellets. After that, 1250rpm centrifuge the
specimen for 7-10 minutes twice. Throw away the
Supernatant, take the pellets out, then add 500l
7 Prepare 60%, 40%, 34%, 26%, and 21% gradients (do
not let them become mixed up)
8 Put the pellets, which has been added with 500l
on the top of gradients.
9 Wing centrifuged with 2750rpm for 30-45 minutes.
10 Take the cells between 60% and 40%.
11 Centrifugation will be resulted in pellets. Wash
with PBS, centrifuge it again with the speed of
2000rpm.
12 Put MK DMEM + 1-2cc.
13 Put on TCD gelatin.
c) Post isolation and cell culture
1)Add AGE-BSA 200g/ml to group 2 and AGE-BSA
200g/ml + Gamma-mangostin 5M to group 3.
2)Homogenisation of each groups.
3)Centrifuge it, then take the supernatan.
4)The result from previous action then will be put
in -80C incubation for 2 weeks.

d) The expression of LHR detection

The expression of LHR detection which measurable by
ELISA methode. Assay procedure :
1) Bring all reagents and samples to room
temperature before use.
2) Centrifuge the sample again after thawing before
the assay.
3) Prepare all reagents and samples as directed in
the previous sections.
4) Determine the number of wells to be used and put
any remaining wells and the desiccant back into
the pouch and seal the ziploc, store unused wells
at 4C.
5) Set a Blank well without any solution.
6) Add 50l of Standard or Sample per well. Standard
need test in duplicate.
7) Add 50l of HRP-conjugate to each well (not to
Blank well), then 50l Antibody to each well. Mix
well and then incubate for 1 hour at 37C.
 8) Aspirate each well and wash, repeating the
process two times for a total of three washes.
Wash by filling each well with Wash Buffer
(200l) using a squirt bottle, multi-channel
pipette, manifold dispenser, or autowasher, and
let it stand for 10 seconds, complete removal of
liquid at each step is essential to good
performance. After the last wash, remove any
remaining Wash Buffer by aspirating ordecanting.
Invert the plate and blot it against clean paper
towels.
9) Add 50l of Substrate A and 50l of Substrate B
to each well, mix well. Incubate for 15 minutes
at 37C. Keeping the plate away from drafts and
other temperature fluctuations in the dark.
10)Add 50l of Stop Solution to each well, gently
tap the plate to ensure thorough mixing.
11)Determine the optical density of each well within
10 minutes, using a microplate reader set to 450
nm.
e) Viability test of Trypan blue assay.
1) Prepare a cell suspension in a balanced salt
solution (e.g., Hanks= Balanced Salts [HBSS],
Product No. H 2513).
2) Transfer 0.5 ml of 0.4% Trypan Blue solution
(w/v) to a test tube.
3) Add 0.3 ml of HBSS and 0.2 ml of the cell
suspension (dilution factor = 5) and mix
thoroughly. Allow to stand for 5 to 15 minutes.
4) Trypan Blue should be sterile filtered before
using it in order to get rid of particles in the
solution that would disturb the counting process.
5) With the cover-slip in place, use a Pasteur
pipette or other suitable device to transfer a
small amount of Trypan Blue-cell suspension
mixture to both chambers of the hemocytometer.
6) Carefully touch the edge of the cover-slip with
the pipette tip and allow each chamber to fill by
capillary action. Do not overfill or underfill
the chambers.
7) Starting with chamber 1 of the hemocytometer,
observe cells under microscope in four 1 x 1 mm
squares of one chamber.
8) Non-viable cells will be blue, viable cells will
be unstained.
III. 8. Data Analysis

Data analysis achieved on data of Luteinizing Hormone

Receptor (LHR) expressions in the Leydig cell culture of

Sprague Dawley rat that induced by Advanced glycation End-

product and Gamma Mangosteen. After that the data which

achieved then analysing by method below :

a Data divided into 3 groups. Group 1 (control, without

treatment), group 2 (leydig cell culture + AGE-BSA

200g/ml), and group 3 (leydig cell culture + AGE-BSA

200g/ml + Gamma Mangosteen 5M). Distribution and

normality data analysed by normality Shapiro-Wilk test.

It is cause of the sample amount every group is <50

sample.
b There is a test of average characteristic comparison on

research subject used one way ANOVA test. If the normal

data distribution and varians as same as on every groups.

