| Part 1:Tanshinone ⅡA regulates atherosclerotic lesions by inhibiting the Hippo-YAP signaling pathway in vitro studyObjective:To investigate the mechanism of tanshinone ⅡA in inhibiting Hippo-YAP signaling pathway in the in vitro culture of human umbilical vein endothelial cells in the regulation of atherosclerotic lesions,and to provide a basis for the following research.Materials and Methods:Human umbilical vein endothelial cells(HUVECs)were selected and stimulated fully by LPS for 12 hours to establish the inflammation model of HUVECs.The Control group,LPS group,TSⅡA+LPS group and PFI-2+LPS group were set respectively.MTT assay was used to detect the proliferative activity and toxicity of each group.The expression levels of TNF-α、IL-1β、IL-6 and IL-8 were detected by ELISA.The expression of NO in supernatant of each group was evaluated.Cell adhesion assay was used to determine the adhesion ability of HUVECs to mononuclear cells(THP-1 cells).The expression of VCAM-1 and ICAM-1 in HUVECs was detected by RT-PCR,Western blot and flow cytometry.Immunoblot analysis of Hippo-YAP transcription factor activity.Results:Cytotoxicity test of HUVECs induced by LPS and Tanshinone ⅡA:With the increase of LPS intervention concentration,the cell activity decreased,and the intervention of LPS on cytotoxicity was significantly enhanced,which reflected the dose dependence.When the LPS intervention concentration was 2.0 g/mL,the difference was statistically significant(P<0.05).With the increase of tanshinone ⅡA intervention concentration,the activity of the cells also decreased.When the concentration of Tanshinone ⅡA was 5.0 and 10.0 μM,the activity of the cells was the lowest,showing a dose dependence,and the difference was statistically significant(P<0.05).Tanshinone ⅡA promoted LPS-induced activity detection of HUVECs cells:LPS addition reduced the proliferation activity of HUVECs cells,while tanshinoneⅡA increased the proliferation activity of HUVECs cells,and the difference was statistically significant(P<0.05).Tanshinone ⅡA inhibits LPS-induced inflammatory factors in HUVECs cells:the addition of LPS solution can significantly promote the intracellular expression of TNF-α、IL-1β、IL-6 and IL-8,and the addition of Tanshinone ⅡA alleviates the release process of inflammatory factors,with statistically significant difference(P<0.05).NO concentration expression:the addition of LPS increased the production of NO,while the addition of Tanshinone ⅡA reduced the production of NO in cells,and the difference was statistically significant(P<0.05).The protein concentration expression of iNOS and eNOS showed an opposite trend.The addition of LPS solution increased the concentration expression of iNOS and decreased the concentration expression of eNOS.The addition of Tanshinone ⅡA reduced the concentration of iNOS and increased the concentration of eNOS(P<0.05).Adhesion detection of HUVECs cells:After LPS solution was added,HUVECs cells’adhesion ability to THP-1 cells was improved,and the expression levels of ICAM-1 and VCAM-1 were increased.After Tanshinone ⅡA solution was added,HUVECs cells’ adhesion ability to THP-1 cells was decreased,and the expression levels of ICAM-1 and VCAM-1 were decreased,with statistically significant difference(P<0.05).Tanshinone ⅡA was detected by inhibiting Hippo-YAP signaling pathway.Cell activity:When LPS was added to the cell solution,the activity of HUVECs cells in each group was decreased,and the addition of tanshinone ⅡA could enhance cell activity.When the Hippo-YAP signaling pathway inhibitor PFI-2 was added,the activity of cells was improved,and the difference was statistically significant(P<0.05).Expression of YAP,LATS1/2 and MST1/2 protein concentrations:The addition of LPS solution could increase the expression level of YAP protein concentrations in cells,decrease the expression levels of MST1/2 and LATS 1/2 protein concentrations,and activate the activity of Hippo-YAP signaling pathway.When tanshinone ⅡA was added,the expression of YAP protein in cells decreased significantly,and the concentrations of MST1/2 and LATS1/2 protein were increased,and the activity of Hippo-YAP signaling pathway was also restricted.In addition,after the addition of Hippo-YAP signaling pathway inhibitor PFI-2,the concentration expression of YAP protein in cells was increased,while the concentration expression of MST1/2 and LATS1/2 protein was decreased,with statistical significance(P<0.05).Conclusions:Tanshinone ⅡA inhibits the Hippo-YAP activity of signaling pathways to regulates HUVECs cell biology,tanshinone ⅡA can significantly inhibit the activity of LPS induced HUVECs cell reduce,inflammatory reaction,NO produce,and the phenomenon such as cell adhesion ability to ascend,to the development trend of foamy cells,inhibit HUVECs cells from in vitro cell based on the provided tanshinone ⅡA suppress the atherosclerosis pathology research foundation,provides the basis for the research of below.Part Ⅱ:In vivo study on the mechanism of tanshinone