| ObjectiveStromal interaction molecule 1(STIM1)plays an important role in the occurrence and development of a variety of diseases.This article intends to investigate the expression of STIM1 in two models of pulmonary arterial hypertension(PAH)induced by hypoxia and monocrotaline in rats.The effect of STIM1 on autophagy and proliferation and its possible mechanism are observed on 293 T human renal epithelial cell line and A7R5 rat Aortic smooth muscle cells,which provided a new idea for the study and treatment of pathogenesis of pulmonary hypertension.Methods1.Induced by oxygen deficit(OD)and monocrotaline(MCT)induced pulmonary hypertension model in rats,measured right ventricular systolic pressure(RVSP)and right ventricular weight index(RVMI)and HE staining observation Pulmonary artery vascular morphology to identify whether the modeling is successful.2.Western blotting analysis technique is used to detect the STIM1 expression in pulmonary artery samples.3.Search for the STIM1 gene encoded by the rat,design gene-specific cloning primers,amplify STIM1 using PCR technology,and connect the above fragments to the PLV lentiviral vector,and perform digestion identification on the PLV-STIM1 recombinant vector.4.Establish STIM1 overexpression model in 293 T cells and A7R5 cells,and adopt western blotting analysis and functional related experiments to explore the effect of STIM1 on cell proliferation.5.In the pulmonary artery tissues,for 293 T cells and A7R5 cells,western blotting analysis is used to analyze the effect of overexpression of STIM1 on the expression of autophagy and inflammatory molecules.Results1.Compared with the Control group,the right ventricular systolic pressure(RVSP)and right ventricular mass index(RVMI)of rats in the OD group and MCT group increased,and HE staining showed pulmonary artery smooth muscle in the OD group and MCT group Hyperplasia,thickening of the tube wall,suggesting that the pulmonary hypertension model is constructed.2.Compared with the Control group,the expression of STIM1 protein in the pulmonary artery tissues of the OD group and MCT group decreased.3.After doubling enzyme digestion,the recombinant vector PLV-STIM1 sequence is corrected.By observing the expression of fluorescent protein in the PLV-EGFP group and western blotting analysis,the STIM1 cell overexpression model is constructed.4.In 293 T cells,compared with the Control group,the expression levels of PCNA and Cyclin D1 are decreased after transfection with PLV-STIM1;after the detection by the CCK-8 method,it is found that the PLV-STIM1 is overexpressed compared with the Control.It can reduce the proliferation ability of cells;in the plate cloning experiment,compared with the Control group,the transfection of PLV-STIM1 can reduce the cloning ability of cells.In A7R5 cells,after detection by CCK-8 method,it is found that compared with the Control group,the overexpression of PLV-STIM1 has no significant effect on cell proliferation.In the plate cloning experiment,compared with the Control group,the PLV-STIM1 infection can reduce the cloning ability of the cells.5.In animal experiments,compared with the Control group,the expression of LC3 in the pulmonary artery tissue of the OD group and the MCT group increased significantly,and the expression of Beclin-1 decreased.In 293 T cells,compared with the Control group,the expression of autophagy-related protein LC3 was significantly decreased,the expression of P62 was increased,and the expression of atg12 was decreased before and after transfection with PLV-STIM1 and before and after EBSS and rapamycin treatment;Compared with the control group,the expression of inflammation-related protein sting after PLV-STIM1 transfection had no statistical significance.The expression of p-sting decreased,and the expression of its downstream protein TBK1 decreased.In A7R5 cells,compared with the control group,the expression of autophagy-related protein LC3 is decreased before and after infection with PLV-STIM1 and before and after treatment with bafilomycin;compared with the Control group,the expression of inflammation-related protein sting after PLV-STIM1 infection had no statistical significance.the p-sting expression level decreased,and the downstream protein NF-κB expression level decreased.Conclusion1.There is a certain connection between STIM1 and pulmonary hypertension2.STIM1 slows down the process of pulmonary hypertension by inhibiting cell proliferation,autophagy and inflammation. |
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