One of the requirements one way ANOVA tes has not fulfill

(abnormal data distribution or varians are not same) used

Kruskal-Wallis test. Then the testing continued for

seeking group which is different meaning by post hoc

analysis. The result of normality test and meaningfull

comparison is p<0,05.

CHAPTER IV. RESULT AND DISCUSSION

 IV. 1. Result

IV. 1.1. Result of Normality Statistical Data.

Since the total of the data in each group is less then

50 subjects, the data that had achieved were firstly tested

with the data normality test, which was Shapiro Wilk test. The

test resulted in normal values, which then concluded that the

data is normal distribution.

IV. 1. 2. The Expression of LHR in The Leydig Cell Culture

Cell culture groups of this research are divided into

3, which are group 1 (control, without treatment), group

2(leydig cell culture + AGE-BSA 200g/ml), and group 3(leydig

cell culture + AGE-BSA 200g/ml + gamma-mangostine 5M). Each

group consist of 3 sample.

After the data normality test was conducted, the

process then continued with the measurement of mean and

standard devitiation in every group. This action followed by

other action such as comparing the means of each group until

the value of p was discovered. After that, the writers of the

research can conclude that the normal distribution and

variance had achieved. Thus, the requirements of one way ANOVA

test have been met.

Table 1. The Mean Value of Research Subject Characteristic.

Characteristic group 1 group 2 group 3 p
(n=3) (n=3) (n=3)
The Level of 7,78+0,00 7,79+0,01 8,06+0,26 0,10
LH Receptor
5
(pg/ 10 /24h)

Explanation: *P value has no significant differences of every

5
groups(P= 0,10 pg/ 10 /24h).
Data presented above were presented in mean+deviation
standard, which value p was required from one way ANOVA test.

Group 1 = control
Group 2 = AGE-BSA 200g/ml
Group 3 = AGE-BSA 200g/ml + Gamma-mangostin 5M

5
Looking on the table above P value is 0,10 pg/ 10 /24h, it is

concluded that the mean of every groups has no significant

differences (p=0,10).

Table 2. The Mean Value of Post-Hoc Least Significant

Difference(LSD) analysis.

Variable Comparison groups P

Expression of LHR Group.1-group.2 0,967
Group.1-group.3 0,60
Group.2-group.3 0,64
Explanation : *P value has no significant(p>0,05)
Group 1 = control
Group 2 = AGE-BSA 200g/ml
Group 3 = AGE-BSA 200g/ml + Gamma-mangostin 5M

After the Post-Hoc Least Significant Difference(LSD)

test was conducted, there is no significant differences of the

mean comparison value in group 1 (control) with group 2 (AGE-

BSA 200g/ml), group 1 with group 3 (AGE-BSA 200g/ml + Gamma-

mangostin 5M) , and group 2 (AGE-BSA 200g/ml) with group 3

(AGE-BSA 200g/ml + Gamma-mangostin 5M).

IV. 2. Discussion

Based on the result The writers concluded that after

giving the treatment (AGE and gamma-mangostin) shows in the

various data. The LHR expression shows in lower level on group

1(7,78 pg/dl) and the highest on group 3(8,06 pg/dl) and group

2(7,79)is in the middle level of it. Based on theory above,

after giving AGE ( group 2) will get a result at the lower

level than control group (group 1). In the group 3 shown

highest level of LHR expression that compered with group 2.

this resulted in a same way with the theory that stated that

gamma-mangosteen is able to hamper the inflamation process by

inhibit LPS-induced increase in the expression of COX-2

protein in a concentarion dependent manner and also have the

ability as an antioxydant.

On Rasheed et al (2011) investigate whether advanced

glycation end products (AGEs) induce the expression of IL-6

and IL-8 through the receptor for AGEs (RAGE)-activated

pathways in human OA chondrocytes. these studies was

explained that AGE-BSA up to 200g/ml had no significant

cytotoxic effects on OA chondrocytes compared with controls

treated with 200g/ml native BSA (P>0.05). then, they treated

primary human OA chondrocytes with AGE-BSA (5100g/ml) for 0

24h and results showed that AGE-BSA significantly up-regulated

the mRNA expression of IL-6 and IL-8. IL-6 is a pleiotropic

cytokine with a wide range of biological activities, including

immunoregulation and that functions in inflammation and the

maturation of B cells. Dysregulated overproduction of IL-6 is

suspected in the systemic inflammatory manifestations.

