| As a common endocrine disease,diabetes mellitus is a systemic metabolic disease whose main characteristics is glucose metabolic disorder that caused by absolute or relative lack of insulin.In recent years,diabetes mellitus becomes the third life long diseases harmful to people's health in developed countries following cardiovascular diseases and cancer.Diabetes mellitus is concerned with damaging and dysfunction of islet ?-cells,and one of the important reasons of glycometabolic disorder is dysfunction of insulin signaling pathway in islet ?-cells.Therefore,it has very important meaning to study insulin signaling pathway in islet ?-cells for prevention and treatment of diabetes and its complications.Streptozotocin(STZ)is a kind of compound which can selectively damage the islet ?-cells,and is a currently recognized drug to establish diabetes rats' models.INS-1 cell lines derived from radiation induced islet?-cell tumor,which is similar to primary-culture islet cells and is widely used to study growth,apoptosis,function,cytotoxicity of islet ?-cells.Sericin is a kind of macromolecular water-soluble protein,which is composed of 18 kinds amino acids.Our early experimental data showed that sericin can reduce blood glucose effectively and prevent increasing of blood glucose of diabetes mellitus rats.However,the specific mechanism is unclear.In this study,the INS-1 cells were damaged by small dose STZ to investigate if sericin has protective effects on STZ-damaged INS-1 cells by regulating insulin PI3K/Akt signaling pathway,and provide experimental data for enriching sericin protecting islet function,as well as provide new idea for prevention and treatment of diabetes mellitus and its complications.Objective:To investigate the regulating effects and possible mechanisms of sericin on STZ-damaged INS-1 cells insulin PI3K/Akt signaling pathway by observing the effects of sericin on expression of insulin receptor(IR),insulin receptor substrate-1(IRS-1),phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt/PKB),forkhead box protein(Fox O1),B-cell lymphoma/leuke-2(Bcl-2)and its family members,Bax and Bad that can promote cell apoptosis.Methods:1.The INS-1 cells were randomly divided into five groups: normal control(NC)group,streptozotocin-damaged(STZ)group,low-dose sericin(LS)group,middle-dose sericin(MS)group and high-dose sericin(HS)group.As a control,the INS-1 cells in NC group were cultured with complete medium without any medicine;the INS-1 cells in STZ group were cultured with complete medium containing 10mmol/L STZ;the INS-1 cells in 3 sericin groups were respectively cultured with complete medium containing 10mmol/L STZ and different dose of sericin: LS group 150?g/m L sericin,MS group 300?g/m L sericin,HS group 600?g/m L sericin.The INS-1cells in each group were all cultured with different drugs for 24 hours.2.The changes of morphological structure of INS-1 cells were observed under inverted microscope.3.The activity of INS-1 cells was detected by CCK8 method.4.The apoptosis was assayed by flow cytometry with Annexin V/PI double staining.5.Western Blotting was used to detect the expression of IR,IRS-1,PI3 K,Akt,P-Akt,Fox O1,Bcl-2,Bax and Bad protein INS-1 cells.6.Real Time PCR was used to measure the expression of IR,IRS-1,PI3 K,Akt,Fox O1,Bcl-2,Bax and Bad mRNA in INS-1 cells.Results:1.The changes of morphological structure of INS-1 cellsNC group: The INS-1 cells were fairly more,grew in monolayer,distributed uniformly;the shape of INS-1 cells is irregular polygon and tended to connect and fuse together.STZ group: After treating with STZ for24 hours,the space between INS-1 cells widened,more cells floated and fell off.The morphological structure of INS-1 cells changed obviously: cells became round and small.Three sericin groups: Compared with INS-1 cells in STZ group,the space between cells became narrow,the floated and falling off cells reduced,and the morphological structure of INS-1 cells tended to be normal.2.The cell viability of INS-1 cellsThe cell viability of INS-1 cells in NC group was(100±0)%;the cell viability of INS-1 cells in STZ group was(53.83±5.76)%,which was significantly lower than NC group(P < 0.05).The cell viability of INS-1cells in LS group,MS group and HS group was respectively(63.53±4.30)%,(71.55±3.13)%,(82.31±5.11)%,which were all obviously higher than STZ group(P<0.05);and the differences about cell viability of INS-1 cells in 3sericin groups was statistically significant(P<0.05).3.Apoptosis of INS-1 cellsThe early apoptosis rate of INS-1 cells in NC group was(5.10±1.64)%;the early apoptosis rate of INS-1 cells in STZ group was(13.73±1.56)%,which was significantly high than NC group(P<0.05).The early apoptosis rate of INS-1 