The Role And Mechanism Of MKP-5 In Glucolipotoxicity Induced Apoptosis And Dysfunction In Mouse Pancreatic ? Cells

Diabetes mellitus is caused by insulin-secreting or insulin resistance,which is caused by a long-term co-action of environment and heredity.Which is a self-metabolic disease with the persistent hyperglycemia.It mainly includes type 1 diabetes and type 2 diabetes,and the type 2 diabetes accounts for more than 90% of the disease.In patients with type 2 diabetes,abnormal glucose metabolism and abnormal lipid metabolism exist side by side.And sugar toxicity and lipid toxicity are not isolated in the development of type 2 diabetes.The two are synergistic.Studies have shown that high blood glucose can promote the apoptosis of the pancreatic islet cells induced by FFA.FFA can further increase glucose concentration in the blood by inhibiting insulin secretion and reducing glucose levels,so as to increase the level of hyperglycemia,thus forming a mixed toxicity of glycolipid.Mitogen-activated protein kinases(MAPKs)are widely distributed in various animals.It plays an important role in a series of physiological activities such as cell growth,development and death.It is one of the signal transduction systems that regulate vital activities.It plays an important role in type 2 diabetes signaling pathway.Mitogen-activated protein kinase phosphatases(MPKs)are also known as bispecific phosphatases.The ability to deactivate MAPKs by dephosphorylation,the negative regulation and regulation effect,this negative regulation plays an important role in cell stress,proliferation,differentiation,apoptosis and other activities.Its abnormal expression is closely related to the occurrence and development of diabetes.MKP5 is one of the MKP family members.Studies have shown that it plays a regulatory role in metabolism and T2 DM.However,the specific mechanism of MKP5 in GP induced islet cell apoptosis and dysfunction remains to be determined.This study explored the role and potential molecular mechanism of MKP5 in the apoptosis and dysfunction of islet cells in mice induced by GP,by building MKP5 and expressing mouse MIN6 cell lines.Purpose: Explore the the role and mechanism of MKP5 in glucolipotoxicity induced apoptosis and dysfunction in mouse pancreatic ? cellsMethods:(1)The MKP5 stable cell line was constructed and identified: pc DNA3.1-MKP5 plasmid and pc DNA3.1 plasmid were successfully transfected into MIN6 cells.To construct the MIN6 stable cell line(MIN6-MKP5)and the control group cell line(MIN6-PC),the expression of MKP-5 protein in two kinds of cells of MIN6-PC and MIN6-MKP5 after transfection was examined by Western Blot.(2)Cell proliferation detection: after the effect of glucolipotoxicity(33.3m M glucose and 0.4Mm PA)on MIN6-PC and MIN6-MKP5 cells 24 h,CCK-8 reagents were used to detect the effect of MKP-5 gene on the proliferation of MIN6 cells.(3)Apoptosis analysis: A.After the effect of glucolipotoxicity on two kinds of MIN6-PC and MIN6-MKP5 cells 24 h,the effect of MKP-5 gene on the apoptosis of MIN6 cells induced by glycolipid toxicity was preliminarily analyzed by Hoechst 33258 staining.B.Annexin V-FICT/PI double staining method combined with flow cytometry was used to analyze the effect of glucolipotoxicity on the apoptosis rate of two kinds of cells MIN6-PC and MIN6-MKP5 after 18 h.(4)Realtime PCR was used to detect the level change of related genes: After the toxic effects of glycolipid on two kinds of MIN6-PC and MIN6-MKP5 cells 12 h,Realtime PCR was used to detect the changes in gene level re lated to apoptosis(Bcl-2,Bax),function(PDX-1),and oxidative stress(NOX4,p22phox).(5)The changes of related protein levels were detected by Western Blot: After the toxic effects of glycolipid on two kinds of MIN6-PC and MIN6-MKP5 cells,0 and 24 h,Western Blot was used to detect the changes of protein expression levels associated with apoptosis(Cleaved-caspase-3,Cleaved-caspase-9)and function(GLUT2).(6)Horizontal detection of cell active oxygen clusters(reactive oxygen species,ROS): DCFH-DA fluorescence probe and fluorescence enzyme labelling instrument were used to analyze the ROS level of two kinds of cells MIN6-PC and MIN6-MKP5 after 24 h glucolipotoxicity.