Whereas, IL-8 induces a massive accumulation of neutrophils,

which produce neutrophil elastase, leading to cartilage

destruction and induces chemotaxis in target cells. Expression

of IL-6 and IL-8 proceeds via the activation of mitogen-

activated protein kinases (MAPKs) and nuclear factor (NF)-B

in response to various stimuli.

The research of Protection Effect of Endomorphins on

Advanced Glycation End Products Induced Injury in Endothelial

Cells, investigated the effects of Endomorphin on the

synthesis and secretion of vasoactive substances induced by

advanced glycation end products in primary cultured human

umbilical vein endothelial cells. By seeing the result from

this study, we can see that incubation with AGEs (100mg/L =

100 g/ml) for 24h led to an significantly increase in the NO,

iNOS production and a decrease in the secretion and the mRNA

expression of eNOS compared to control group. In explanation,

NO produced by eNOS is described as low output pathway

whereas iNOS generates NO in a high output manner which

causes cell or organ dysfunction and apoptosis. In other side,

Endomorphin treatment can enhance NOS activity by up-

regulating eNOS expression and decreasing iNOS production,

which then leads to a health production and bioavailability

of NO. It is indicated that Endomorphin can attenuate the

dysfunction of NOS induced by AGEs. In this research article,

the writer argumented two things, which are first, with AGE

100 mg/L (100g/ml) dose, it is effective enought to create

oxidative stress reaction and sell destructions. Secondly,

Endomorphin appeared as an effective cell to hamper or inhibit

the reaction that caused by AGE in the exception of gamma-

mangosteen. (Liu et al., 2013).

Basically, a cell has survival of adaptation mechanism

to handle the stress to which it is exposed. Cell survival and

daeth depending on the level and mode of stress exposure. If

stressstimuly is unresolved, then cell activate death

signaling pathway to eliminate these damage.

For example on the research by Sivakumar et al (2006) about

Radiation Exposure Impairs Luteinizing Hormone Signal

Transduction and Steroidogenesis in Cultured Human Leydig

Cells explained that Leydig cell surface LH/hCG receptor

concentration signicantly decreased, following higher doses

of radiation exposure (6 Gy and above). However, lower doses

(2 and 4 Gy) did not signicantly affect the receptor

concentration. LH receptor mRNA expression was increased

slightly in Leydig cells following 2 Gy of radiation exposure.

The increased expression of LH receptor mRNA in Leydig cells

at 2 Gy of exposure may be the adaptive response of Leydig

cells to radiation.

Based on theory, AGE has destruction effect on the DNA

cells via Reactive Oxygen Species (ROS). DNA double strand

breaks (DSBs) and single strand breaks (SSBs) are considered

as key lesions that initiate the activation of the DNA damage

response. DNA damage initiates one of several mammalian DNA

repair pathways, which eventually restore the continuity of

the DNA double strand. The former constitutes the predominant

DNA repair pathway in mamalian and involves DNA repair

proteins such as DNA-PK, Ku70, and Ku80. Base damage can be

repaired either by enzyme-catalyzed reversal or alternatively

via excision repair . Mismatch repair is responsible for the

removal of incorrectly paired nucleotides.

To adapt with damage cells caused by ROS, cells also has

mechanism to defens these exposure by producing aintioxidan.

These include several detoxifying enzyme, for example

catalase, GSH peroxidase, and superoxide, and superoxide

Dismutase (SOD). When the cells antioxidant defenses are

overwhelmed, ROS can induce cell death. It can be concluded

that on this study administrati on of AGE 200g/ml is not

possible cause the damage of leydig cell. Because the leydig

cell is still in in adaptation or survival phase. So, there is

no decline in the LH receptor expression (Fulda et al., 2010).

 The Gamma Mangostin concentration also give effect to

the result. the insignificant (P>0.05) data after giving gamma

mangostin is may possibly due to the lack of Gamma Mangostin

concentrated that was exposed. The Gamma Mangostin

concentration that was used in this research is the 5M,

where in the previous research that was conducted by Nakatani

et al. (2004) used the 10M, which compound (Gamma Mangostin)

showed a 70% inhibition of the LPS-induced increase in

expression of COX-2 protein in C6 rat glioma cell. In other

words, it is proven that in such dose the Gamma Mangosteen has

the ability to detain the inflamation process.

