| With the rapid development of social economy and the aging of population,the incidence of type 2 diabetes mellitus?T2DM?is increasing rapidly.The main pathogenesis of type 2 diabetes mellitus is insulin resistance and islet?-cells secretion defects.It is found that one of the important causes of insulin resistance is the disorder of the signaling transduction pathway induced by the combination of insulin and its receptors.Most scholars believe that insulin exerts its function of regulating blood glucose mainly through the PI3K/Akt signaling pathway,which plays an important role in regulating the number and function of islet?-cells.Therefore,it is of great significance to study the expression of cell cycle related factors of pancreatic islet?-cells and the insulin PI3K/Akt signaling pathway for the clinical treatment and prevention of type 2 diabetes and related complications.Silk consists of two parts.The part made up of silk collagen in the periphery of silk is called sericin,which mainly contains 18 kinds of amino acids.Our research group found that sericin played a significant role in type 2diabetic rats,which can effectively reduce its blood sugar,and to a certain extent,it can prevent the increasing of blood glucose,but its specific mechanism is not clear yet.Rat islet cell tumor cells?INS-1 cells?have the physiological characteristics of rat islet?-cells,and can be expressed stably over 80 generations.Therefore,it is widely used in the study of insulin secretion.In this study,streptozotocin?STZ?was used to establish INS-1 cells injury to investigate whether sericin affects the cell cycle of INS-1 cells through insulin PI3K/Akt signaling pathway,in order to provide new methods for clinical prevention and treatment of type 2 diabetes and related complications.Objective:To explore whether sericin affects the cell cycle of INS-1 cells through insulin PI3K/Akt signaling pathway by observing the changes of insulin receptor?IR?,insulin receptor substrate-1?IRS-1?,phosphatidylinositol3-kinase?PI3K?,protein kinase B?Akt/PKB?,glycogen synthase kinase-3?GSK3??,CyclinD1,ribosomal protein S6 kinase?P70 S6K?,phosphorylation of P70 S6kinase?P-S6K1?,and mammalian target of rapamycin?m-TOR?expression in INS-1 cells induced by STZ.Methods:1.The INS-1 cells were cultured in 37?and 5%CO2 incubator with RPMI-1640 culture medium containing 10%FBS and 50?mol·L-1?-mercaptoethanol.INS-1 cells were divided into 5 groups randomly:normal group?N group?,diabetic model group?DM group?,low concentration sericin group?LC group?,medium concentration sericin group?MC group?and high concentration sericin group?HC group?.The INS-1 cells in N group were cultured in RPMI-1640 complete medium containing 10%fetal bovine serum and 50?mol·L-1?-mercaptoethanol without any other treatment;the INS-1cells in DM group were cultured with complete culture medium containing10mmol·L-11 STZ;the cells in LC group,MC group and HC group were cultured in complete medium with 10mmol·L-11 STZ and different concentrations of sericin,the final concentrations of sericin in the treatment groups are 150?g·mL-1,300?g·mL-1 and 600?g·mL-11 respectively.The INS-1cells were cultured with different drugs for 24 hours.2.The growth and morphological changes of INS-1 cells in each group were observed under inverted microscope.3.The proliferation activity of cells in each group was detected by CCK-8 method.4.The expression of IR,IRS-1,PI3K,Akt,GSK-3?,CyclinD1 and m-TOR mRNA in each group was detected by real-time fluorescence quantitative PCR?Real Time PCR?.5.The expression of IR,IRS-1,PI3K,Akt,P-GSK3?,GSK3?,CyclinD1,P70 S6K,and P-S6K1 protein in each group was detected by Western BlottingResults:1.The morphological structure of INS-1 cells in each group:N group:The INS-1 cells were flat,irregular and polygonal,with good refraction and quantity,monolayer growth,and tendency to connect and fuse;DM group:Compared with the N group,the number of adherent cells decreased obviously,and the cells fell off or half adherent,cells shrinked,the shape of cells became round,and the volume became smaller.LC,MC,HC group:Compared with DM group,the number of adherent cells in each group increased,the distribution was more uniform and the shape close to normal cells.2.The proliferation activity of INS-1 cells in each group:The cell survival rate of INS-1 cells in N group was?100±0?%;the cell survival rate of INS-1 cells in DM group was?68.50±6.14?%,which was significantly lower than N group?P<0.01?.The cell survival rates of INS-1cells in LC group,MC group and HC group were respectively?75.09±6.49?%,?82.57±2.96?