Er-xian Drug Serum Inhibits Ox-LDL-induced Proliferation Of Coronary Smooth Muscle Cells Through Estrogen Receptors

Objective:Coronary atherosclerosis sex heart disease,is caused atherosclerosis coronary artery vascular cavity stenosis or occlusion,coronary arterial blood supply to relative or absolute lack of myocardial ischemia and hypoxia caused by change,its mechanism is very complicated,abnormal smooth muscle cell proliferation is one of the main reason,which type Oxidized low density lipoprotein can cause endothelial injury and release a variety of inflammatory mediators,Promote monocyte into lipid formation of macrophage cell lipid necrotic core,surrounded by smooth muscle cells and appreciation of lipid core become smooth muscle fiber cap promote vascular smooth muscle cells(VSMC)proliferation packages lipid necrotic core,eventually become the artery atheromatous plaque clogging cause myocardial ischemia,hypoxia or necrosis in the lumen.Studies have suggested that estrogen has an intervention effect on atherosclerotic plaques,but its use is limited due to its carcinogenic,thrombogenic and other risks.In recent years,studies have found that phytoestrogens,such as icariin and baicalin,can simulate the effects of estrogen with few side effects.Therefore,the development of similar phytoestrogens has become a need.Previous studies in this study showed that Erxien’s blood injection components contain a variety of phytoestrogens and can activate GPER30(G protein-coupled estrogen-related receptor)and downstream ERK1/2,Akt and other signaling pathways to reduce myocardial reperfusion injury.Based on this previous study,this study aimed to explore the effect of Erxian drug-containing serum on the proliferation of coronary smooth muscle cells induced by oxidized low density lipoprotein(Ox-LDL)at the cellular level,and to explore its mechanism.Methods:1.Cell culture: primary rat coronary smooth muscle cells were cultured in complete medium,and the experimental cells were the 4-6 generation cells(all in logarithmic growth phase).2.Experimental procedures: Firstly,different concentrations of Ox-LDL were given to induce cells,and CCK-8 was used to select the optimal concentration of Ox-LDL at50mg/L.The optimal concentration of 10% Er-xian drug serum was selected.The formal experimental groups were divided into 9 groups: normal cell group,model group(Ox-LDL),positive control group(17-β-estradiol),negative control group(rat negative serum),Er-xian drug serum group,ICI182780 group,G15 group,Er-xian drug serum +ICI182780 group,Er-xian drug serum +G15 group,cell morphology was observed under inverted microscope,cell proliferation was detected by CCK-8method,calcium concentration was determined by flow cytometry(FLU-3),m RNA expression of GPER30 ERK1/2,Akt in cells was detected by fluorescence quantitative PCR,The expression of GPER30 ERK1/2,Akt protein was determined by ELISA and Western blot.Results:1.Morphological effects of Er-xian drug serum on Ox-LDL-induced smooth muscle cells were observed with inverted microscope and compared with normal RCSMCs under the action of Ox-LDL,the cell bodies of RCSMCs became larger and irregular from findles,while the cell bodies became smaller after the intervention of Er-xian drug serum,and the effect of the addition of ICI182780 and G15 drug serum of Er-xian was weakened.2.The effect of the drug serum of Er-xian on the proliferation of Ox-LDL-induced smooth muscle cells was detected by CCK-8 method Ox-LDL significantly promoted cell proliferation compared with normal cells,while Ox-LDL was inhibited after the intervention of Er-xian drug serum,and this effect of Er-xian drug serum could be blocked by ICI182780 and G15.3.The effect of Er-xian drug serum on the level of calcium ion in Ox-LDL induced smooth muscle cells was detected by flow cytometry.Compared with model group,the Methods:concertration of calcium in RCSMCs in Ex-xian drug serum group wa significantly decreased.Compared with Erxian drug serum group,Er-xian drug serum group+ICI182780 and Er-xian drug serum group +G15 blocked the inhibitory effect of Er-xian drug serum on smooth muscle proliferation.4.Fluorescence PCR method was used to detect the effect of Er-xian drug serum on Ox-LDL-induced smooth muscle cells Gper30 ERK1/2,Akt Compared with the model group,the m RNA expression of GPER30 in Er-xian serum group was significantly increased,but the m RNA expression of ERK1/2,Akt was not significantly affected.Compared with the Ex-xian drug serum group,the m RNA expression of GPER30 in the Ex-xian drug serum +ICI182780 group and the Ex-xian drug serum+G15 group was significantly decreased,while the m RNA expression of ERK1/2.Akt was not significantly changed,respectively.These results indicated that Er-xian drug serum promoted the expression of GPER30 gene but had no obvious effect on ERK1/2,Akt at the gene level,and this effect of Er-xian drug serum could be detected by ICI182780 and G15-blocking.5.ELISA on Ox-LDL-induced smooth muscle cell GPER30 Compared with model group,the expression level of GPER30 protein in Er-xian drug serum group was significantly increased,while the expression level of ERK1/2,Akt protein was significantly decreased.Compared with the Ex-xian drug serum group,the expression levels of GPER30 protein in the Ex-xian drug serum +ICI182780 group and Ex-xian drug serum+G15 group were significantly decreased,while the expression levels of ERK1/2,Akt protein were significantly increased;These results indicated that Er-xian drug serum significantly promoted the expression of GPER30 protein and inhibited the expression of ERK1/2,Akt protein at the protein level,and this effect of Er-xian drug serum could be detected by ICI182780 and G15 blocking.6.Western blot method to detect the effect of Er-xian drug serum on Ox-LDL-induced GPER in smooth muscle cells.Compared with model group,the expression level of GPER30 protein in Er-xian drug serum group was significantly increased,while the expression level of ERK1/2,Akt protein was significantly decreased.Compared with Er-xian drug serum group,the expression levels of GPER30 protein in the Er-xian drug serum+ICI182780 group and Er-xian drug serum +G15 group were significantly decreased,while the expression levels of ERK1/2,Akt protein were significantly increased;These results indicated that Er-xian drug serum significantly promoted the expression of GPER30 protein and inhibited the expression of ERK1/2,Akt protein at the protein level,and this effect of Er-xian drug serum could be blocked by ICI182780 and G15.