Another research explained that the expression of LH

receptor that used Adult male (90 to 120 days old) Sprague

Dawley rats with two experimental paradigms: the rat treated

with ethylene dimethan esulfonate(EDS) and the EDS-rat treated

with a high dose of testosterone (EDS+T). The pattern of LHR

expression was evaluated in the two experimental paradigms by

means of binding assay and Northern hybridization analysis.

The administration of the cytotoxic drug ethylene dimethane

sulfonate (EDS) induces selective destruction of mature Leydig

cells. In EDS-treated rats, specific testicular binding

of[125I]iodo-hCG,asestimate of the total number of LHRs, fell

to undetectable levels on 5 days and 15 days. Then, appearing

recovery condition continuously and 20 days after treatment

happens recovering partially (;10% of control levels), and

returned to control levels 45 days after treatment with EDS.

In EDS+Testosterone rats, a similar drop in LHR binding was

detected on days 5 and 15 after treatment. However, under the

LH suppressed conditions, no recovery of [125I]iodo-hCG

binding was detected on days 20 and 45. Because on 5 days

EDS+Testosterone rats detects serum T significantly increased,

Serum LH levels decreased gradually 5, 15, and 20 days,and

Serum FSH levels also decreased. Then on EDS-treated rats on

the contrary.

The multiple species of LHR mRNA, with four major bands

of 6.8, 4.2, 2.7, and 1.8 kb size, were detected on Northern

hybridization analysis of total testicular RNA from control

animals. In EDS treated rats, persistent expression of the

1.8-kb band of LHR mRNA was detected 5 and 15 days after EDS,

whereas the longer forms (6.8, 4.2, and 2.7 kb) of the message

disappeared. On 20 and 45 days after EDS, the pattern of

hybridization of these splice variants gradually returned to

that resembling the controls. In EDS+Testosterone rats, a

pattern similar to that of EDS rats, i.e. persistent

expression of the 1.8-kb form with the absence of the other

transcripts, was detected on days 5 and 15 after treatment.

However, no recovery in the expression of the longer forms of

the LHR message was observed, whereas the intensity of

expression of the 1.8-kb band gradually increased along the

experimental period. On the research appears an ability for

recovering continuously on LH receptor expression in the

testis Sprague Dewley rats by given treatment that trigger

cell damage.(Tena et al., 1997).

Viability assay is important test to determinethe

ability of cells to maintain or recover viability. Viability

can be distinguishedfrom the all or nothing statesof life and

death by use of a quantifiable index. This study already used

Trypan blue test that show selectively colour dead tissues or

cells blue(diazo dye). We didnt count specifically in the

percentage value, but we done with observation of the cell

structure. Live cells with intact cell membranes are not

coloured. Since cell are very selective in the compounds that

pass through the membrane, in a viable cell trypan blue is not

absorbed. However, it traverses the membrane in a dead cell.

The dead cell are shown as a distinctive blue colour under

microscope. This methode is commonly used in microscopy and in

laboratory rat and mice for assessment of cell viability. For

example in the research by Morgan et al(2015) about apotosome

activation, an important molecular instigator in 6-

mercapturopurine induced leydig cell death. For viability

assays, Leydig cells were cultured at a density of 106 cells

per ml in six-well plate and incubated with indicated drugs

for 12 hours. Subsequently, the cells were harvested and

immedietely assessed trypan blue dye exclussion.

Other example of viability assay that already applied

to the leydig cell culture in the research by Huo et al (2012)

about Effect of norepinephrine and acetylcholine on the

development of culture leydig cells in mice is MTT

tetrazolium cell viability assay. MTT tetrazolium assay has

been widely adopted and remains popular in academic labs as

evidenced by thousands of published article. The MTT

substrate,is prepared in physiologically balanced solution,

added to cells in culture, usually at a final concentration of

0,2-0,5 mg/ml, and incubated for 1 to 4 hours. Then, it is

measured by recording changes in absorbance at 570 nm using a

plate reading spectrophotometer. Viable cells with active

metabolism convert MTT into puple colored. When cells die,

they lose the ability to convert MTT into purple colored. Thus

color formation serves as a useful and convinient marker of

only the viable cells. The exact cellular mechanism is not

well understood, but likely involves reaction with NADH or

similar reducing molecules that transfer electrons to MTT.

