| Although there are great differences between type 1 diabetes and type 2diabetes(T2DM)in nature,islet β cell dysfunction plays a key role in the pathogenesis of both.In T2 DM,insulin resistance(IR)can lead to "overwork death" of β cells.When diabetes occurs,mitochondrial dysfunction,oxidative stress and endoplasmic reticulum stress(ERS)are the main causes of β cell dysfunction and apoptosis.Recently,some researchers have proposed that autophagy can promote the survival and maintenance of β cell through its adaptive response to prevent and alleviate the adverse effects of ers,mitochondrial dysfunction and oxidative stress.Under these consensus,the regulation of autophagy on β cells is an important topic in the treatment of type 2 diabetes.Sericin is a kind of spherical water-soluble glycoprotein in silk worm cocoon,which has biodegradability and biocompatibility.Our group found that sericin can effectively improve blood glucose and multiple organ injury in T2 DM rats.In this study,we observed the regulatory effect of sericin on mitochondrial autophagy signal transduction pathway of INS-1 cells in order to explore the possible mechanism of sericin restoring insulin secretion function and cell viability of INS-1 cells,and provide more theoretical basis for sericin treatment of T2 DM and related complications.Objective:Discuss to investigate whether sericin can reduce the oxidative stress,ROS level and apoptosis of INS-1 cells injured by STZ through mitochondrial autophagy signal transduction pathway.Method:1.Grouping and treatment of INS-1 cellsINS-1 cells were divided into 5 groups and treated with drugs for 24 hours:(1)Normal group(NC group): INS-1 cells were cultured in complete medium.(2)Model group(MG group): INS-1 cells were cultured in complete medium containing 10mmol/L streptozotocin(STZ).(3)Low dose sericin group(LC group): INS-1 cells were cultured in complete medium containing 10mmol/L STZ and 150μg/ml sericin.(4)Medium dose sericin group(MC group): INS-1 cells were cultured in complete medium containing 10mmol/L STZ and 300μg/ml sericin.(5)High dose sericin group(HC group): INS-1 cells were cultured in complete medium containing 10mmol/L STZ and 600μg/ml sericin.2.Western blotting was used to detect protein expression of Silent information regulator 2 related enzyme 1(SIRT1),Phosphatase and tensin homolog deleted on chromosome ten induced kinase 1(PINK1),Autophagy related protein 6(ATG6/BECLIN1),Autophagy related protein 5(ATG5),Sequestosome1(SQSTEM1/P62),Microtubule associated protein light chain3B-II(LC3B-II),Microtubule associated protein light chain 3B-I(LC3B-I),B cell lymphoma-2(Bcl-2),P53.3.The m RNA expressions of SIRT1,PINK1,BECLIN1,ATG5,P62,LC3BII/I,Bcl-2 and P53 in INS-1 cells were detected by real time PCR.4.The mitophagy detection kit was used to confirm the phenomenon of mitochondrial autophagy fluorescence in INS-1 cells.5.The content of reactive oxygen species(ROS)in INS-1 cells was determined by DCFH-DA fluorescence probe under fluorescence microscope.Result:1.The expression of SIRT1 in INS-1 cells of each experimental group was as follows:Compared with INS-1 cells in NC group,the expression of SIRT1 protein and m RNA in INS-1 cells in MG group was significantly decreased(P <0.05).Compared with MG group,the expressions of SIRT1 protein and m RNA in INS-1 cells of LC group,MC group and HC group were significantly increased(P<0.05),and the differences among the three groups were statistically significant(P<0.05).2.The expression of PINK1 in INS-1 cells of each experimental group was as follows:Compared with NC group,the expression of PINK1 protein and m RNA in INS-1 cells in MG group was significantly decreased(P<0.05);compared with MG group,the expression of PINK1 protein and m RNA in INS-1 cells in LC group,MC group and HC group was gradually increased(P<0.05),and there was significant difference in the expression of PINK1 protein and m RNA among the three groups(<0.05).3.The expression of BECLIN1 in INS-1 cells of each experimental group was as follows:Compared with NC group,the expression of BELIN1 protein and m RNA in INS-1 cells of MG group was significantly lower(P<0.05);compared with MG group,the expression of Beclin1 protein and m RNA in INS-1 cells of LC group,MC group and HC group was gradually increased(P<0.05),and the expression of BECLIN1 protein and m RNA in INS-1 cells of three groups was statistically significant(P<0.05).4.The expression of ATG5 in INS-1 cells of each experimental group was as follows:Compared with NC group,the expression of ATG5 protein and m RNA in INS-1 cells of MG group was significantly lower(P<0.05);compared with MG group,the expression of ATG5 protein and m RNA in INS-1 cells of LC group,MC group and HC group was gradually increased(P<0.05),and there was significant difference in the expression of ATG5 protein and m RNA among the three groups(P<0.05).5.The expression of P62 in INS-1 cells of each experimental group was as follows:Compared with NC group,the expression of P62 protein and m RNA in INS-1 cells of MG group was significantly lower(P<0.05);compared with MG group,the expression of P62 protein and m RNA in INS-1 cells of LC group,MC group and HC group was gradually increased(P<0.05),and the expression of P62 protein and m RNA in INS-1 cells of three groups was statistically significant(P<0.05).6.The expression of LC3B-II/I in INS-1 cells of each experimental group was as follows:Compared with NC group,the expression of LC3B-II/I protein and LC3 B m RNA in INS-1 cells of MG group was significantly lower(P<0.05);compared with MG group,the expression of LC3B-II/I protein and LC3 B m RNA in INS-1 cells of LC group,MC group and HC group was gradually increased(P<0.05),and the expression of LC3B-II/I protein and LC3 B m RNA in INS-1 cells of three groups were statistically significant(P<0.05).7.The expression of Bcl-2 in INS-1 cells of each experimental group was as follows:Compared with NC group,the expression of Bcl-2 protein and m RNA in INS-1 cells of MG group was significantly lower(P<0.05);compared with MG group,the expression of Bcl-2 protein and m RNA in INS-1 cells of LC group,MC group and HC group was gradually increased(P<0.05),and there was significant difference between the three groups(P<0.05).8.The expression of P53 in INS-1 cells of each experimental group was as follows:Compared with NC group,the expression of P53 protein and m RNA in INS-1 cells of MG group was significantly increased(P<0.05);compared with MG group,the expression of P53 protein and m RNA in INS-1 cells of LC group,MC group and HC group was gradually decreased(P<0.05),and the expression of P53 protein and m RNA in INS-1 cells of three groups was statistically significant(P<0.05).9.the mitochondrion autophagy fluorescence of INS-1 cells in each experimental group was observed:Compared with NC group,the autophagy fluorescence of INS-1 cells in MG group was significantly decreased;compared with MG group,the autophagy fluorescence of INS-1 cells in LC group,MC group and HC group was increased.10.ROS content of INS-1 cells in rats of each experimental group:Compared with INS-1 cells in NC group,ROS content of INS-1 cells in MG group was significantly increased(P<0.05);compared with MG group,ROS content of INS-1 cells in LC group,MC group and HC group was significantly reduced(P<0.05),and the expression of ROS in the three groups was statistically significant(P<0.05).Conclusion:Sericin can activate mitochondrial autophagy pathway by regulating the expression of SIRT1-PINK1-PARKIN signal transduction pathway SIRT1,PINK1,BECLIN1,ATG5,P62,LC3B-II/I,Bcl-2,P53,related factors in INS-1cells injured by STZ,reduce oxidative stress of INS-1 cells,and protect INS-1cells. |
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