| Background:Endothelial cells play an important role in physiological and pathological processes.Oxidative stress injury(OSI)of vascular endothelial cells(VEC)is an important component of cardiovascular diseases such as atherosclerosis,hypertension and diabetes,prevention and treatment of endothelial oxidative stress injury has important clinical significance.However,the pathophysiological mechanism of oxidative stress injury has not been clarified so far due to the involvement of many cytokines and signaling pathways in oxidative stress injury.The PI3K/Akt signaling pathway is a signaling pathway widely distributed between tissue cells,which regulates signaling between cells,signal transduction in the cytoplasm,and transcription of genes in the nucleus.In addition,the PI3K/Akt pathway plays an extremely important role in cell proliferation,migration,growth,differentiation and death,and is a highly conserved signaling pathwayGeniopicroside(GPS)belongs to the split-ring iridoid glycoside compound and its molecular formula is C16H20O9.GPS is the main active ingredient of gentian gentian gentian.A large number of clinical and experimental studies have found that:GPS not only has anti-inflammatory and analgesic effects,liver and gallbladder,and antagonizes tumors,but also has anti-oxidative damage,inhibits the proliferation and proliferation of aortic smooth muscle cells,and the cardiovascular protective effect of preventing and treating atherosclerosis.However,whether GPS has a protective effect on oxidative stress damage of vascular endothelial cells has rarely been reported,and further research is needed.Objective:Oxidative stress plays an important role in the pathogenesis of endothelial dysfunction,which precedes the development of many cardiovascular or non-cardiovascular diseases.In this study,hydrogen peroxide(H2O2)was used to simulate oxidative stress damage in cells,and the protective effect of gentiopicroside(GPS)on oxidative stress injury of human umbilical vein endothelial cells(HUVECs)and its potential in injury therapy were investigated.Mechanism research.Method:Experiment 1:Human umbilical vein endothelial cells were treated with different concentrations of H2O2(100,200,400 μmol/L)for 2,4,and 8 h.The purpose was to detect the toxicity gradient range of H2O2 and establish a suitable oxidative stress injury cell model.We used H2O2 interference to study the survival rate,apoptosis rate and mobility of human umbilical vein endothelial cells with GPS in oxidative stress injury.HUVECs cultured in vitro were divided into 4 groups:Group of control,Group of H2O2,Group of H2O2+740Y-P and Group of 740Y-P.The H2O2 group was treated with H2O2 culture medium at a concentration of 400 μmol/L;the H2O2+740Y-P group was treated with 500 mg/mL 740Y-P solution for treatment of HUVECs 2 h before treatment with 400 μmol/L H2O2 medium;740Y-P group Only 500 mg/mL of 740Y-P solution was added to the medium.Each group of cells was cultured for 6 hours at a constant temperature of 37℃ n a saturated humidity cell incubator of 5%CO2.After the above treatment,the cell survival rate was detected by MTT,the apoptosis rate was detected by TUNEL staining,and the migration rate of vascular endothelial cells was detected by scratch test.Experiment 2:First,gentiopicroside(GPS)was pre-dissolved in DMSO and diluted to the corresponding concentration with cell culture solution.Firstly,different concentrations of GPS(10,20,40 μmol/L)were used to treat human umbilical vein endothelial fine 2,4,8 h.The purpose is to detect whether there is a range of toxicity gradients in GPS,to explore whether GPS has cytotoxic effects,and to establish a suitable oxidative stress damage cell model.Umbilical vein endothelial cells were divided into 4 groups,and the cells were divided into 4 groups:Group of control,Group of H2O2,Group of GPS+H2O2 and Group of GPS.The Control group was treated without serum and cultured in serum-free DMSO(0.01%);the H2O2 group was treated with 400 μmol/L H2O2 medium;the GPS+H2O2 group was treated with 400 μmol/L,H2O2.Add 1 mol/L GPS;the GPS group only added 1 mol/L GPS to the Control group.Each group of cells was cultured for 6 hours at a constant temperature of 37℃ in a saturated humidity cell incubator of 5%CO2.After the above treatment,the cell survival rate was detected by MTT,the apoptosis rate was detected by TUNEL staining,the mobility of vascular endothelial cells was detected by scratch test,and the release rate of lactate dehydrogenase(LDH)in each group was detected by a special kit.And oxygen free radical(ROS),SOD,GSH-Px and MDA content,immunofluorescence and West-blot detection of each group of cells The expression levels of each protein Bcl-2,Caspase3,Bax,cytochrome C.Experiment 3:The