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EXPERIMENTAL RESEARCH
Inhibitory Effect of Kangjia Pill () on Thyrocyte Proliferation in Rat Goiter Model
HAN Yong ( )1, ZHOU Jing ( )2, YU Shu-jing ()2, CUI Bin ( )3, ZHANG Hai-qing ()1, GAO Ling ( )3, and ZHAO Jia-jun ()1
ABSTRACT Objective: To investigate the inhibitory effects of Kangjia Pill (, KJP) on the cell proliferation

in rat goiter model induced by methimazole (MMI). Methods: Fifty-six Wistar rats were randomly divided into four groups: the normal group, MMI model group (MMI), low dose of KJP group (LKJP), and high dose of KJP (HKJP). Except the normal group (20 rats), the other groups (12 rats in each) were given 0.04% (w/v) MMI through the drinking water until the end of the experiment. One week later, the rats in the LKJP and HKJP groups were given KJP by gastrogavage at the dose of 250 mg/ (kg d) and 1 000 mg/ (kg d), respectively for 12 weeks. The relative thyroid weight (mg/100 g body weight) of each rat was accessed. The expression of proliferating cell nuclear antigen (PCNA) was determined by immunohistochemistry, and the correlation analysis between the PCNA positive thyrocytes and the relative thyroid weight was performed. The expressions of PCNA and cyclin D1 were examined with Western blotting. Results: After KJP treatment for 12 weeks, compared with the MMI group, the relative thyroid weight of the HKJP group decreased significantly, and the positive thyrocyte populations of PCNA in the two KJP groups reduced markedly (all P <0.05). The correlation analysis showed that PCNA was closely correlated with thyrocyte proliferation (r =0.685, P <0.05). KJP significantly decreased the protein expression of PCNA and cyclin D1 in the thyroid specimens (P <0.05), the high dose showed better effects. Conclusion: KJP played a therapeutic role via inhibiting cell proliferation in the rat goitrous glands. KEY WORDS goiter, Chinese medicine, cell proliferation, proliferating cell nuclear antigen, cyclin D1

Goiter genesis results from the regulatory imbalance between thyrocyte apoptosis and proliferation (1, 2) . Inhibition of cell proliferation in goiter is an effective strategy for treating goiter. The proliferating cell nuclear antigen (PCNA), a cofactor of DNA polymerase delta, has widely been accepted as a hallmark of cell proliferation, because of its important role in DNA-replication and DNA-repair machinery(3-5). It has been reported protein expression of PCNA was enhanced in goiter, no matter in rat or in human(1, 2, 6). Cyclin D1 is a central G1 phase cell cycle protein(7). It is considered to be a contributory factor to the thyrocyte proliferation(8, 9). The expression of cyclin D1, however, has not been investigated in goiter specimens. Kangjia Pill (, KJP) is an approved hospital prescription for the treatment of goiter with the function of enhancing qi, promoting blood circulation, and removing blood stasis, as well as softening hard mass. So far, the toxicity or clinical side effect associated with KJP has not been observed. Although applied clinically

for many years, its underlying mechanism in treating goiter still remains unclear. The aim of this study was to explore the potential treatment mechanism through examining the expression of cyclin D1 and PCNA by immunohistochemistry and Western blotting from the view of cell proliferation in thyroid tissues in a rat goiter model, induced by methimazole (MMI). The results were reported as follows.

METHODS
Reagents
MMI was obtained from Xingjian Fangtian
Supported by the National Natural Science Foundation of China (No. 30672748), the Natural Science Foundation of Shandong Province (No. 22003C02), and the Shandong Administration of Traditional Chinese Medicine (No. 2005-070) 1.Department of Endocrinology, Provincial Hospital Affiliated to Shandong University, Jinan (250021), China; 2.Department of Endocrinology of Zaozhuang City Hospital, Shandong (277102), China; 3. Scientific Centre, Shandong Provincial Hospital Affiliated to Shandong University, Jinan (250021), China Correspondence to Prof. ZHAO Jia-jun, Tel: 86-531-87065127, Fax: 86-531-87037758, E-mail: jjzhao@medmail.com.cn DOI: 10.1007/s 11655-009-0284-8

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Company Ltd. (Beijing, China). Antibody to PCNA was purchased from Boster Biotechnology Co. Ltd. (Wuhan, China). The polyclonal antibody to cyclin D1 for Western blotting was from Upstate Biotechnology, Inc. (NY, USA).

for 24 h, embedded in paraffin, and sectioned at 5 m for immunohistochemistry. The other was frozen in the liquid nitrogen for Western blot assay.

