Berberine Protects Inflammatory Injury Of Pancreatic Beta Cells Induced By Cytokines

Objective We reported that berberine stimulates insulin secretion without any impact on insulin biosynthesis,but the underlying mechanism has not been explored.Protective effect of berberine on inflammatory injury of pancreatic ? cells induced by cytokines has also been evaluated in this study.Methods Insulinoma cell line INS-1 cells were cultured in 1640 medium with different concentrations of berberine(0,2.5,5,and 10 ?M)for 48 hours.Levels of phosphorylation of CREB and AMPK were detected in the cell extracts by Western blot.Nuclear location of CRTC2 was also analyzed.After cultured in 6-well plates for 24 hours,the medium were changed to a culture medium containing 0.2% BSA to mimic starvation for 8 hours,and then treated with the following chemicals including DMSO(vehicle)for 3h,0.5mM AICAR for 3h,10?M Compound C for 3h,5?M Berberine(BBR)for 48 h,5?M BBR plus 0.5mM AICAR,5?M BBR plus 10?M Compound C.Signaling pathway molecules related to insulin secretion such as pAMPK,AMPK,CRTC2,p-CREB and CREB were determined by Western blot.INS-1 cells were treated and grouped as control group,cytokine mixture group(5ng/ml IL-1?,10ng/ml TNF-?,100ng/ml IFN-?)Group,Berberine group(5?M),BBR plus cytokine group.The level of NF-?B in nucleus,L-type calcium channel protein(Cav1.2),Bcl-2 and Cleaved-caspase3 were detected by Western blot.The phosphorylation of AKT,ERK1/2,and AMPK were also investigated.Cell viability was applied to MTT assay.Glucose stimulated insulin secretion was measured by insulin ELISA.Results INS-1 cells treated with 2.5?M,5?M,and 10?M of berberine promoted the activity of PKA,the levels of phosphorylation of CREB,AMPK and inhibited nuclear translocation of CRTC2.Western blot showed that the phosphorylation of CREB could be enhanced by berberine but inhibited by inhibitor H-89.Berberine activated the phosphorylation of AMPK that lead to phosphorylate CRTC2 and keep it in the cytoplasm.When the phosphorylation of AMPK was inhibited by Compound C,CRTC2 dephosphorylated and translocated to the nucleus.Compared to the control group,cytokine mixture improved phosphorylation of AKT and induced nuclear transport of NF-?B in the INS-1 cells.But berberine could reverse the phosphorylation of AKT and nuclear transport of NF-?B significantly.Berberine did not show any impacts on cytokine-induced ERK1/2 signaling pathway.Berberines reversed the decrease of cytokine-induced anti-apoptotic protein Bcl-2 expression and increase the hydrolysis level of pro-apoptotic protein caspase3.The expression of Cav1.2 in the cytokine group was decreased,but increased after adding berberine.MTT showed that cell activity in the cytokine group was significantly reduced.BBR could improve it to some extent,but there was no statistically significant difference.Glucose-stimulated insulin secretion was reduced in the cytokine mixture group.Even though the values have shown some protective effect of BBR to insulin secretion,statistical analyses did not show significance between the BBR+cytokine mixture group and the cytokine mixture group.Conclusion Based on our previous study,berberine may increase the expression of Cav1.2 by activating cAMP/PKA cascade to improve the insulin secretion of INS-1 cells.Meanwhile berberine decreases the nuclear tranfer of CRTC2 by activating AMPK,thereby inhibiting the transcriptional process of downstream related insulin genes.Berberine can reduce NF-?B translocation by inhibiting the phosphorylation of AKT or activating the phosphorylation of AMPK to protect INS-1 cells against cytokine induced inflammatory injury,but no impact on the MAPK/ERK pathway.Berberine can also reverse the decrease of the cytokine-induced anti-apoptotic protein Bcl-2 and increase the hydrolysis level of pro-apoptotic protein caspase3.The reduction of insulin secretion by cytokines may be related to lower expression of Cav1.2.