cells in LS group,MS group and HS group was respectively(10.74±1.64)%,(8.57±1.60)%,(7.12±1.56)%,which were all obviously lower than STZ group(P<0.05);and the differences about early apoptosis rate of INS-1 cells in 3 sericin groups was statistically significant(P<0.05).4.IR expression in INS-1 cellsCompared with INS-1 cells in NC group,the IR protein and mRNA expression in INS-1 cells of STZ group obviously decreased(P < 0.05).Compared with INS-1 cells in STZ group,the IR protein and mRNA expression in INS-1 cells of LS group,MS group and HS group obviously increased(P<0.05).In addition,the differences about IR protein expression of INS-1 cells in 3 sericin groups was statistically significant(P<0.05);theIR mRNA expression in INS-1 cells of HS group was significantly higher than LS and MS group group(P<0.05).5.IRS-1 expression in INS-1 cellsCompared with INS-1 cells in NC group,the IRS-1 protein and mRNA expression in INS-1 cells of STZ group obviously decreased(P < 0.05).Compared with INS-1 cells in STZ group,the IRS-1 protein and mRNA expression in INS-1 cells of LS group,MS group and HS group obviously increased(P < 0.05).In addition,the differences about IRS-1 protein expression of INS-1 cells in 3 sericin groups was statistically significant(P< 0.05);the IRS-1 mRNA expression in INS-1 cells of HS group was significantly higher than LS group(P<0.05).6.PI3 K expression in INS-1 cellsCompared with INS-1 cells in NC group,the PI3 K protein and mRNA expression in INS-1 cells of STZ group obviously decreased(P < 0.05).Compared with INS-1 cells in STZ group,the PI3 K protein and mRNA expression in INS-1 cells of LS group,MS group and HS group obviously increased(P < 0.05).In addition,the differences about PI3 K protein and mRNA expression of INS-1 cells in 3 sericin groups was statistically significant(P<0.05).7.Akt and P-Akt expression in INS-1 cellsThere was no statistical significance about Akt protein and mRNA expression in INS-1 cells of each group(P>0.05).Compared with INS-1cells in NC group,the P-Akt protein expression in INS-1 cells of STZ group obviously decreased(P<0.05).Compared with INS-1 cells in STZ group,the P-Akt protein expression in INS-1 cells of LS group,MS group and HS group obviously increased(P < 0.05).In addition,the differences about P-Akt protein expression of INS-1 cells in 3 sericin groups was statistically significant(P<0.05).8.Fox O1 expression in INS-1 cellsCompared with INS-1 cells in NC group,the Fox O1 protein and mRNA expression in INS-1 cells of STZ group obviously increased(P < 0.05).Compared with INS-1 cells in STZ group,the Fox O1 protein and mRNA expression in INS-1 cells of LS group,MS group and HS group obviously decreased(P < 0.05).In addition,the differences about Fox O1 protein expression of INS-1 cells in 3 sericin groups was statistically significant(P< 0.05);the Fox O1 mRNA expression in INS-1 cells of HS group was significantly lower than LS group(P<0.05).9.Bcl-2 expression in INS-1 cellsCompared with INS-1 cells in NC group,the Bcl-2 protein and mRNA expression in INS-1 cells of STZ group obviously decreased(P < 0.05).Compared with INS-1 cells in STZ group,the Bcl-2 protein and mRNA expression in INS-1 cells of LS group,MS group and HS group obviously increased(P < 0.05).In addition,the differences about Bcl-2 protein expression of INS-1 cells in 3 sericin groups was statistically significant(P<0.05).10.Bax expression in INS-1 cellsCompared with INS-1 cells in NC group,the Bax protein and mRNA expression in INS-1 cells of STZ group obviously increased(P < 0.05).Compared with INS-1 cells in STZ group,the Bax protein and mRNA expression in INS-1 cells of LS group,MS group and HS group obviously decreased(P < 0.05).In addition,the differences about Bax protein expression of INS-1 cells in 3 sericin groups was statistically significant(P< 0.05);the Bax mRNA expression in INS-1 cells of HS group was significantly lower than LS group(P<0.05).11.Bad expression in INS-1 cellsCompared with INS-1 cells in NC group,the Bad protein and mRNA expression in INS-1 cells of STZ group obviously increased(P < 0.05).Compared with INS-1 cells in STZ group,the Bad protein and mRNA expression in INS-1 cells of LS group,MS group and HS group obviously decreased(P < 0.05).In addition,the differences about Bad protein expression of INS-1 cells in 3 sericin groups was statistically significant(P<0.05).Conclusions:Sericin can inhibit INS-1 cells apoptosis in a dose-dependent manner by regulating insulin PI3K/Akt signaling pathway in STZ-damaged INS-1 cells and influencing the expression of related apoptosis factors;This maybe one of the mechanisms of sericin reducing blood glucose. |
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