Effect Of Tongxinluo On High Glucose-induced Proliferation And Apoptosis Of Cardiac Fibroblasts

ObjectiveMyocardial fibroblasts cultured with high glucose to simulate the pathological environment of myocardial fibrosis induced by diabetes,to Investigate the effect of different doses Tongxinluo on the proliferation and apoptosis of cardiac fibroblasts induced by high glucose,to explore the possible mechanism of Tongxinluo inhibiting myocardial fibrosis.MethodsPrimary culture and Subculture of neonatal SD rat cardiac fibroblasts(CFs).Immunofluorescence staining was performed to identify CFs.Used high glucose medium(DMEM with 25mmol/L glucose)to culture CFs for different time points(6,12,24,48,72h),and each time point was cultured with low glucose medium(DMEM with 5.5mmol/L glucose)as control group.The proliferation activity of CFs in high glucose culture group and low glucose culture group at different time points was detected by MTT,which could be showed by absorbance(OD).We could get the OD peak of high glucose culture group,the time of OD peak was the time strongest proliferation activity of CFs in high glucose culture.The experiment were divided into control group(cultured with low-glucose DMEM),model group(cultured with high-glucose DMEM),20?g/ml TXL group(cultured with high-glucose DMEM+TXL at 20?g/ml TXL),80?g/ml TXL group(cultured with high-glucose DMEM+TXL at 80?g/ml TXL),320?g/mlTXL group(cultured with high-glucose DMEM+TXL at 320?g/ml TXL).The proliferation activity of CFs each group was detected by MTT at the time of OD peak in high glucose culture group.The expression contents of type?collagen,type ? collagen and TGF-?1 each group were detected by ELISA.Western blotting was used to detected the expression of bax,bcl-2 each group.Results1.The operation time of CFs cultured with high glucose detected by MTT.The OD of high glucose group increased gradually with the prolonging of incubation time,reached a peak at 24 h and the OD reduced gradually with the prolongation of the culture time after 24 h,suggestting that the proliferation activity of CFs cultured with high glucose is strongest at 24 h.The OD of low glucose group increased gradually with the prolonging of incubation time,reached a peak at 48 h and the OD reduced gradually with the prolongation of the culture time after 48 h.The OD of high glucose group at each time point were higher than that of low glucose group,especially 24 h is the most obvious.2.The proliferation activity of CFs each group was detected by MTT.Compared with the control group,the OD in model group was significantly increased;Compared with the model group,the OD in 20?g/m LTXL group did not decrease obviously,while the OD in 80 and 320?g/m LTXL group decreased significantly.The most obvious reduction was in 320?g/m LTXL group.3.ELISA was used to detect the expression of type ? collagen,type ?collagen and TGF-?1.the expression of type ? collagen,type ? collagen and TGF-?1 in model group was significantly higher than that of control group.While the expression of each protein in 20,80 and 320?g/m LTXL group decreased significantly compared with the model group,especially the decrease in the320?g/m LTXL group is most obvious4.Western blotting was used to detect the protein expression of Bax andBcl-2.Compared with the control group,the expression of Bcl-2 in model group increased significantly,while the expression of Bax was decreased but not obvious.Compared with the model group,the expression of Bcl-2 was decreased and the expression of Bax was increased in the 20,80 and 320ug/m L TXLgroup.Conclusions1.High glucose culture environment can enhance the proliferation activity of CFs,make it over secrete type ? collagen,type ? collagen and TGF-?1,and reduce the apoptosis of CFs by increasing the expression of Bcl-2.2.Different doses of TXL can inhibit the proliferation activity of CFs,reduce the secretion of type ? collagen,type ? collagen and TGF-?1,also can promote the apoptosis of CFs by increasing the expression of Bax and inhibiting expression of Bcl-2,so as to play its role in inhibiting myocardial fibrosis.The effects above exist dose dependent manner.