To Explore The Molecular Mechanism Of Erchen Decoction Regulating Foam Cell Pyroptosis Based On Nrf2 Pathway

Purpose:The aim of this study was to explore the possible targets and signaling pathways of ErChen Tang in the prevention and treatment of coronary atherosclerotic heart disease(CHD)using a network pharmacological analysis.It was also validated by cellular experiments to investigate the molecular mechanism of ErChen Tang in regulating the level of oxidative stress injury-induced foam cell scorch death.Material and method:1.Screening potential molecular targets of ErChen Tang to prevent and treat coronary heart disease based on network pharmacological analysisThe main compounds of ErChen Tang and its action targets were screened using TCMSP database;GenCLip3 database was searched for coronary heart disease-related proteins;the intersection of ErChen Tang and coronary heart disease-related proteins was obtained by Venny2.1;the core targets were screened by STRING database,and then enrichment analysis was performed by KEGG database to predict the action of ErChen Tang in treating coronary heart disease mechanism.2.To explore the molecular mechanism of ErChen Tang to reduce the level of oxidative stress-induced foam cell focal death based on Nrf2 signaling pathwayWe established a foam cell model by oxidizing low-density lipoprotein(ox-LDL)to induce oxidative stress injury in THP-1 cells,a model group(10% fetal bovine serum culture + 2%ox-LDL),a low concentration dosing group(5% ErChen Tang low concentration drug-containing serum + 10% fetal bovine serum + 2% ox-LDL),a high concentration dosing group(5% ErChen Tang high concentration drug-containing serum + 10% fetal bovine serum+ 2% ox-LDL),and a blank serum group(5% blank serum + 10% fetal bovine serum + 2% ox-LDL).,and detected the apoptosis of foam cells under the effect of different concentrations of ErChen Tang-containing serum by flow cytometry to select the optimal concentration of ErChen Tang-containing serum.QRT-PCR and Western Blot methods were used to detect the levels of Nrf2,NLRP3 mRNA and the expression levels of Nrf2,NLRP3,caspase-1 and caspase-10 proteins in the foam cells and Erchen Decoction-containing serum.Results:1.A total of 208 components of ErChen Tang were obtained from TCMSP database,258target proteins of ErChen Tang and 946 related proteins of coronary heart disease were predicted,and 37 target proteins were obtained after intersection.Constructing protein interaction network(PPI)of intersecting target proteins of ErChen Tang and coronary heart disease by STRING database.2.Then further GO and KEGG analyses were performed,and the results showed that they mainly involved common pathways of oxidative stress such as apoptosis and NOD-like receptor pathway.3.According to the results of flow cytometry assay,the apoptosis ratio was significantly decreased in the high concentration dosing group(5% ErChen Tang high concentration containing serum + 10% fetal bovine serum + 2% ox-LDL)compared with other dosing groups(p<0.01).q RT-PCR assay was performed to detect the expression of Nrf2 and NLRP3 mRNA in cells of blank control group(10% fetal calf serum culture),model group(10% fetal calf serum culture + 2% ox-LDL),blank serum group(5% blank serum + 10% fetal calf serum + 2% ox-LDL)and drug-containing serum group(5% dichen tang high concentration drug-containing serum + 10% fetal calf serum + 2% ox-LDL).The protein expression of Nrf2,NLRP3,caspase1 and caspase10 were detected by Western blot method in the above four groups.caspase1 and caspase10 protein expression were significantly increased(p<0.01)and Nrf2 mRNA and protein expression were significantly decreased(p<0.01);compared with the model group and blank serum group,NLRP3 mRNA and protein expression were significantly decreased(p<0.01)and caspase1 and caspase10 protein expression were significantly decreased(p<0.01)in the serum group.The expression of caspase1 and caspase10 were significantly decreased(p<0.01),and the expression of Nrf2 mRNA and protein were significantly increased(p<0.01).Conclusions:The results of network pharmacology and cytology experiments showed that ErChen Tang could effectively attenuate ox-LDL-induced oxidative stress damage in foam cells and reduce the level of cell scorching by regulating the Nrf2 pathway,suggesting that Nrf2/NLRP3 may be the molecular target of ErChen Tang for the treatment of coronary artery disease.