I.D. Zweifel1, R. Onoszko2, E. Weber1,2 1Indiana University School Of Medicine, Indianapolis, IN, USA 2Methodist Hospital, Department Of Surgery, Indianapolis, IN, USA
Introduction: Three Schwann cell states are important for peripheral nerve regeneration: the myelinating Schwann cell (mSC), the repair Schwann cell (rSC), and the chronically denervated Schwann cell (cdSC). When a peripheral nerve is damaged, the mSC converts to an rSC, upregulating Sonic Hedgehog (Shh) and c-Jun expression, which is necessary for nerve regeneration. Within 1-3 months of injury, the rSC deteriorates to the cdSC, reflected as a decrease in c-Jun expression and reduced axonal regeneration rates. In patients with proximal peripheral nerve injuries, the supportive rSC state deteriorates much before regeneration is complete and may, in part, contribute to poorer outcomes. Beta-caryophyllene (BCP) is a naturally occurring, non-toxic, FDA-approved compound that upregulates Shh. We hypothesize that treating Schwann cells with BCP will upregulate Shh and, subsequently, c-Jun, maintaining the rSC state and prolonging regeneration. To test this, we first examined the effects of BCP on 293 cells expressing the primary BCP receptor, cannabinoid receptor 2 (CB2).
Methods: 293 and CB2-293 cells were grown on coverslips for immunofluorescence staining (IF) or 6-well plates for Western blot (WB). Cells were grown to either 40% or 80% confluency. BCP was added in increasing concentrations (0, 10, 20, 50 μM) and cells were harvested after 24 or 48 hours. The coverslips were stained with antibodies to c-Jun, Shh, and DAPI. WB were probed with antibodies to c-Jun, Shh, and Cyclophilin B.
Results: c-Jun was consistently expressed in both 293 and CB2-293 cells when grown to 40% confluency and, therefore, still actively dividing. The addition of BCP did not increase c-Jun expression. When cells were grown to confluency, c-Jun expression was eliminated and the addition of BCP produced a dose-dependent increase in c-Jun expression, most notable in CB2-293 cells but also present in 293 cells. 50 μM BCP was toxic to all cells.
Conclusion: c-Jun is a cell cycle protein necessary for progression from the G1 to S phase during proliferation. The addition of BCP to proliferating cells did not impact c-Jun expression. However, BCP did drive c-Jun expression in non-dividing cells, though the effect may not have occurred solely through the CB2 receptor. cdSCs are senescent Schwann cells that we hypothesize will be re-activated by BCP treatment. Future studies will examine the mechanism of BCP upregulation of c-Jun and the effect of BCP in primary Schwann cells and a rodent model of peripheral nerve regeneration.