Nicotine Increase The Sensitivity Of Apoptosis And Fibrosis Induced By High Glucose And High Fat Through ERK5 Translocation In H9c2 Cells

Obj ective:Smoking is a risk factor for type 2 diabetes and an important factor in the development of cardiovascular disease.In this study,the effects of nicotine on H9c2 cells in a high-sugar and high-fat condition were studied in vitro.The injury model of cardiomyocytes in diabetic patients was simulated,and the damage mechanism of H9c2 cells induced by nicotine under high glucose and high fat conditions was examined.ERK signaling proteins clarify their effects on heart damage.Methods:1.To test the effect of nicotine on apoptosis under high glucose and high fat conditions.H9c2 cells were divided into normal control group,nicotine group(6×10-8M,6×10-7M,6×10-6M),high glucose and high fat group(HGHL,glucose 33mmol/L+sodium palmitate 500μmol/L).Nicotine is combined with a high-sugvb ar and high-fat group(6×10-8 M+HGHL,6×10-7 M+HGHL,6×10-6M+HGHL).After 2 hours of nicotine intervention,high glucose and high fat treatment for 24 h,Western blot was used to detect the related proteins cleaved caspase-3,cleaved caspase-8,cleaved caspase-9,cleaved caspase-11,cleaved caspase-The expression of 12 was analyzed by flow cytometry to detect the apoptosis rate of H9c2 cells.2.To test the effect of nicotine on cell fibrosis under high glucose and high fat conditions.This part of the experiment was divided into four groups:normal control group,nicotine group(NIC,6×10-7M),high glucose and high fat group(HGHL),nicotine combined with high sugar and high fat group(NIC+HGHL).The expression of MMP2,Collagen Ⅰ,Collagen Ⅲ,Smad3,TGF-p and other genes related to H9c2 cell fibrosis was detected by Real-Time PCR.3.To examine the effect of ERK family on apoptosis and fibrosis of H9c2 cells.1)The experimental group was divided into normal control group,nicotine group(NIC,1×10-7M,2×10-7M,4×10-7M),high glucose and high fat group(HGHL),nicotine combined with high glucose and high fat group(1×10-7M NIC+HGHL,2×10-7M NIC+HGHL,4×10-7M NIC+HGHL)Western Blot was used to detect the protein expression of ERK1/2,ERK3 and ERK5;2)Experimental grouping was:normal Group(CON),nicotine group(NIC,1×10-7M),nicotine+ERK5 inhibitor group(BIX02189+NIC),nuclear translocation of ERK5 was observed by immunofluorescence;3)experiment was divided into:normal control group,Nicotine group(NIC,1×10-7M),high glucose and high fat group,nicotine+high glucose and high fat group,PD98059/BIX0218 group,nicotine+PD98059/BIX02189 group,high glucose and high fat+PD9805 9/BIX02189 group,nicotine+High sugar and high fat+PD9805 9/BIX02189 group.The effects of ERK1/2 inhibitor(PD98059)and ERK5 inhibitor(BIX02189)on apoptosis and fibrosis expression were investigated by Western Blot and PCR.Results:1.Under high glucose and high fat conditions,nicotine increases the activation and apoptosis rate of cleaved caspase-9 and cleaved caspase-3,and promotes apoptosis of H9c2 cells.Nicotine alone increases cleaved caspase-11 associated with coke death and Endoplasmic reticulum stress-related protein expression of cleaved caspase-12.Compared with the normal control group,cleaved caspase-3,cleaved caspase-8 and cleaved caspase-9 significantly increased the expression of high glucose and high fat and nicotine combined with high glucose and high fat protein(P<0.01),among them,high glucose and high fat.Compared with the group,the expression of cleaved caspase-3 and cleaved caspase-9 in 6×10-6M nicotine combined with high glucose and high fat increased significantly(P<0.01);the apoptosis rate of cleaved caspase-3 protein was the same;Compared with the control group,the expression of cleaved caspase-11 and cleaved caspase-12 nicotine histone associated with apoptosis of endoplasmic reticulum was significantly increased(P<0.01).2.Under high glucose and high fat conditions,nicotine increases the expression of MMP2,MMP8,IL-6,Collagen Ⅰ,Collagen Ⅲ,SMAD3,TGF-β and AKT,which in turn promotes the transformation of H9c2 cells into myofibroblasts,resulting in myocardial Cell fibrosis.Compared with the normal control group,high glucose and high fat group and nicotine combined with high glucose and high fat group MMP2,MMP8 and the expression of IL-6 was significantly increased(P<0.01);the expression of Collagen Ⅰ,Collagen Ⅲ,Smad3,TGF-β and AKT was significantly increased in the nicotine group and nicotine combined with high glucose and high fat group(P<0.01).The gene expression of MCP-1 in nicotine combined with high glucose and high fat group was significantly increased(P<0.01).3.ERK5 inhibitor(BIX02189)reduces cleaved caspase-9 and cleaved caspase-3 protein expression and expression of genes involved in fibrosis.At 30 min and 1 h,the expression of pERK5 in high glucose and high fat group,nicotine combined with high glucose and high fat group was significantly lower than that in nicotine group(P<0.01),and at 30 min,pERKl/2 protein expression was identical to pERK5 protein expression.The effect(P<0.01)suggests that pERKl/2 and pERK5 are involved in the damage of H9c2 cells by nicotine under high glucose and high fat conditions.Compared with high glucose and high fat group,PD98059+high glucose and high fat and PD98059+nicotine+high glucose and high fat cleaved-caspase3 and cleaved-caspase9 protein expression were significantly increased(P<0.001);compared with high glucose and high fat group,BIX02189+The expression of cleaved-caspase3 and cleaved-caspase9 protein in high glucose and high fat group and BIX02189+nicotine+high glucose and high fat group were significantly decreased(P<0.001),indicating that ERK5 inhibitor can reduce the apoptosis of H9c2 cells.Compared with high glucose and high fat group and nicotine combined with high glucose and high fat group,BIX02189 can significantly reduce the expression of MMP2 and MMP8 genes(P<0.001).Compared with the nicotine group and nicotine combined with high glucose and high fat group,BIX02189 can significantly reduce the expression of TGF-P,AKT,Collagen Ⅰ and Collagen Ⅲ genes(P<0.001).Meanwhile,PD98059 can reduce the MMP2,MMP8 and SMAD3 genes.Expression(P<0.001).Conclusion:1.Nicotine increases the apoptosis of H9c2 cells under high glucose and high fat conditions.Among them,nicotine alone is associated with increased cell apoptosis and endoplasmic reticulum stress.2.Under high glucose and high fat conditions,nicotine increases the expression of genes involved in fibrosis in H9c2 cells,which in turn promotes the transformation of H9c2 cells into myofibroblasts.3.Under high glucose and high fat conditions,nicotine increases the activation of ERK family,especially ERK5,which reduces the apoptosis and fibrosis of H9c2 cells by inhibiting the phosphorylation of ERK5 into the nucleus.