The Research For Mechanisms In The Prevention Of Heat Stress-induced Endothelial Cell Apoptosis By Sodium Tanshinone IIA Sulfonate

Objective:heat stroke HS is a life-threatening disease,heat stress interaction in various cytokines and inflammatory factors,early endothelial cells can be activated and impaired,and the injury of endothelial cells can further aggravate the inflammatory reaction,which eventually led to the occurrence of multiple organ failure.Tanshinone IIA sulfonate(Tanshinone,IIA,sulfonate,STS)has many roles in the protection of vascular endothelium.Current studies have shown that STS can inhibit the apoptosis of human umbilical vein endothelial cells and alleviate the inflammatory reaction induced by hyperthermia in rats.Therefore,it is of theoretical and practical significance to investigate the effects of STS treatment on heat stroke(HS)endothelial cells and to prevent heat stroke and elucidate the mechanism of high heat damage to endothelial cells.To sum up,this study aimed to explain the effects of tanshinone IIA sulfonate(Tanshinone,IIA,sulfonate,STS)on heat stroke(HS)endothelial cells and the mechanism of cellular response to STS treatment.Methods:HUVEC cell CCK-8 assay detected the 1.exposure cell activity is not the same length at 42 DEG C,Annexin V/PI flow cytometry and heat induced effect on the apoptosis rate of HUVEC cells,changes of HUVEC cell apoptosis related molecule Western-blotting detection after heat induction of heat induced apoptosis of HUVEC cells.The activity of 2.HUVEC cells changed under different concentrations of STS,and the activity of HU-VEC cells was changed at different time after heat induction,in order to screen the concentration of STS and the recovery time after heat induction.The changes of HUVEC cells was detected in 3.CCK-8 cells in the presence of STS activity,to study the effect of STS on heat induced HUVEC cell apoptosis rate of Annexin V/PI flow cytometry,using caspase assay kit to detect the apoptotic effect of Caspase-3 enzyme activity.To investigate the protective effect of STS on HUVEC cells in heat induced environment.4.to investigate the mechanism of STS induced apoptosis in HUVEC cells induced by heat.The nitrate/nitrite colorimetric analysis method was used to detect the effect of STS induced nitrite production and HUVEC cells to heat,Western phosphorylation of blot detected at different time points of eNOS,blocking eNOS phosphorylation by eNOS inhibitor L-NMMA,to study the effect of STS on blocking,eNOS in HUVEC cells after eNOS phosphorylation of HUVEC cell viability and cell apoptosis of STS on the high temperature caused by the rate of.To investigate the correlation between NO and apoptosis induced by heat shock in HUVEC cells.5.to investigate whether phosphorylation of eNOS in heat induced HUVEC cells is regulated by phosphorylation of Akt.By using Western blot STS on the detection of heat induced phosphorylation of Akt at different time points in HUVEC cells,STS on the effect of blocking eNOS phosphorylation of Akt in HUVEC cells after using Akt inhibitor MK-2206,using Western blot STS on the detection of heat induced phosphorylation of Akt HUVEC cells in different time points.6.to investigate whether STS induced phosphorylation of Akt or phosphorylation of eNOS is controlled by the PI3 kinase dependent regulatory mechanism.The changes of phosphorylated Akt and phosphorylated STS in HUVEC cells induced by eNOS were detected by PI3K inhibitors Wortmannin and Western blot.Effects of inhibitors MK-2206 and Wortmannin on apoptosis of HUVEC cells induced by heat.Results:1.HUVEC cells at 42 degrees C and exposed cell activity decreased with time prolonging,the heat induced and the apoptosis rate of HUVEC cells increased,cleaved-caspase-3 heat induction in HUVEC cells with time and protein expression level increased.2.the concentration of STS was 2μV/ml,the activity of HUVEC cells induced by heat is obviously improved;in 2 hours when the protective role of STS has emerged;when the recovery