Immunohistochemistry for PCNA

Immunohistochemistry for PCNA was performed based on the manufacturer's instructions. The results were observed under a Leica photograph microscope. The numbers of PCNA positive nucleuses were counted in five continuous fields in each section by the same observer who was blinded to the protocol. The total number of PCNA positive nucleuses was expressed as the average per 103 cells.

Composition and Preparation of KJP

KJP formula consists of eight Chinese herbs, Radix Astragali 30 g, Radix Ginseng 20 g, Rhizoma Sparganii 15 g, Rhizoma Curcumae 15 g, Semen Coicis 15 g, Fructus Ligustri Lucid 20 g, Radix Glycyrrhizae 6 g, and Radix Polygoni Multiflori 20 g. They were bought from Jianlian drug store (Jinan, China) and authenticated by the pharmaceutical preparation section of Shandong Provincial Hospital, Shandong University (Jinan, China). Technologies of water extracting and alcohol precipitation were used. The extraction rate was 23.4% (w/w). The extracts were stored in a dry vessel (4 ) and diluted with distilled water before use.

Western Blotting Analyses for PCNA and Cyclin D1

The proteins (80 g per lane) from thyroid tissues were resolved with 12% SDS-PAGE and transferred to nitrocellulose membranes. After being blocked with 5% nonfat milk, the membranes were incubated with the primary antibody against PCNA (1:1 000) or cyclin D1 (1: 1 000) at 4 overnight, followed by HRP-conjugated IgG (1:5 000) for 1 h at room temperature. The blots were then visualized with an ECL-Plus Western blotting detection kit (GE Lifesciences Co., USA) and then developed on Kodak films. Finally, the membranes were reblotted with an anti--actin monoclonal antibody (Abcam, UK). The intensities of the immunoblot bands were detected by densitometry with AlphaImager TM 2000 software (Alpha Innotech, USA). All results were normalized to -actin levels in the same sample.

Animals and Treatment

All procedures were approved by the animal care committee of Shandong University. A total of 56 male Wistar rats (eight weeks old, weighing 180-220 g) were purchased from the Experimental Animal Center of Shandong University. Rats were housed at 23 in a 12-h light-dark cycle and humidity-controlled (60%) environment. After they were acclimatized for one week, the animals were randomly divided into four groups: the normal group, MMI model group (MMI), low dose of KJP (LKJP), and high dose of KJP (HKJP) groups. Except the normal group (20 rats), all groups (12 rats in each group) were given 0.04% (w/v) MMI through the drinking water (1, 2, 10). One week later, rats in the LKJP and HKJP groups were administrated with KJP at 250 mg/ (kg d) and 1 000 mg/(kg d) (equal to 4-fold and 20-fold clinical adult doses) in 1 mL distilled water by gastrogavage, respectively. Rats in the normal and MMI groups were given an equal volume of distilled water. The individual dose of KJP was adjusted based on body weight changes, once a week. At the end of the 12-week treatment, all rats were sacrificed under pentobarbital anesthesia (30 mg/kg, intraperitoneal injection). The thyroid gland tissues were weighed to calculate the relative weight (mg/ 100g body weight). Each thyroid gland sample was divided into two portions. One was fixed with 4% paraformaldehyde

Statistical Analysis
All statistical analyses were performed with SPSS 13.0 software. All the variables were tested for normality, and data with a normal distribution were analyzed with one-way ANOVA followed by Post Hoc test (Tukey when equal variances assumed or Dunnett's T3 when equal variances not assumed). The data were expressed as s.

RESULTS
Effect of KJP on Relative Thyroid Weight of Goiter Rats
The relative thyroid weight was significantly heavier in the MMI group than that in the normal group (P <0.01). Compared with the MMI group, the relative thyroid weight decreased by 10% in the LKJP group (P =0.067) and by 21% in the HKJP group (P <0.05). No

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80 70 60 50 40 30 20 10 0 Normal MMI LKJP HKJP

PCNA posive cells per 103 cells

significant difference was found between the two KJP groups (P = 0.052, Figure 1).
Relative thyroid weight (mg/100g body weight)

25 20 15 10 5 0 Normal MMI LKJP HKJP

Figure 3.

