Effects Of Astragalus-miltiorrhiza And Its Components On Atherosclerosis And Pericytes Inflammation

Objective:In this study,ApoE^-/-mouse model of atherosclerosis was established to observe the effects of Astragalus miltiorrhiza and its components on aortic plaque area,serum lipids and expressions of inflammatory factors as well as the number of pericytes in each group of mice,and to explore the therapeutic effect of Astragalus miltiorrhiza and its components on atherosclerosis.The pericytes inflammatory injury model was established in vitro to study the regulatory effect of Piezo1 on YAP/JNK and inflammatory factors and the alleviating effect of the combination of Astragalus miltiorrhiza and its components on inflammatory response,so as to reveal the internal mechanism of the combination of Astragalus miltiorrhiza and its components against atherosclerosis by inhibiting inflammatory response.Methods:In the animal experiment,ApoE^-/-mice were fed with high fat diet to establish mouse model of atherosclerosis,and ApoE^-/-mice were interfered with the combination of Astragalus miltiorrhiza and its components.The specific groups were control group（0.9%Na Cl）,model group（0.9%Na Cl）,salvianolic acid B group（Sal-B30mg/kg）,astragaloside IV group（AS-Ⅳ 20mg/kg）,astragalus miltiorrhiza mixture group（Sal-B 30mg/kg+AS-Ⅳ 20mg/kg）,astragalus miltiorrhiza granules group（Astragalus miltiorrhiza 1.56g/kg+Saltiorrhiza 0.78g/kg）and rosuvastatin group（rosuvastatin0.41g/kg）.The area of atherosclerotic plaque in the aorta of mice was determined by gross oil red O staining,HE staining and oil red staining.The serum of mice was detected by enzyme linked immunosorbent assay.The number of pericytes was observed by immunofluorescence double staining.The pericytes were divided into control group（without any special treatment）,model group（50μg/m L ox-LDL）,si RNA group（25n M of si RNA+0.5 n M of Li PO3000）and negative control group（25n M of negative control si RNA（NC si RNA）+0.5 n M of Li PO3000）.The expression of Piezo1 in pericytes was detected by PCR and immunofluorescence assay,and the differences in the expression of Piezo1 in the control group and the model group were observed.PCR,immunofluorescence,calcium influx assay and patch clamp were used to determine whether Piezo1 could be successfully knocked down in pericytes.Cell scratch assay,Transwell migration assay was used to detect the migration of pericytes in different groups.The expression levels of Piezo1,YAP/TAZ and inflammatory factors（JNK,NF-κB,TNF-α）in pericytes of different groups were determined by PCR,WB and immunofluorescence.In cell experiment,the pericytes were intervened by Astragalus and Salvia Miltiorrhiza components.The pericytes were divided into control group（without special treatment factors）,model group（50μg/m L ox-LDL）,si RNA group（50μg/m L ox-LDL+25n M si RNA+0.5 n M LIPO3000）,salvianolic acid B group（50μg/m L ox-LDL+25μg/m L SAL-B）,astragaloside IV group（50μg/m L ox-LDL+15μg/m L AS-Ⅳ）and Astragalus miltiorrhiza mixture group（50μg/m L ox-LDL+25μg/m L Sal-B+15μg/m L As-IV）.Cell scratch assay,Transwell migration assay was used to observe the effects of Astragalus miltiorrhiza component and its combination on pericyte migration.PCR,WB and immunofluorescence experiments were used to investigate the effects of Astragalus miltiorrhiza component and its combination on the m RNA and protein expression of Piezo1,YAP/TAZ and inflammatory related markers（JNK,NF-κB and TNF-α）.The effects of components of Astragalus miltiorrhiza on calcium ion influx were observed by calcium influx experiment.In addition,PCR,WB and immunofluorescence experiments were conducted to detect the effects of combination of Astragalus miltiorrhiza components and Piezo1 knockdown method on the expression levels of YAP/TAZ and inflammatory related markers in pericytes.Results:Animal experiments confirmed that Astragalus-miltiorrhiza and its components can effectively reduce the mice aortic atherosclerotic plaque area,regulating blood lipid levels and reducing inflammatory factor expression,which Astragalus-miltiorrhiza components effect is most obvious,atherosclerosis can be significantly antagonized.The number of pericytes in the control group was more,while the number of pericytes in the model group was reduced.However,Astragalus miltiorrhiza and its components could effectively promote the proliferation of pericytes,so this topic decided to take pericytes as the research object of cell experiments.PCR and immunofluorescence experiments confirmed that Piezo1 could be expressed in pericytes,and the expression of Piezo1 was increased in model group compared with the control group.In addition,PCR,immunofluorescence,patch clamp and calcium influx experiments confirmed that Piezo1 in pericytes could be successfully knocked down.After Piezo1 protein knockdown,cell scratch assay,Transwell migration assay confirmed that the migration ability of pericytes was impaired.PCR,WB and immunofluorescence experiments confirmed that,compared with the control group,the expression of YAP/TAZ and inflammatory factors were increased in the model group.The expression levels of Piezo1,YAP/TAZ and inflammatory factors in the Piezo1-si RNA group were decreased compared with the model group after knockdown of Piezo1.Cell scratch assay,Transwell migration assay confirmed that the Astragalus miltiorrhiza components could improve the migration ability of pericytes and improve the biological function of pericytes.Combined with PCR,WB and immunofluorescence experiments,it was confirmed that the m RNA and protein expressions of Piezo1,YAP/TAZ and inflammatory related markers were significantly decreased in the combination group of Astragalus salvia miltiorrhiza compared with the model group.In addition,the calcium influx experiment confirmed that the combination of Astragalus miltiorrhiza could effectively reduce the calcium ion influx.In PCR,Western Blot and immunofluorescence experiments,the combination of Astragalus salviae and Piezo1knockdown was used in pericytes.It was found that the combination of Astragalus salviae and Piezo1 knockdown could further reduce the expression of YAP/TAZ and inflammatory markers.Conclusion:The combination of Astragalus miltiorrhiza and its components can improve the inflammatory response by interfering PIEZO1/YAP/JNK signal axis and play an anti-atherosclerosis effect in pericytes,thus providing a new direction for the development of atherosclerosis drugs.