(7)MAPK signaling pathway phosphorylation level detection: After the toxic effects of glycolipid on two kinds of MIN6-PC and MIN6-MKP5 cells,0,1,3,6,9,and 12 h,Western Blot was used to detect the changes in the phosphorylation level of three signal pathway proteins of P38,ERK and JNK.Results:(1)Western results showed that MKP-5 protein expression in MIN6-MKP5 cells increased significantly compared with MIN6-PC cells.The experimental results show that the MKP-5 MIN6 cell line was successfully constructed.(2)CCK-8 results showed that cell viability of MIN6-PC group decreased by 20%,which was statistically significant(P<0.05).The cell viability of MIN6-MKP5 glycolipid CO treatment group was(73.27 + 2.33)%,which was significantly higher than that of MIN6-PC glycolipid CO treatment group(42.70 + 2.47)%.The cell viability of the MIN6-MKP5 ethanol control group increased by 40%(P<0.05)than that in the MIN6-PC ethanol control group.This experiment showed that MPK-5 could alleviate the inhibitory effect of high glucose and high fat on the proliferation of islet beta cells.(3)Results of apoptosis analysis:A: Hoechst results indicate that MPK-5 may play a role in the apoptosis of islet beta cells induced by glycolipid toxicity.B: Annexin-V-FICT/PI reagent and flow cytometry were used to detect apoptosis.The results showed that the apoptosis rate of MIN6-MKP5 glycolipid treatment group was significantly lower than that of MIN6-PC glycolipid treatment group.The results were basically consistent with the results of Hoechst 33258 staining in detecting apoptosis of MIN6 cells.(4)The results of realtime PCR showed: The ratio of Bcl-2 to Bax in the MIN6-PC glycolipid treatment group was 47% lower than that in the ethanol control group,which was significantly lower than that in the MIN6-MKP5 glycolipid treatment group(P<0.05),and the expression of NOX4 and p22 phox gene m RN A in the MIN6-MKP5 group was significantly lower than that in the MIN6-PC group(P<0.05).The experimental results showed that MKP-5 could effectively inhibit the expression of ROS related genes NOX4 and p22 phox,and Western Blot results showed that the expression of Caspase-3 protein in the MIN6-MKP5 group was always at a lower level,and the m RNA expression of PDX-1 gene in the MIN6-PC glycolipid treatment group was significantly lower than that of the MIN6-MKP5 glycolipid treatment group.(5)The results of Western Blot showed that the activation level of Caspase-3 and Caspase-9 in the cells of group MIN6-PC and group MIN6-MKP5 was significantly higher than that in the control group of the control group.The Caspase-3 and Caspase-9 protein H water in group MIN6-MKP5 were significantly lower than those in MIN6-PC group.In group MIN6-PC and group MIN6-MKP5,the expression of GLUT2 protein in the glycolipid treatment group was significantly decreased,but the expression of GLUT2 protein in the blank control group and the glycolipid treatment group was higher than that of the MIN6-PC group.(6)ROS results showed that compared with the ethanol control group,the MIN6-MKP5 glycolipid treated cells did not increase significantly.The level of ROS in the MIN6-PC glycolipid treated group was significantly higher than that in the ethanol control group(P<0.05).The level of ROS in MIN6-MKP5 glycolipid treated group was significantly lower than that in MIN6-PC glycolipid treated group(P<0.05).The results showed that MKP-5 could effectively inhibit the increase of ROS level in pancreatic beta cells induced by glycolipid toxicity.(7)Western Blot detected the phosphorylation level of MAPK signaling pathway.The results showed that the MKP-5 gene could inhibit the activation of P38 and JNK proteins.Unlike the P38 protein and JNK protein,there was no significant difference in the activation level of ERK protein between MIN6-PC and MIN6-MKP5 two cells in the process of synergistic treatment of sugar and lipid,indicating that MKP-5 is in the cell.The downregulation of JNK and P38 pathway in MAPK family is obvious,and the regulation of ERK pathway is not significant.Conclusions:(1)MKP-5 through p38 and JNK pathway were involved in the regulation of islet ? cell apoptosis;(2)MKP-5 may relieve glucoselipotoxicity-induced apoptosis and dysfunction of pancreatic islet beta cells through mitochondrial pathway.