CHAPTER V. CONCLUSION AND SUGGESTION

V. 1. Conclusion

Based on the result of this research, the writer conclude:

1. The Expression of Luiteinizing Hormone Receptor (LHR) level

is higher on the leydig cell culture which given Advanced

Glycation End-products compere with control (no treatment).

2.After giving gamma-mangostin, the expression of Luiteinizing

Hormone Receptor (LHR) level is higher then leydig cell

culture which only given Advanced Glycation End-product.

3. The mean of LHR levels in the all group, include in the

Expected levels.(3.9 pg/ml)

V. 2.Suggestion

1.The researcher suggested to know how is the system of LH

receptor damage specifically. Then, after knowing the

mechanism of the process could find a way to give a

prevention for the damage of LH receptor.

2.The reseacher suggest to effort in the indentification of

method or the level of substances that this study

used(Advanced Glycation End-product and gamma-mangostin). To

 find out there is an eror in this study or justify the

result of this study.

3.The other of researchers suggest to find out the prevention

of destruction on organ that caused by the effect of

accumulation of Advanced Glycation End-product in the body.

CHAPTER VI. REFERENCES

Ames, BN, Shigenaga, MK, Hagen, TM, 1993, Oxidants,

antioxidants, and the degenerative diseases of aging. Proc
Natl Acad Sci U S A, vol.90, no.17, pp.7915-7922.

Basta, G, Schmidt, AM, Caterina, RD 2004, Advanced Glycation

End Products and Vascular Inflammation: Implications for
Accelerated AtherosclerosisiIn Diabetes. J Cardiovasc,
vol.63, pp.582 592.

Bayne, JW, Thorpe, SR 1999, Role of oxydative stress in

diabetic complication a new perspective on an old
papadigm. J Diabet, vol.48, pp.1-9.
Bierhaus, A, Chevion, M, Hofmann, M, Quehenberger, P, Illmer, T, Luther, T,
Berentshtein, E, Muller, M, Wahl, P, Ziegler R, Nawroth, 1997, Advanced
Glycation End Product-Induced Activation of NF-KB IS Suppressed by a-
Lipoic Acid in Cultured Endothelial Cells. Int med, vol.46 pp.1481-1489.

Boel, E, Selmer, J, Flodgaard, HJ, Jensen, T 1995, Diabetic

late complicatioms: will aldose reductase inhibitor or
inhibitors of advanced glysosylation end-product formation
hold promise?, J Diabet Complication, vol.9, pp.104-129.

Brownlee, M 1995, Advanced protein glycosylation in diabetes

and aging. Ann Rev Med, vol.46, pp.223234.

Brownlee, M, Vlassara, H, Cerami, A 1985, Nonenzymatic

glycosylation products on collagen covalently trap low-
density lipoprotein. J Diabet, vol.34, pp.938941.

Bumrungpert, A, Ruchaneekorn, W, Kalpravidh,

Chitchumroonchokchai, C, Chuang, C, West, T, Kennedy A,
McIntosh, M 2009. Xanthones from Mangosteen Prevent
Lipopolysaccharide-Mediated Inammation and Insulin
Resistance in Primary Cultures of Human Adipocytes. Dep
Nutri, vol.139 pp.1185-1191.

Ceriello, A 1999, Hyperglycaemia: the bridge between non-

enzymatic glycation and oxidative stress in the
 pathogenesis of diabetic complications. Diabet Nutri Met,
vol.12, pp.42-46.

Chang, H, Yang, L 2012. Gamma-Mangostin, a Micronutrient of

Mangosteen Fruit, Induces Apoptosis in Human Colon Cancer
Cells. Pharmacol, vol.17 pp.8010-8021.
Chen, H, Matthew, P, Hardy, Barry, R, Zirkin 2002, Age-Related
Decreases in Leydig Cell Testosterone Production Are Not
Restored by Exposure to LH in Vitro. The Endocrinol soc,
vol. 143, no.5, pp.16371642.
Chin, YA. Kinghorn, D, 2008, Structural Characterization, Biological Effects, and
Synthetic Studies on Xanthones from Mangosteen (Garcinia mangostana), a
Popular Botanical Dietary Supplement. Rev Org Chem, vol.5, no.4, pp.355
364.

