The Inhibitive Effect Of Salidroside On Apoptosis Of EA.hy926 Cells Induced By H₂O₂ In Simulated Microgravity

ObjectiveMicrogravity or simulated microgravity can induce cardiovascular dysfunction and vascular endothelial dysfunction plays an important role in it.Studies have shown that simulated microgravity induces vascular endothelial dysfunction,the increasing of oxidative stress level has a very important effect in it.The purpose of this experiment is to study the effects of hydrogen peroxide?H₂O₂?on human umbilical vein endothelial cells?EA.hy926?under simulated microgravity,and to explore its effect on apoptosis and its related mechanism,and discuss the cell protection effects of salidroside and the underlying mechanism.MethodsEA.hy926 cells were cultured in cell culture medium with 10% fetal bovine serum in DMEM high glucose culture solution,and performed in a cell incubator with 5%CO2 at 37?,when the cells were passaged into the four generations,they were randomly divided into control group?Con72?and rotating group?Z72?,and then after 72 hours of cell rotation,the two groups were randomly divided into two groups and gave the following treatment at the same time:?1?not stimulated by H₂O₂?Con72 and Z72 group?;?2?give stimulation by H₂O₂ for 5 hours?Con72+ H₂O₂ and Z72+ H₂O₂ group?;?3?give stimulation by H₂O₂ for 5 hours and meanwhile give salidroside intervention?C+ H₂O₂+Sal and Z+ H₂O₂+Sal group?;?4?give stimulation by H₂O₂ for 5 hours and meanwhile give salidroside and PI3 K pathway inhibitor LY-294002 intervention?C+ H₂O₂+Sal+LY and Z+ H₂O₂+Sal+LY group?.Detect the apoptosis by flow cytometry method and take the secondary sequencing technology to conduct the transcriptome sequencing of cells in four groups include Con72,Con72+H₂O₂,Z72 and Z72+ H₂O₂,screening differential genes related to apoptosis,and analyze GO and KEGG with bioinformatics technology,verify the screening differential genes through RT-q PCR technology.On this basis,RT-q PCR technology was used to detect the effect of salidroside on the expression of differential genes,and analyze the molecular mechanism of H₂O₂ induced apoptosis under simulated microgravity.ResultsThe result of apoptosis measured by flow cytometry with Annexin V-FITC / 7-AAD method shows that EA.hy926 cells could induce obvious apoptosis treated by hydrogen peroxide stimulation under simulated microgravity,salidroside can inhibit cell apoptosis induced by hydrogen peroxide under simulated microgravity and plays a protecting role in EA.hy926 cells.There are six significant differential genes screened by transcriptome sequencing including BCL-2A1?FAM196B?TMEM158?PPP1R16B?CXCL8?PPP1R3B.Through the application of RT-q PCR technology,it can be found that BCL-2A1 and PPP1R16 B were consistent with the transcriptome sequencing results,the BCL2A1 is the anti-apoptotic gene and PPP1R16 B can promote cell proliferation and inhibit cell apoptosis,FAM196 B gene is the promoting factor that can promote cell proliferation.At the same time,it was found that BCL2A1 and FAM196 B genes were significantly increased after salidroside intervention,which indicated that salidroside can inhibit apoptosis by regulating these 2 genes and protect endothelial cells apoptosis induced by hydrogen peroxide under simulated microgravity.ConclusionsThe apoptosis rate of EA.hy926 stimulated by H₂O₂ was significantly increased under simulated microgravity,the effective active ingredient of Rhodiola,salidroside can inhibit cell apoptosis induced by H₂O₂ stimulation under simulated microgravity,and the mechanism was related to salidroside regulates the genetic expression of BCL2A1 and FAM196 B.