| Objective:With the improvement of people’s living quality,the incidence rate of cardiovascular diseases（CVD）is increasing in recent years.Under the background of high incidence rate and high mortality of Arteriosclerosis（AS）,it is the main pathological characteristic of CVD and the core pathological basis for progression.In many researches on the pathogenesis of AS,endothelial cell injury is known as an early critical step in the progression of AS,and apoptotic positive endothelial cells can be found in almost any atherosclerotic plaque.The apoptosis of endothelial cells may promote the formation of atherosclerotic plaque,which is not conducive to the stability of plaque and the rupture of endothelium caused by coronary plaque acute coronary syndrome（ACS）occurs,but the mechanism of endothelial cell apoptosis remains to be explored.MicroRNA-199-5p（miR-199-5p）is one of the most widely studied carcinogenic miRNA,which usually produces high levels of expression in many types of cells and tissues.In recent years,many studies indicate that miR-199-5p plays an important role in the development of cardiovascular system and the occurrence and development of CVD.It has been proved to be involved in cell apoptosis,vascular inflammation,neointima formation,proliferation of vascular smooth muscle cells and other cell types.Yet,there are few studies on the role of miR-199-5p in the apoptosis of endothelial cells exposed to ox-LDL at home and abroad reports.Salvia miltiorrhiza functions as promoting blood circulation and reducing blood stasis,calming and calming nerves.Tanshinol,as the main active component of Salvia miltiorrhiza,has the functions of protecting heart,inhibiting thrombosis,anti-inflammatory and protecting blood vessels.However,whether Tanshinol is involved in the regulation of endothelial cell function and whether Tanshinol is involved in endothelial cell apoptosis has not been reported.In this paper,the apoptosis model of human umbilical vein endothelial cells（HUVECs）exposed to ox-LDL was established to explore the pathogenesis of endothelial cell apoptosis.Methods:This experiment was divided into three parts.1.The role and mechanism of miR-199-5p in the apoptosis of human umbilical vein endothelial cells（HUVECs）exposed to ox-LDL.（1）The establishment of apoptosis model:50μg/ml and 100μg/ml of ox-LDL stimulated HUVECs for 12 h,24 h,36 h and 48 h,and then collected cells.The apoptosis rate of HUVECs cells treated by different concentrations of ox-LDL at different time points was detected by flow cytometry to assess the apoptosis model.（2）To detect the change of apoptosis rate after miR-199-5p mimics transfection:the cells were randomly divided into 4 groups:（1）control group:DMEM with only 10%fetal bovine serum;（2）model group:DMEM with 10%fetal bovine serum+50μg/ml ox-LDL;（3）miR-NC+ox-LDL group:transfection of miR-NC+50μg/ml ox-LDL.（4）miR-199-5p mimics+ox-LDL group,transfection of miR-199-5p mimics+50μg/ml ox-LDL.After 24 hours,the cells were collected,the apoptosis rate of each group was detected by flow cytometry,the expression of mitochondrial fission related protein and cell signal pathway protein was detected by Western blot,and the expression of miR-199-5p and AKAP1 m RNA were detected by real-time quantitative PCR.（3）The target relationship between miR-199-5p and AKAP1 was detected by luciferase reporter gene assay.The cells were randomly divided into three groups:p GL3 group,p GL3/AKAP1-UTR group,p GL3/AKAP1-UTR+miRNA-NC group and p GL3/AKAP1-UTR+miRNA mimics group.Luciferase reporter gene was used to detect the activity of luciferase and explore the targeting relationship between miR-199-5p and AKAP1.2.The effect of Tanshinol on miR-199-5p and target gene expression.HUVECs were cultured in vitro and randomly divided into:（1）Control group:DMEM medium containing only 10%fetal bovine serum.（2）Model group:DMEM medium+final concentration of 10%fetal bovine serum was 50μg/ml ox-LDL.