Withaferin A inhibits NF-kB in beta cells and protects inflammatory damage to transplanted islets

J. SoRelle¹, T. Itoh², H. Peng¹, M. Lawrence³, K. Sugimoto², M. Shimoda², S. Matsumoto², M. Levy⁴, B. Naziruddin^5
1 Institute of Biomedical Studies, Waco, TX, USA; ² Baylor Reserach Institute Fort Worth Campus, Fort Worth, TX, USA; ³ UT Southwestern Medical Center, Dallas, TX, USA; ⁴ Annette C and Harold C Simmons Transplant Institute, Dallas, TX, USA; ⁵ Baylor University Medical Center, Transplant Services, Dallas, USA

Background: Beta cell death triggered by pro-inflammatory cytokines plays a central role in the pathogenesis of type1 diabetes and loss of transplanted islets. NF-kB signaling pathway is the key regulator of beta cell stress response, survival and apoptosis. We now show that Withaferin A (WA), a steroidal lactone derived from the plant Withania somnifera effectively blocks NF-kB activation in beta cells, minimizes cytokine-induced cell death and improves survival of transplanted islets.

Method: Min-6, INS-1 cells, mouse and human islets were treated with species-specific cytokine cocktail (IL-1β, TNF-α and INF-γ) ± WA. NF-kB activation was measured by immunoblots and p65 nuclear translocation by immunofluorescence. Cell viability was analyzed by Hoe342/PI staining and islet function by glucose stimulated static incubation. Intraportal transplantation of a marginal mass (200 IEq) of syngeneic islets isolated from C57BL/6 mice was performed to test the in vivo protective effect of WA.

Results: Treatment of cytokine cocktail for 30 min rapidly degraded up to 90% IkB-α chain in MIN-6, INS-1 and human islets and the treatment with 1 µg/ml WA prevented the reduction of IkB to the level observed in control cells. Similarly, immunofluorescence staining with p65 and insulin antibody demonstrated complete inhibition of translocation to nucleus. Treatment with 0.5-1.0 ug/ml WA prevented cytokine-induced islet cell death up to 96 hours (94.9% vs 65.5%; p<0.00005), while showing no significant change in islet potency. Syngeneic islet transplantation restored normoglycemia in 4/5 diabetic C57BL/6 mice treated with WA when compared to 0/5 in control group. Islet function tested by IPGTT further confirmed superior islet function in WA treated animals.

Conclusion: Withaferin A is a strong inhibitor of NF-kB in beta cells, protects against pro-inflammatory cytokine induced cell damage and also improves survival of transplanted islets. Withaferin A could improve islet transplant outcome by preventing inflammatory response.