cells were uniformly seeded in a 96-well plate at 1 × 108/L.The HUVECs cells were first divided into two groups:Control cells were treated with siRNA,and cultured in serum-free DMSO(0.01%),PI3K siRNA.The group added 1 mol/L of PI3K protein molecule to the Control group.The cells in both groups were cultured in a constant temperature of 37℃,5%CO2 in a humidified cell culture incubator for 4 hours.The SOD,GSH-Px and MDA contents and PI3K activity were observed.Whether the PI3K/Akt signaling pathway was involved in signal transduction Play a role.A group of cells were uniformly inoculated into 96-well plates at 1 × 108/L,and the cells were divided into three groups:H2O2 group,GPS+H2O2 group and GPS+H2O2+PI3K siRNA group.The H2O2 group was treated with 400 μmol/L H2O2 medium for interference;the GPS+H2O2 group was treated with 400 μmol/L H2O2 medium and 1 mol/L GPS;the GPS+H2O2+PI3K siRNA group was based on the GPS+H2O2 group.Add 1 mol/L of PI3K siRNA.Each group of cells was cultured for 6 hours at a constant temperature of 37℃ in a saturated humidity cell incubator of 5%CO2.After the above treatment,the cell survival rate was detected by MTT,the apoptosis rate was detected by TUNEL staining,the mobility of vascular endothelial cells was detected by scratch test,and the release rate of lactate dehydrogenase(LDH)in each group was detected by a special kit.And oxygen free radical(ROS),SOD,GSH-Px and MDA content,immunofluorescence and West-blot detection of each group of cells The expression levels of each protein Bcl-2,Caspase3,Bax,and cytochrome C.Results:In experiment 1,the survival rate and mobility of human umbilical vein endothelial cells treated with H2O2 were obviously decreased,and the apoptosis rate was obviously increased.The expression levels of intracellular protein molecules PI3K and P-AKT were significantly reduced.It is in a concentration-and time-dependent effect relationship.After intervention of HUVECs with PI3K/Akt signaling pathway-specific agonist 740Y-P,cell viability and migration were significantly increased,and apoptosis rate was significantly decreased.Western-blot results showed that 740Y-P obviously down-regulated of Caspase3 and Bax,and the expression of P-Akt and Bcl-2 was significantly up-regulated.It is further suggested that PI3K/Akt signaling pathway plays a very important role in oxidative stress injury.Activation of PI3K/Akt signaling pathway can antagonize oxidative stress damage in vascular endothelial cells and protect the normal function of endothelial cells.In experiment 2,The HUVECs were not significantly reduced after the intervention of different concentrations of GPS and time.It is suggested that GPS has no obvious toxic effect on HUVECs.The survival rate,migration rate and intracellular SOD and GSH-Px levels of human umbilical vein endothelial cells treated with H2O2 were significantly lower than those of control group.Apoptosis rate and LDH and MDA release were significantly increased compared with control group.After GPS pretreatment,compared with H2O2 group,the survival rate,migration rate and intracellular SOD and GSH-Px content of GPS+H2O2 group were significantly decreased,and apoptosis rate and LDH and MDA release were significantly increased Western-blot results showed that GPS could obviously dn-regulate the expression of Caspase3 and Bax in H2O2,and up-regulate the low expression of PI3K,P-AKT and Bcl-2 in H2O2.It is suggested that GPS can effectively antagonize oxidative stress damage in vascular endothelial cells,and its specific signal transduction pathway may be related to PI3K/Akt.In experiment 3,vascular endothelial cells were transfected with PI3K siRNA,and the level of PI3K protein was detected by Western Blot.The expression of PI3K protein was significantly decreased.This indicates that PI3K siRNA successfully inhibits the expression of PI3K protein in cells,and the inhibition rate is over 70%.Human umbilical vein endothelial cells were transfected with PI3K siRNA prior to the administration of gentiopicroside and H2O2.Western blot results showed that the expression levels of HUVEC Caspase3 and Bax were significantly increased with PI3K siRNA,expression of P-Akt and Bcl-2 was obviously decreased.It is further suggested that PI3K/Akt signaling pathway is involved in the protective effect of gentiopicroside-mediated H2O2 injury on HUVEC.Conclusion:PI3K/Akt signaling pathway plays an important role in oxidative stress injury.Inhibition of PI3K/Akt signaling pathway can antagonize oxidative stress damage in vascular endothelial cells.The drug can be effectively antagonized by oxidative stress damage in vascular endothelial cells and regulated by PI3K/Akt signaling pathway. |
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