Esser, K, Stadtman, ER, Wiley, C, Martin, GM 1995, The status

of oxidatively modified proteins as a marker of aging,
Eds. Mol Aspects of Aging, pp.129-144.

Fu, MX, Wells-Knecht, KJ, Blackledge, JA, Lyons, TJ, Thorpe,

SR, Baynes, JW 1994. Glycation, glycoxidation, and cross-
linking of collagen by glucose kinetics, mechanisms, and
inhibition of late stages of the Maillard reaction. J
Diabet, vol.43, pp.676683.

Fulda, S, Gorman, AM, Hori, S, Samali,A 2010, Cellular Stress

Responses: Cell Survival and Cell Death. Inter J Cell
Biol, Vol.2010, Article ID.214074, pp. 23.
http://dx.doi.org/10.1155/2010/214074 (accessed on 1
december 2015).

Goldin, A, Beckman, JA, Schmidt, AM, Creager, MA, 2015,

Advanced Glycation End Products Sparking the Development
of Diabetic Vascular Injury. Cardiovasc Division, Boston
pp.4-5.
Goluza, 2014, Macrophages and Leydig Cells in Testicular
Biopsies of Azoospermic Men, Biol Med Research Inter.
http://dx.doi.org/10.1155/2014/828697 (accessed on 1 April
2015)

Huebschmann, AG, Regensteiner, JG, Vlassara, H, Reusch, JEB

2006, Diabetes and Advanced Glycoxidation End Products.
Diabet Care, vol.29, pp.1420.

Huo, S, Zhong, X, Wu, X, Li, Y 2012, Effect of norepinephrine

and acetylcholine on the development of culture leydig
cells in mice. J Biol med and Biotech, vol.2012, Article
ID.503093, pp.8
Irina, G, Tikhonova, Costanzi, S 2009, Unraveling the
structure and function of G protein-coupled receptors
through NMR spectroscopy. Lab Biol Mod, Vol.15, No.35,
pp.40034016.
Li, J, Schmidt, AM 1997, Characterization and functional analysis of the promoter
of RAGE, the receptor for advanced glycation end products. J Biol Chem
vol.272 pp.1649816506.

Linn, S, Esser, K, Martin, GM, Chichester, W 1995, DNA repair

in mitochondria: How is it limited? What is its function?
Eds. Mol Aspects of Aging, pp.199-225.
Liu, G, Duranteu, L, Claue, J, Monroe, J, Daniel, A, Doyle,
Shenker, A 1999, Leydig-cell Tumors Caused by an
Activating Mutation of Gene Encoding The Luteinizing
Hormone Receptor. Brief Report vol.341, pp.1731-1735.
Liu, J, Yan, L, Niu, R, Tian, L, Zhang, Q, Quan, J, Liu,
H, Wei, S, Guo, Q 2013, Protection Effect of Endomorphins
on Advanced Glycation End Products Induced Injury in
Endothelial Cells. J Diabet Research, Vol.2013,
Article.ID 105780, pp.9.

Loo AEK, Huang D, 2007, Assay-guided Fractionation study of -

amylase inhibitors from Garcia Mangostana pericarp. J
Agricult Food Chem, Vol.55, pp.98059810.
Morgan, JA, Linch, J, Schuetz, JD 2015, Apotosome activation,
an important molecular instigator in 6- mercapturopurine
induced leydig cell death. Dep Pharmacol sci, vol.5
pp.16488.
Nakatani, K, Nakahata, N, Arakawa, T,Yasuda, H, Ohizumi, Y,
2002, Inhibition of cyclooxygenase and prostaglandin E2
synthesis by -mangostin, a xanthone derivative in
mangosteen, in C6 rat glioma cells. J Biochem Pharmacol,
vol.63, pp.7379.
Nakatani, K, Yamakuni, T, Kondo, N, Arakawa, T, Oosawa, K,
Shimura, S, Inoue, H, Ohizumi, Y 2004, Mangostin
inhibits Inhibitor-kB kinase activity and decreases
lipopolysaccharide-induced cycloocygenase-2 gene
expression in C6 rat glioma cells. J Mol Pharmacol,
vol.66, pp.667-674.

