Based On Network Pharmacology To Explore Antioxidant Stress Effect Of Stachydrine

Objective:Oxidative stress is a kind of imbalance between oxidative stress and oxidative stress.Excessive oxidative stress is considered to be the cause of many diseases,such as lung disease,hypertension,diabetes and so on.Therefore,the research on antioxidant stress is of great significance for the prevention and delay of diseases.Stachydrine,as one of the main active components of motherwort,a traditional Chinese medicine,has played an important role in the treatment of myocardial fibrosis,cardiovascular diseases and anti-inflammatory diseases in recent years,but there is little research on its antioxidant stress.Therefore,this paper aims to explore the possible antioxidant stress injury effect and its mechanism of stachydrine based on network pharmacology.Methods:1.Network pharmacology analysis methods.CTD database was used to retrieve oxidative stress related proteins;TCMSP,BATMAN-TCM,Pharm Mapper database and swisstargetprediction platform were used to predict stachydrine target proteins;Venny online platform obtained the same proteins of the above two parts;Use STRING to build interaction network between proteins;Obtain GO enrichment and KEGG pathway analysis by Metascape.2.Experimental validation methods.The oxidative stress injury models of A549 and HUVEC cells were constructed by H₂O₂;Detect the effects of different concentrations of stachydrine on the activity of A549 and HUVEC cells by CCK-8;Screen the optimal concentration of stachydrine for the protection of A549 and HUVEC cells from oxidative stress injury by CCK-8.The biochemical indexes SOD and MDA of stachydrine against cellular oxidative stress were detected.The effects of stachydrine on the expression of antioxidant gene Nrf2,HO-1,NQO1 and GSTP1 m RNA in A549 and HUVEC cells were detected by q PCR;The effects of stachydrine on the expression of antioxidant genes HO-1,NQO1and GSTP1 in A549 and HUVEC cells were detected by Western blot.Results:1.Network pharmacology analysis results.765 proteins related to oxidative stress were retrieved,156 stachydrine prediction target proteins were obtained,and 18 proteins were obtained after intersection.These proteins are related to oxidative stress.The interaction network between stachydrine and oxidative stress protein was constructed in STRING database.14 proteins were in the same interaction network and had interaction relationship,and the other two proteins also had interaction.Go enrichment results include biological processes such as response to oxidative stress,amino acid metabolism of glutamine family,response to reactive oxygen species and molecular function of amide binding;KEGG pathway analysis showed arginine and proline metabolism,relaxin signaling pathway,fluid shear stress and atherosclerosis.We selected Nrf2 pathway,which is closely related to oxidative stress in fluid shear stress and atherosclerosis pathway,to study the antioxidant stress of stachydrine.2.Experimental valiation results.（1）Construction of oxidative stress injury model of H₂O₂cellsWe used A549 and HUVEC cells to construct oxidative stress injury model.Compared with the normal control group without H₂O₂treatment,the survival rate of A549 cells treated with 3mmol/L H₂O₂for 1 hour was 49.81±7.5%（p<0.05）;When treated with 1mmol/L H₂O₂for 1 hour,the survival rate of HUVEC cells was47.38±2.7%（p<0.05）.Therefore,the above concentration was selected to construct the cell oxidative stress injury model.（2）The effects of different concentrations of stachydrine on cell activity were detectedUse different concentrations（0,10,20,40,80μmol/L）stachydrine acted on A549and HUVEC cells for 24 hours respectively.Compared with the normal control group without stachydrine treatment,the cell activities of A549 and HUVEC increased in a concentration dependent manner（p<0.05）,indicating that stachydrine at the above concentration had no toxic effect on cells.（3）To screen the best concentration of stachydrine for the protection of cells from oxidative stress injuryUse 20μmol/L stachydrine pretreatment of A549 and HUVEC cells for 24 hours,the viability of A549（p<0.05）and HUVEC（p<0.05）cells increased significantly compared with H₂O₂oxidative damage model group.（4）Detection of biochemical indexes of stachydrine against cellular oxidative stressCompared with 3mmol/L H₂O₂oxidative damage model group,after pretreatment of A549 cells with 20μmol/L stachydrine for 24 hours,SOD activity increased significantly（p<0.05）and MDA content decreased significantly（p<0.05）Compared with 1mmol/L H₂O₂oxidative damage model group,after HUVEC cells were pretreated with 20μmol/L stachydrine for 24 hours,SOD activity increased significantly（p<0.05）and MDA content decreased significantly（p<0.05）.（5）The effect of stachydrine on the expression of Nrf2 signal pathway related genes was detected by q PCRCompared with the blank control group,when A549 and HUVEC cells were treated with 20μmol/L stachydrine for 24 hours,the expression levels of HO-1decreased（p<0.05）,and the expression levels of NQO1,GSTP1 and Nrf2 increased significantly（p<0.05）;Compared with the oxidative damage model group,after pretreatment of A549 and HUVEC cells with 20μmol/L stachydrine for 24 hours,the expression levels of HO-1 decreased（p<0.05）,and the expression levels of NQO1,GSTP1 and Nrf2 increased significantly（p<0.05）.In addition,we also detected the effect of stachydrine on the expression of Nrf2signal pathway related genes at different times.In A549 cell group,the expression level of HO-1 increased significantly at 2 and 12 hours（p<0.05）;The expression level of NQO1 increased significantly at 2 hours and 4 hours（p<0.05）;The expression level of GSTP1 decreased significantly at 2 and 4 hours（p<0.05）,rise to near normal value at 8 hours（p>0.05）and increased significantly at 12 hours（p<0.05）;The expression level of Nrf2 increased at 2 hours,4 hours,8 hours and 12 hours（p<0.05）.In HUVEC cell group,the expression levels of HO-1 and NQO1 increased at 2 hours,4 hours,8 hours and 12 hours,and generally increased first and then decreased;The expression level of GSTP1 decreased significantly at 2 and 4 hours（p<0.05）,and increased significantly at 8 and 12 hours（p<0.05）;The expression level of Nrf2increased at 2 hours,8 hours and 12 hours（p<0.05）.（6）The effect of stachydrine on the expression of Nrf2 signal pathway related proteins was detected by Western blot.A549 cells and HUVEC cells,compared with the blank control group,after 24hours of treatment with 20μmol/L stachydrine alone,the expression of HO-1 protein did not change significantly（p>0.05）,and the expression of NQO1 and GSTP1protein increased（p<0.05）;Compared with the H2O2 damage model group,20μmol/L stachydrine pretreatment group and A549 cell group,the expression of HO-1 and GSTP1 protein decreased（p<0.05）,and the expression of NQO1 protein increased（p<0.05）;In HUVEC cell group,the expression of HO-1 protein did not change significantly（p>0.05）,but the expression of NQO1 and GSTP1 increased（p<0.05）.Conclusion:Stachydrine may have anti oxidative stress effect,and activating Nrf2 signaling pathway may be an important way for stachydrine to exert its anti oxidative stress effect.