%and?89.04±1.55?%,which were all significantly higher than DM group?P<0.05?;and the difference about cell survival rate of INS-1 cells in LC group,MC group and HC group was statistically significant?P<0.05?.3.The expression of IR in each group of INS-1 cells:The expression of IR mRNA and protein in DM group were significantly lower than that in N group?P<0.05?.Compared with DM group,IR mRNA and protein expression were significantly higher in LC group,MC group and HC group INS-1 cells?P<0.05?.Compared with LC and MC groups,IR mRNA expression was significantly higher in HC group?P<0.05?.The expression of IR protein in three groups of LC,MC and HC cells was compared,and the difference was statistically significant?P<0.05?.4.The expression of IRS-1 in each group of INS-1 cells:The expression of IRS-1 mRNA and protein in DM group were significantly lower than that in N group?P<0.05?.Compared with DM group,the expression of IRS-1 mRNA and protein were significantly higher in LC group,MC group and HC group INS-1 cells?P<0.05?.Compared with LC group,the expression of IRS-1 mRNA in HC group was significantly higher?P<0.05?.The expression of IRS-1 protein in three groups of LC,MC and HC cells was compared,and the difference was statistically significant?P<0.05?.5.The expression of PI3K in INS-1 cells:The expression of PI3K mRNA and protein in DM group were significantly lower than that in N group?P<0.05?.Compared with DM group,PI3K mRNA and protein expression were significantly higher in LC group,MC group and HC group?P<0.05?.In addition,the expression of PI3K mRNA and protein in three groups of LC,MC and HC were compared,and the differences were statistically significant?P<0.05?.6.The expression of P-Akt in INS-1 cells:The expression of P-Akt protein in DM group was significantly lower than that in N group?P<0.05?.Compared with DM group,the expression of P-Akt protein was significantly higher in LC,MC,and HC group?P<0.05?.The expression of P-Akt protein in three groups of LC,MC and HC was compared,and the difference was statistically significant?P<0.05?.7.The expression of GSK3?and P-GSK3??Tyr216?in INS-1 cells:The expression of GSK3?mRNA and protein in DM group were significantly higher than that in N group?P<0.05?.Compared with DM group,the expression of GSK3?mRNA and protein significantly decreased in LC group,MC group and HC group?P<0.05?.The expression of GSK3?mRNA in three groups of LC,MC and HC was compared,and the difference was statistically significant?P<0.05?.The expression of P-GSK3??Tyr216?protein in DM group was significantly higher than that in N group?P<0.05?.Compared with DM group,the expression of P-GSK3??Tyr216?protein significantly decreased in LC group,MC group and HC group.?P<0.05?.8.The expression of CyclinD1 in each group of INS-1 cells:The expression of CyclinD1 mRNA and protein in DM group were significantly lower than that in N group?P<0.05?.Compared with DM group,the expression of CyclinD1 mRNA and protein were significantly higher in LC group,MC group and HC group?P<0.05?.Furthermore,the expression of CyclinD1 mRNA in LC,MC,and HC was compared and the difference was statistically significant?P<0.05?.The expression of CyclinD1 protein in two groups of LC and HC was compared with MC group,and the difference was statistically significant?P<0.05?.9.The expression of P-S6K1 in INS-1 cells:The expression of P-S6K1 protein in DM group was significantly higher than that in N group?P<0.05?.Compared with DM group,the expression of P-S6K1 protein significantly decreased in LC,MC,and HC groups?P<0.05?.The expression of P-S6K1 protein in LC,MC,and HC was compared,and the difference was statistically significant?P<0.05?.10.The expression of P70 S6K in INS-1 cells:The expression of P70 S6K protein in DM group was significantly higher than that in N group?P<0.05?.Compared with DM group,P70 S6K protein expression significantly decreased in LC group,MC group and HC group?P<0.05?.11.The expression of m-TOR in INS-1 cells:The expression of m-TOR mRNA in DM group was significantly lower than that in N group?P<0.05?.Compared with DM group,m-TOR mRNA expression was significantly higher in LC group,MC group and HC group?P<0.05?.The expression of m-TOR mRNA in three groups of LC,MC and HC was compared,and the difference was statistically significant?P<0.05?.Conclusions:Sericin can regulate the expression of cell cycle related factors in INS-1cells induced by streptozotocin and promote cell proliferation by affecting PI3K/Akt signaling pathway,so as to exert the effect of reducing blood glucose. |
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