（3）Tanshinol group:Tanshinol 10μmol/L+10%fetal bovine serum DMEM medium+final concentration of 50μg/ml ox-LDL.After 24hours,the apoptosis rate of each group was detected by flow cytometry.The expression of miR-199-5p and AKAP1 m RNA was detected by q PCR,and mitochondrial fission related proteins and cell signaling pathway proteins were detected by Western blot.3.The effect of Tanshinol on atherosclerosis in ApoE-/-mice.114-6-week-old male wild-type C57BL/6J mice and 22 ApoE-/-mice（atherosclerosis model mice）with the same genetic background.They were divided into three groups:control group 11,model group 11,Tanshinol group11.The atherosclerotic model was established in the high-fat feeding group and the Tanshinol group（ApoE knockout mice）for 12 weeks,and the Tanshinol group was administrated with drugs.The expression level of miR-199-5p and AKAP1 m RNA was detected by real-time quantitative PCR.Results:1.In this experiment,HUVECs were stimulated with 50μg/ml ox-LDL for 24 h as the apoptosis model.（1）Compared with the control group,the apoptosis rate of endothelial cells induced by ox-LDL was increased（P<0.05）,the down-regulation of miR-199-5P and up-regulation of AKAP1 in the Model Group（P<0.05）,in the model group,the expression of PDRP1 was significantly increased（P<0.05）,but the expression of DRP1 was not significantly changed,and the expression of p-/JNK and p-ERK1/2/ERK1/2 in HUVECs was significantly increased after ox-LDL treatment,however,miR-199-5p mimic transfection inhibited the phosphorylation of these proteins（P<0.05）.（2）Human umbilical vein endothelial cells were transient transfected with miR-199-5p mimics and then treated with ox-LDL:The expression of miR-199-5p was significantly increased（P<0.05）,the apoptosis rate was significantly decreased（P<0.05）,while the expression of AKAP1 m RNA was significantly decreased（P<0.05）and the expression of mitochondrial fission related protein（AKAP1,p-Drp1/Drp1）was significantly decreased（P<0.05）.The p-ERK1/2 phosphorylation expression of apoptosis-related signal pathways increased significantly（P<0.05）,whereas the total expression of ERK,JNK and p-JNK did not change significantly（P>0.05）.（3）AKAP1was one of the target genes of miR-199-5p confirmed by luciferase reporter gene detection.2.In the model of endothelial cell apoptosis induced by ox-LDL,compared with the model group,the apoptosis rate of endothelial cell in Tanshinol group decreased（P<0.05）,the expression of miR-199-5p increased significantly（P<0.05）,and AKAP1 m RNA decreased significantly（P<0.05）.The expression of mitochondrial fission related protein（AKAP1,p-Drp1/Drp1）was significantly decreased（P<0.05）.Apoptosis signal p-ERK1/2 expression were also significantly reduced（P<0.05）,and there was no difference in total ERK and JNK protein expression（P>0.05）.3.The atherosclerotic model of apo E knockout mice was successfully established after high-fat feeding 12 weeks.After the experiment,the blood lipid level increased significantly,but there was no significant difference between the Tanshinol group and the model group（P>0.05）;The result of HE staining showed that the intima of the normal group was smooth,without plaque;there was significant plaque formation in the model group,the plaque area of the Tanshinol group decreased significantly.Compared with the control group,a large number of AKAP1 positive cells were observed in the arteries of the model group,especially in the plaques,after Tanshinol treatment,some AKAP1 positive staining cells could be seen in the plaque location,but the number of AKAP1 positive staining cells was significantly reduced.The expression of miR-199-5p in Tanshinol group was significantly higher than that in the control group（P<0.05）,and AKAP1 m RNA was significantly lower（P<0.05）.Conclusions:AKAP1 is the target gene of miR-199-5p,which may affect the phosphorylation of p-Drp1 and p-ERK1/2 and inhibit the apoptosis of endothelial cells through its target gene AKAP1,and Tanshinol may play an anti apoptosis role by up regulating the expression level of miR-199-5p,inhibiting the expression of AKAP1 and affecting the p-Drp1 and p-ERK1/2signal pathway. |
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