Nieschlag, E, Behre, HM, 2010, Textbook of Male Reproductive

Health and Dysfunction, 3th edn, Springer, Berlin.

Ogiwara, K, Fujimori, C, Rajapakse, C, Takahashi, T 2013,

Characterization of Luteinizing Hormone and Luteinizing
Hormone Receptor and Their Indispensable Role in the
Ovulatory Process of the Medaka. PLos ONE, 8(1), p.e54482.
Rajfer , J 2003, Decreased Testosterone in the Aging Male. J Rev of Urol, 5(Suppl
1):S1-S2.

Ran, O, Gu, M, Van Remmen, H 2006. Glutatione peroxidase 4

protects cortical neurons from oxidative injury and
amyloid toxicity. J Neuro Sci Res, vol.84, pp.202-208.
Rasheed, Z, Akhtar, N, Tariq, Haqqi, M 2011, Advanced glycation
end products induce the expression of interleukin-6 and
interleukin-8 by receptor for advanced glycation end
product-mediated activation of mitogen-activated protein
kinases and nuclear factor-B in human osteoarthritis
chondrocytes. J Rheumatol (Oxford), vol. 50, no.5 pp. 838
851.

Rizal, DM 2014, Kadar Testosteron, Ekspresi COX-2, Reseptor

Advanced Glycation End Products, dan Reseptor Luteinizing
Hormone Pada Kultur Sel Leydig Tikus Sprague Dawley yang
diinduksi Advanced Glycation End Products dan diberi
Gamma-mangostin, Proposal Disertasi Doktoral. Universitas
Gadjah Mada, Yogyakarta.

Roess, DA, Brady, CJ, Barisas, BG, 2000, Biological function

of the LH receptor is associated with slow receptor
rotational diffusion. Vol.1464.

Rothstein, M, Lippman, RD, Liss, AR 1983, Lipid peroxidation

and metabolism in aging. Rev Biol Research in Aging,
vol.1, pp.315-342.
Sasaki, N, Fukatsu, R, Tsuzuki, K, Hayashi, K, Yoshida, T,
Fujii, N, Wakayama, I, Yanagihara, R, Garutto, R, Amano,
N, Makita, Z, 1998, Advanced Glycation End-products in
Alzheimers Disease and Other Neurodegenerative Disease.
Am J Pathol, Vol.153, no.4, pp.11491155.

Schmidt AM, Yan SD, Yan SF, Stern, DM 2001, The multiligand
receptor RAGE as a progression factor amplifying immune
and inflammatory responses. J Clin Invest, vol.108,
pp.949955

Schmidt, AM, Mora, R, Cao, R, Yan, SD,Brett , J, Ramakrishnan,

R, Tsang, TC, Simionescu, M, Stern, D 1994, The
endothelial cell binding site for advanced glycation end
products consists of a complex: an integral membrane
protein and a lactoferrin-like polypeptide. J Biol Chem
vol.269, pp.98829888.

Schmidt, AM, Vianna, M, Gerlach, M, Brett, J, Ryan, J, Kao, J,

Esposito, C, Hegarty, H, Hurley, W, Clauss, M, Clauss, FW,
Yu-Ching, EP, Tsang, TC, Stern, D 1992, Isolation and
 characterization of two binding proteins for advanced
glycosylation end products from bovine lung which are
present on the endothelial cell surface. J Biol Chem,
vol.267, pp.1498714997.

Schmidt, AM, Yan, SD, Wautier, JL, Stern, D 1999, Activation

of receptor for advanced glycation end products: a
mechanism for chronic vascular dysfunction in diabetic
vasculopathy and atherosclerosis. Circ Resp, vol.84,
pp.489497.

Sherwood, L. 2011. Textbook of Fundamentals Human physiology,

4th edn, USA, West virginia.

Shibata, MA, Matoba, Y, Tosa, H, Iinuma, M, 2013, Effects of

Mangosteen Pericarp Extracts Against Mammary Cancer.
Laboratory of Anatomy and Histopathology. Altern
Integrative Med, vol.2, No.8, pp.2327-5162. Vailable on
http://dx.doi.org/10.4172/2327-5162.1000139.

Singh, R, Barden, A, Mori, T, Beilin, L 2001, Advanced

glycation end-product. Rev Diabetologia, vol.44, pp.129-
146.

