Investigation of the use of Glycyrrhetinic Acid to Mitigate Unloading Induced Bone Loss

Andrew Walrond, Andrew Krause, Henry Donahue

Division of Musculoskeletal Sciences, Department of Orthopaedics and Rehabilitation, Penn State College of Medicine

Introduction
Previous mouse model studies regarding the effects of unloading-induced bone loss have revealed that Connexin 43 (Cx43), a protein which mediates gap junction intercellular communication (GJIC), plays a vital role in bone remodeling1. Studies show that when Cx43 is deficient in bone, loss due to unloading is attenuated. GJIC can also be inhibited by Glycyrrhetinic acid (GA), an antiinflammatory compound2. Our hypothesis is that, like Cx43 deficiency, GA will attenuate unloading-induced bone loss both in vivo and in vitro.

no treatment HLS, GA treatment HLS, and olive oil control treatment HLS. Mice were monitored by veterinarian as well as research staff observations. Bone density, volume, and thickness changes from baseline were measured via microCT analysis. Histology, three point bending, and NMR were also used to acquire data.

on cytodex-3 collagen beads, and rotated under three conditions: normal media as described in petri dish model, addition of GA suspended in DMSO, and addition of DMSO as a GA control. Media was adjusted to 1%calf serum, 1% fetal bovine serum, and 1% penn strep.

Methods
For in vivo studies ten C57BL/6J mice were injected with GA daily for three weeks. A new set of mice were then hind-limb suspended (HLS) and injected with GA every other day. GA was suspended in olive oil at 20mg/kg and administered subcutaneously. Mice were split into five groups: ground control, ground control with GA treatment,
Figure1: Image of mouse hind-limb suspended after day 1 of the 3 week study animals suspended at roughly 30 degrees

the GJIC inhibiting properties of GA, we predict suspended mice receiving GA will show an attenuation of bone loss due to unloading relative to vehicle controls. Trabecular bone volume in the proximal femur will significantly decrease in mice subjected to HLS without treatment of GA relative to those treated. Cortical bone structure from the mid-shaft of the femur will be expected to remain similar amongst groups. We predict exposure to simulated microgravity in vitro will lead to an increase in genes associated with bone resorption and a decrease in those associated with bone formation. Exposure to GA will attenuate these responses.

Figure 41: Trabecular (left) and cortical (right) microCT analysis. Previous result demonstrate that trabecular degradation of microstructure as a result of HLS is attenuated in Cx43 deficient mice.

Conclusions
If our hypothesis is supported and GA, like Cx43 deficiency, attenuates unloading-induced bone loss in vivo as well as in vitro, using GA could prove to be a promising front. This data could prove to be a gateway into new methods to decrease bone loss from reduced activity, space travel, and even osteoporosis. Furthermore it may lead to advanced research into the physiological effects of GA.

For in vitro studies MLO-Y4 cells were rotated in a High Aspect Ratio Vessel3 (HARV) as well as a normal petri dish environment. Petri dish cells were grown in media containing 2.5% calf serum, 2.5% fetal bovine serum, and 1% penn strep. HARV environment cells were cultured

References
Figure 2: Image of High Aspect Ratio Vessel with no vessels attached
1. Lloyd, S. et al., Connexin 43 Deficiency Attenuates Loss of Trabecular Bone and Prevents Suppression of Cortical Bone Formation During Unloading. JBMR, 2012 Vol. 27, No. 11: p. 2359-2372 Davidson, J., et al., Reversible Inhibition of Intercellular Junctional Communication by Glycyrrhetinic Acid. Biochemical and Biophysical Research Communications, 1986 Vol. 134: p. 29-36. Educational Brief NASAs Bioreactor: Growing cells in a Microgravity Environment. National Aeronautics and Space Administration, EB-2002-12-187-MSFC Bucaro, M., "Bone cell survival in microgravity: The effects of vector-averaged gravity on the osteoblast" (January 1, 2006). ETD Collection for Thomas Jefferson University. Paper AAIDP18521.

Results
Suspended mice we expect will show results similar to previous Cx43 knockout studies1. Due to
Figure 3: Graph displaying predicted changes in trabecular bone volume fraction of control subjects as well as HLS subjects with or without treatment of GA

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