The Role Of SET-Rac1 Pathway In Pancreatic β Cells Apoptosis And Dysfunctions Induced By Arsenic

Objective:Impaired insulin synthesis and secretion caused by pancreaticβ-cell dysfunction is one of the essential pathological features of type 2 diabetes（T2D）.The research group’s previous studies have shown that the pancreaticβ-cell apoptosis induced by inorganic arsenic can regulate the pancreaticβ-cell dysfunction,but its specific molecular mechanism is still unknown.The abnormal expression of small GTPase Rac1（ras-related C3 botulinum toxin substrate 1）and nuclear cancer factor SET（Suvar 3-9 enhancer-of-zeste trithorax）will induce apoptosis.However,the specific role and molecular mechanism in pancreaticβ-cell dysfunction caused by arsenic exposure is still unclear.Therefore,this study will explore the role and possible mechanism of Rac1 and SET in the apoptosis and dysfunction of isletβcells induced by inorganic arsenic exposure.Methods:In vivo experiment:Adult male SD rats（200-250g）were randomly divided into two groups,treated with（0,5 mg/kg）sodium arsenite（Na As O₂）for 5months,and then killed and dissected for following experiments.Western blot were used to detect the expression of small GTPase Rac1,nuclear cancer factor SET,MAPK cascade pathway target protein（p-P38MAPK,P38MAPK）,cytoskeleton-related protein Cofilin-1 and the apoptosis-related protein（Bax,Bcl-2,Caspase-3,cleaved-caspase3）;Immunofluorescence was used to detect the dysfunction of pancreaticβ-cells.Based on the above results,the effects of arsenic exposure on the expression of Rac1 and SET,cytoskeleton rearrangement,apoptosis and isletβ-cell dysfunction were explored.In vitro research:Min6 cells were used as the research object.Transfection with1.2 n M overexpressed SET plasmid or 25 n M small interfering RNA si Rac1 for 16 h and then 4μM Na As O₂ for 24 h.Western Blot was used to detect the expression of SET,Rac1,MAPK cascade pathway target proteins（p-P38MAPK,P38MAPK）and apoptosis-related proteins（Bax,Bcl-2,caspase3,Cleaved-Caspase-3）to explore the regulatory relationship between SET and Rac1 in pancreaticβ-cell dysfunction caused by apoptosis.Western Blot was used to detect the cytoskeleton-related protein Cofilin-1and immunofluorescence was used to detect the depolymerization of F-actin.ELISA was used to detect insulin secretion;From the above results,the mechanism of SET-Rac1 in cytoskeleton rearrangement,apoptosis and dysfunction induced by arsenic exposure will be discussed.Results:The results of Western blot and Immunofluorescence jointly showed that chronic arsenic could reduce the expression of nuclear cancer factor SET and improved the expression of small GTPase Rac1 in rat pancreatic tissue.In addition,the immunofluorescence results also showed that chronic arsenic lead to the lower insulin levels.In the corresponding in vitro experiment,arsenic also reduced the expression of the tumor factor SET and improved the expression of small GTPase Rac1 in Min6 cells.The insulin release test showed that after 16 h of pretreatment with the expression of 1.2n M SET plasmid or 25 n M si Rac1,the insulin secretion deficiency was relieved.Western Blot results showed that chronic arsenic activated the target protein of MAPK cascade pathway in rat pancreas and lead to apoptosis.In the corresponding in vitro experiment,the overexpression of SET plasmid in Min6 cells were transfected for 16hours before treated with 4μM Na As O₂ for 24 hours.After above process,the up-regulation of Rac1,activated MAPK cascade pathway and apoptosis caused by arsenic were alleviated.Also si Rac1 was transfected for 16 h and then treated with 4μM Na As O₂ for 24 hours.The down-regulation of SET caused by arsenic was unchanged,but the activated MAPK cascade pathway and apoptosis were alleviated.The expression of cytoskeleton-related protein Cofilin-1 was increased due to the arsenic in rat pancreatic tissue or Min6 cells.After transfected with 1.2 n M overexpressed SET plasmid or 25 n M si Rac1 for 16 hours,4μM Na As O₂ were used for 24 hours.WB results showed that the up-regulation of cytoskeleton-related protein Cofilin-1 caused by arsenic was alleviated and the damage rearrangement of F-actin was restored in Min6cells.Conclusion:Na As O₂ injured the cytoskeleton rearrangement through SET-Rac1pathway,and finally lead to the Pancreatic-β-cells apoptosis and dysfunctions.