Sivakumar, R, Sivaraman, BP, Mohan-Babu, N, Jainul-Abideen,

IM, Kalliyappan, P, Balasubramanian, K, 2006, Radiation
Exposure Impairs Luteinizing Hormone Signal Transduction
and Steroidogenesis in Cultured Human Leydig Cells. Dep
Endoc, vol.91, no.2, pp.550556.
Sugaya, K, Fukagawa, T, Matsumoto, K, Mita, K, Takahashi, E,
Ando, A, Inoko, H, Ikemura, T 1994, Three genes in the
human MHC class III region near the junction with the
class II gene for receptor of advanced glycosylation end
products, PBX2 homeobox gene and a notch homolog, human
counterpart of mouse mammary tumor gene int-3. J Genom,
vol.23, pp.408419.

Tena-sempere, M, Rannikko, A, Kero, J, Zhang, FP, Huhtainiemi,

IT 1997, Molecular Mechanisms of Reappearance of
Luteinizing Hormone Receptor Expression and Function in
Rat Testis after Selective Leydig Cell Destruction by
Ethylene Dimethane Sulfonate. Dep Physiol, vol.138, no.1,
pp.3340-3348.

Valko, M, Leibfritz, D, Moncol, J, Cronin, MT, Mazur, M,

Telser, J 2007, Free radicals and antioxidants in normal
physiological functions and human disease. Inter J Biol
Chem Cell, vol.39, no.1 pp.44-84.

Wickens, AP 2001, Ageing and the free radical theory. Resp

Physiol, vol.128, no.3, pp.379-391.
Yan, SF, Ramasamy, R, Naka, Y, Schmidt, AM 2003, Glycation,
inflammation, and RAGE: a scaffold for the macrovascular
complications of diabetes and beyond. Circ Resp, vol.93,
pp.11591169.
CHAPTER VII. ATTACHMENT

ATTACHMENT 1.

Detail of Data Statistic

Descriptives
LH Receptor

N Mean Std. Deviation

control 3 7,7887667 ,00055

AGE-BSA 200g/ml
3 7,7939333 ,01158

AGE-BSA 200g/ml +
Gamma-mangostin 5M 3 8,0680000 ,25646

Total 9 7,883566667 .08953

ATTACHMENT 2.

Normality of Data Distribution Test

Tests of Normality

Shapiro-Wilk

Kelompok Statistic df Sig.

LHR_expression Kontrol ,997 3 ,90

AGE-BSA 200g/ml ,776 3 ,05

AGE-BSA 200g/ml +
,999 3 ,94
Gamma-mangostin 5M

Normality test of Shapiro-Wilk resulting the value :

probability (p) 0, 900, on control group, 0, 058 on group of
AGE-BSA 200g/ml, and 0, 943 on group of AGE-BSA 200g/ml +
Gamma-mangostin 5M. Because of p>0,05. So, the conclusion on
all groups are Normal Distribution data.
 ATTACHMENT 3.

One way ANOVA Test

ANOVA
Expression_of_LHR

Sum of Squares df Mean Square F Sig.

Between Groups ,153 2 ,077 3,485 ,099

Within Groups ,132 6 ,022
Total ,285 8

Test of Homogeneity of Variances

Expression_ofi_LHR

Levene Statistic df1 df2 Sig.

4,210 2 6 ,072

Test of Variance Homogeneity resulting the value

probability (p) is 0,072. Because of p>0,05 the conclusion
data variance is normal.
 `

ATTACHMENT 4.

Result of Post Hoc Statistical Analysis

Multiple Comparisons

Dependent Variable:
Expression_of_LH_
Receptor
LSD

95% Confidence Interval

(I) Kelompok (J) Kelompok Mean Difference (I-J) Std. Error Sig. Lower Bound Upper Bound

LH Kontrol LHR + AGE -.0051667 .1210226 .967 -.301298 .290965

LH R+ AGE + GM 5 -.2792333 .1210226 .060 -.575365 .016898

LH + AGE LHRKontrol .0051667 .1210226 .967 -.290965 .301298

LHR+ AGE + GM 5 -.2740667 .1210226 .064 -.570198 .022065

LH + AGE + GM 5 LHR Kontrol .2792333 .1210226 .060 -.016898 .575365

LH + AGE .2740667 .1210226 .064 -.022065 .570198