Populations of PCNA Positive Thyrocytes

Notes: P<0.05, compared with the normal group; P<0.05, compared with the MMI group;

P <0.05, compared with the

Figure 1. Comparison of the Relative Thyroid Weight among Groups

LKJP group; the same in the below figures

Effect of KJP on PCNA Protein Expression in Rat Goiter

As shown in Figure 2 and 3, the numbers of positive thyrocytes were more in the MMI group than those in the normal group. They were obviously lower in the two KJP groups than those in the MMI group. In the PCNA positive cells, further quantitative analyses indicated a significant decrease in both doses of KJP groups compared with the MMI group (10.833.87, 6.492.61 vs 16.564.25 respectively, P <0.05), and also, the high dose was lower than the low dose. Moreover, the correlation analysis suggested that the PCNA numbers were closely correlated with thyrocyte proliferation (r = 0.685, P < 0.05). Western blotting results for PCNA were consistent with those data obtained from immunohistochemistry. The expression of PCNA in the MMI group significantly increased compared with the normal group (P <0.05). However, compared with the MMI group, the PCNA protein expression decreased by 45% (P < 0.05) and by 58% (P <0.05) in the LKJP and HKJP groups, respectively (Figure 4).

Effect of KJP on Cyclin D1 Protein Expression

The cyclin D1 expression was markedly higher in the MMI group than that in the normal group. Compared with the MMI group, it decreased by 25% and 39% in the LKJP and HKJP groups, respectively (P <0.05) (Figure 5).
PCNA -actin Ratio of PCNA /-actin 1.6 1.2 0.8 0.4 0 Normal MMI LKJP HKJP

Figure 4. Relative Expression of PCNA at Protein Level

cyclin D1 -actin Ratio of cyclin D1/-actin 1.6 1.2 0.8 0.4 0

Normal

MMI

LKJP

HKJP

C
Figure 2. PCNA Immunostaining (400)

Figure 5. Relative Expression of Cyclin D1/-actin at Protein Level

DISCUSSION
The present study showed that thyrocyte proliferation in goiter rats induced by MMI was

Note: A: normal group B: MMI group; C: LKJP group; D: HKJP group; arrows show PCNA positive cells

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obviously increased. Moreover, KJP exerted an inhibitory effect on the thyrocyte proliferation so that it contributes to the decrease of thyroid mass. PCNA is a cell-cycle related protein, reaching the maximal concentration in S- and G2-phases of cell cycle (11). PCNA interacts with various proteins involved in cell cycle control including p21, Gadd45, cyclins, and replication factor C, etc(12). Extracellular signals for promoting cell proliferation, such as insulinlike growth factor1 (IGF-1) and epidermal growth factor (EGF), may elevate PCNA expression(13, 14). In many diseases related to cell proliferation, such as tumors and Grave's thyroid glands, PCNA expresses actively(15). Several clinical studies demonstrated that PCNA expression increased in goitrous thyroid(16,17). Experimental studies further confirmed the correlation between the goiter occurrence and the change in PCNA expression (1,2,18,19). Our present results also showed an increase of PCNA expression in the goiter tissue which significantly decreased after KJP treatment. PCNA has a relatively long half life, so that the antigen could still be detected in a long duration after its protein production(20, 21). Besides, in M-phase, PCNA staining could also be seen in the cytoplasm due to a break of nuclear membrane or aberrant cytoplasmic synthesis(22). In order to avoid the false positive of PCNA, a 12-week KJP treatment was designed; on the other hand, expression of another representative proliferation protein, cyclin D1, was also observed. Cyclin D1 belongs to the D group of cyclins, which has been found expressed in almost all mammalian cells. In general, cyclin D1 is ratelimiting for cell cycle progression, and its abundance varies with the cell cycle, peaking in mid G 1(7,23-26). Currently, studies on cyclin D1 and thyroid have been focused on thyroid tumor(27-30), but there are very few on goiter. Our present results exhibited that cyclin D1 expression at protein level in goiter tissue intensively elevated compared with the normal thyroid. After KJP treatment, the protein expression of cyclin D1 significantly decreased. In addition, the changes of cyclin D1 in the four groups were consistent with the changes of PCNA expression, which might be due to their integration(31). Several evidences supported our results and implied that PCNA and cyclin D1 are good indexes for accessing cell proliferation(32, 33). In summary, our main findings demonstrated

that cell proliferation in goiter induced by MMI was inhibited after KJP treatment, lending supporting evidence for the use of KJP in treating patients with goiter in clinic. However, it is necessary to further explore the mechanism of the inhibitory effect of KJP on cell proliferation in goiter.

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