Effects Of Vaspin On Hyperglycemia And Hyperpalmitic Acid-induced INS-1 Cells Oxidative Stress Via Nrf2/ARE Signaling Pathway

Objective:1.To investigate the effect of vaspin on oxidative stress level and the secretary function of INS-1 cells induced by the hyperglycemia and hyperpalmitic acid.2.To investigate the effect of vaspin on Keap1-Nrf2-ARE signaling pathway and its regulatory mechanism in INS-1 cells which were cultured with hyperglycemia and hyperpalmitic acid.Methods:INS-1 cells cultured in vitro were divided into normal control group(NC),hyperglycemia and hyperpalmitic acid group(HG+PA),HG+PA+vaspin(80 ng/ml),HG+PA+vaspin(160 ng/ml),HG+PA+vaspin(320 ng/ml),HG+PA+vaspin(640 ng/ml).After intervention,glucose-stimulated insulin secretion assay(GSIS)is used to detect the basal and high-glucose-stimulated insulin secretion function,the levels of reactive oxygen species(ROS)in cells were detected by flow cytometry after staining with DCFH-DA fluorescent probe,oxidative stress related indicators were examined by colorimetry and ELISA,flow cytometry was used in examining apoptotic rate in each group,and western blot was used to detect the level of Akt,p-Akt,p38 MAPK,p-p38 MAPK,plasma Nrf2 and nuclear Nrf2 protein.In order to investigate whether vaspin can mediate Nrf2 translocation into nucleus through PI3K/Akt and p38 MAPK signaling pathways,INS-1 cells cultured in vitro were divided into NC group,HG+PA group,HG+PA+640 ng/ml vaspin,HG+PA+640 ng/ml Vaspin+ LY 294002/SB 203580 group,HG+PA+ LY 294002/SB 203580 group.Western blot was used to detect the level of Akt,p-Akt,p38 MAPK,p-p38 MAPK,plasma Nrf2 and nuclear Nrf2 protein.Results:(1)GSIS test showed that compared with HG+PA group,insulin secretion levels stimulated by low glucose and high glucose were significantly improved(P < 0.05)in 640 ng/ml vaspin group.(2)Compared with HG+PA group,the intracellular content of ROS,malondialdehyde,8-hydroxy-2 deoxyguanosine were decreased significantly,the levels of total antioxidant capacity and antioxidant enzymes,such as superoxide dismutase and glutathione peroxidase,were significantly increased in HG+PA+vaspin(640 ng/ml)group(P < 0.05).(3)Results from flow cytometry analysis showed that compared with HG+PA group,the apoptotic level of vaspin intervention group could significantly decrease in a concentration-dependent manner.(P < 0.05).(4)Western blot analysis showed that the expression level of Nrf2 in cytoplasm increased with the increase of vaspin intervention concentration,and this in nucleus was only higher than that in 640 ng/ml vaspin intervention group(0.798±0.080 vs.0.579±0.065,P=0.039).(5)The phosphorylated Akt and nuclear Nrf2 protein levels decreased significantly after the intervention of hyperglycemia and hyperpalmitic acid.With the increase of vaspin intervention concentration,the ratio of p-Akt to Akt increased gradually,and the relative content of Nrf2 translocation into nucleus also increased significantly.After preconditioning with PI3 K inhibitor LY 294002,the effect of vaspin on the expression of p-Akt was significantly weakened.At this time,the level of Nrf2 in nucleus was also significantly reduced.(6)After the intervention of hyperglycemia and hyperpalmitic acid,the p-p38 MAPK level was significantly increased and the nuclear Nrf2 protein level was decreased.The levels of p-p38 MAPK protein were significantly decreased and nuclear translocation of Nrf2 increased in vaspin intervention group and p38 MAPK inhibitor SB 203580 alone.The combined effects were more remarkable than that of single treatment.Conclusion:Vaspin can regulate Nrf2/ARE signaling pathway by PI3K/Akt and p38 MAPK to improve the antioxidant capacity,suppress oxidative stress injury induced by hyperglycemia and hyperpalmitic acid and finally reduce apoptosis,and improve the islet cell secretory function.