ⅡA regulating atherosclerotic injury by inhibiting Hippo-YAP signaling pathwayObjective:To explore the mechanism of tanshinone ⅡA regulating atherosclerosis injury by inhibiting Hippo-YAP signaling pathway on the basis of animal model,and to provide theoretical basis for the clinical treatment of atherosclerosis with traditional Chinese medicine.Materials and Methods:ApoE-/-mice were selected as the research subjects.The mice were fed with a high-fat diet containing 21%fat and 0.15%cholesterol for about 12 weeks.The success of the atherosclerosis model was determined by staining blood lipid,cholesterol and aortic root.The mice were divided into the following groups:Control group,AS model group,TSⅡA group,PFI-2 group,and TSⅡA+PFI-2 group.The mice were treated with intragastric administration for 4 weeks.The mice in each group were sacrificed for item analysis:to determine the pathology of aorta;The levels of TNF-α,IL-1β,IL-6 and IL-8 in blood were analyzed by ELISA.The thickening of aorta wall and the degree of lumen were analyzed.Analysis n of lipid deposition and accumulation;The concentration and expression of VCAM-1,ICAM1 and Hippo-YAP in aortic tissues were detected by RT-PCR and Western blot.Results:Atherosclerosis:the aortic plaque area of AS group was the most widely distributed and the largest.After tanshinone ⅡA and PFI-2 inhibitor were added,the plaque area decreased.After the intervention of tanshinone ⅡA and PFI-2 inhibitor,the plaque area distribution was further reduced,and the changes of vascular plaque were slight.HE and Movat staining:After the atherosclerosis model was established,the smooth muscle layer at the vascular terminal was thinned.Movat staining indicated that the intimal plaques were composed of old yellow-stained collagen fibers and purple foam cells near the lumen.After the intervention of tanshinone ⅡA and Hippo-YAP pathway inhibitor PFI-2,the degree of aortic plaques was reduced,the amount of luminal plaques was significantly reduced,and the percentage of purple foam cells was also decreased.The degree of aortic plaques in mice was further reduced after the two interventions.After the establishment of the model,the vascular stenosis rate was significantly increased,the proportion of lipids and foam cells outside the mouse aorta was increased,and the proportion of collagen fibers was decreased.After the intervention of tanshinone ⅡA and Hippo-YAP pathway inhibitor PFI-2,the vascular stenosis rate was relieved,and the proportion of collagen fiber was increased,with statistical significance(P<0.05).Tanshinone ⅡA lipid levels in ApoE-/-mice:the lowest concentration of expression,the control model of atherosclerosis,TC,LDL,TG concentration factor expression increased significantly,tanshinone ⅡA and Hippo-YAP pathway inhibitor PFI-2 intervention both reduce the concentration of the blood fat factor expression,tanshinone ⅡA and Hippo-YAP pathway inhibitor PFI-2 joint intervention group of decreasing blood fat factor expression level in mice,statistically significant difference(P<0.05);Effects of tanshinone ⅡA on vascular inflammatory response in ApoE-/-mice:After the establishment of atherosclerosis model,the concentrations of TNF-α,IL-1β,IL-6 and IL-8 increased,and the intervention of tanshinone ⅡA and Hippo-YAP pathway inhibitor PFI-2 could significantly reduce the concentrations of the above-mentioned inflammatory response factors,with statistical significance(P<0.05).Tanshinone ⅡA in ApoE-/-mice vascular cell adhesion effect:the control group mice vascular adhesion factor VCAM-1 and ICAM-1 expression is low,after model establishment,VCAM-1 and ICAM-1 expression concentration increased,tanshinone ⅡA and Hippo-YAP pathway inhibitor PFI-2 intervention can reduce vascular adhesion factor VCAM-1 and the expression of ICAM-1 concentration(P<0.05),statistically significant difference;The inhibitory activity of tanshinone ⅡA on Hippo-YAP pathway was detected:the expression of YAP in aortic tissues of mice in control group was the lowest,and the expression of MST1/2 and LATS 1/2 were higher.After the establishment of the model,the expression of YAP protein in tissues of mice was increased,and the expression of MST1/2 and LATS 1/2 were decreased.The intervention of tanshinone ⅡA significantly reduced the expression concentration of YAP and increased the expression concentrations of MST1/2 and LATS1/2 proteins,with statistical significance(P<0.05).Conclusions:During the physicochemical development of mouse atherosclerosis,tanshinone ⅡA can affect the biological behavior of cells by inhibiting the activity of Hippo-YAP pathway.After tanshinone ⅡA intervention,the activity of Hippo-YAP pathway decreased,the accumulation of plaque in mouse aorta decreased,the adhesion between cells decreased,the inflammatory response of the body decreased,and the plaque was in a stable state,which inhibited the development of atherosclerosis,providing certain clinical enlightenment. |
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