time is getting longer,no cell activity in the role of the drug will have a certain degree of recovery,and even very low concentration of STS(0.002μg/ml)can protect cells.3.after exposure to 42 DEG C for 1 hours after the change can be observed and off phenomenon in heat induced cells,and STS(2μg/ml)after the treatment of this phenomenon has been significantly reduced.From the detection of cell activity,1 hours after exposure to 42 DEG C,we could observe the decrease of cell activity after heat induction,and the recovery of cell activity to a certain extent after treatment with STS(2μg/ml).In the use of STS(2μg/ml)after the cells were exposed to 42 DEG C for 1 hours,compared with the untreated group of STS cells induced by heat,the apoptosis of HUVEC cells after STS treatment so that the heat induced rate was significantly reduced,the result is a statistically significant difference.After heat induced HUVEC cell action in STS,the apoptotic effector enzyme Caspase-3 activity decreased.To investigate the protective effect of STS on HUVEC cells in heat induced environment.4.in the absence of STS treatment,the amount of nitrite production in HUVEC cells increased significantly at 0.5 h,decreased sharply at 1 and 2 h,and nitrite production was at baseline at 2 h.While the STS(2 g/ml)treatment of HUVEC cells with nitrite in heat induced in cells after production gradually increased,reached a steady state in 1 hours was significantly higher than the same period without nitrite STS in HUVEC cells treated by level.Phosphorylation of eNOS protein in HUVEC cells induced by heat after 15 minutes and 30 minutes increased,and then decreased in 1 hours and 2 hours after the phosphorylation of eNOS;STS(2 g/ml)after the treatment,HUVEC cells from heat induced phosphorylation of eNOS after 15 minutes to 1 hours was sustained increase.Phosphorylation of eNOS protein in HUVEC cells induced by heat after 15 minutes and 30 minutes increased,and then decreased in 1 hours and 2 hours after the phosphorylation of eNOS,STS(2 g/ml)after the treatment,HUVEC cells from heat induced phosphorylation of eNOS after 15 minutes to 1 hours was sustained increase.After pretreatment with L-NMMA,the phosphorylation level of HUVEC was not changed even after eNOS was induced by STS.In addition,pretreatment with L-NMMA attenuated the activity of HUVEC cells induced by hyperthermia and did not improve after STS.Similarly,pretreatment with L-NMMA increased the expression of cleaved-caspase-3 in HUVEC cells induced by hyperthermia,and this effect was not altered by the action of STS.5.in the absence of STS treatment,Akt phosphorylation was elevated in HUVEC cells at 15 min and 30 min after heat induction 1 h and 2 h,compared with heat induction.However,if STS(2μg/ml)acts,phosphorylation of Akt continues to increase over time and tends to stabilize at 1 hours.After MK-2206 pretreatment,eNOS phosphorylation of heat induced HUVEC cells can inhibit STS induced in MK-2206 after pretreatment enhanced,similarly,MK-2206 pretreatment significantly inhibited the effect of STS on nitrite production in the HUVEC cells under high temperature.6.Wortmannin inhibited the phosphorylation of eNOS and Akt in HUVEC cells induced by STS in a high temperature environment.In addition,Wortmannin pretreatment also inhibited the increase of nitrite content in HUVEC cells when STS induced high temperature.MK-2206 pretreatment resulted in an increase in the proportion of heat induced apoptosis in HU-VEC cells,which could not be altered whether or not it was added to STS.MK-2206 pretreatment resulted in an increased expression of Caspase-3 in heat induced HU-VEC cells,which was similarly unaffected by STS.Conclusions:1.Heat stress induces apoptotic cell death in human umbilical vein endothelial cells(HUVEC).2.STS suppresses heat stress-induced apoptosis of HUVEC.3.STS treatment stimulated the PI3K/AKT pathway and led to increased eNOS phosphorylation,which resulted in increased nitric oxide(NO)production in HUVEC under heat stress.