| Purpose:1.Predict the main components,targets and pathways of Gualou Xiebai Banxia Decoction in the treatment of coronary heart disease based on the method of network pharmacology.2.Use ox-LDL to induce the foaming model of human THP-1 macrophages,observe the effect of Gualou Xiebai Banxia Tang medicated serum on the expression of foam cell apoptosis-related genes and proteins,and explore the regulation of Gualou Xiebai Banxia Tang to regulate apoptosis The mechanism of death.Material and methods:1.Search and combine the literature on the TCMSP platform to collect the chemical components of the three traditional Chinese medicines in Gualou Xiebai Banxia Tang,search and collect the targets of the active ingredients of the drugs in PubChem and SwissTargetPrediction databases,and use the DisGeNET database to collect the targets of coronary heart disease Then use STRING10.5 and Cytoscape 3.7.1 software to draw the protein interaction(PPI)network,select the core target,and enter it into the DAVID 6.8 database for GO analysis and KEGG pathway analysis,and then construct the"component-target"-The network diagram of the "Access".2.Divide 9 SD rats into 3 groups randomly,namely the blank group and the Gualou Xiebai Banxia Tang low and high concentration groups,with 3 rats in each group.Gualou Xiebai Banxia Tang low and high concentration groups were given low and high concentration(0.295g/mL,0.59g/ml)Gualou Xiebai Banxia Tang Chinese medicine decoction by gavage,the blank group was filled with equal volume of distilled water.All rats were given intragastric administration for 7 days,2 times a day,2 ml each time.At the end of 7 days,the rats were fasted for 24 hours.Blood was taken from the abdominal aorta and centrifuged.The upper serum was collected and inactivated at 56? for 30 minutes.Then the serum was filtered with a 0.22um water-based filter and placed in a-80? refrigerator.Saved in,for later use.3.After PMA induces adherence of human THP-1 cells,the cells are treated with 50 ?g/mL ox-LDL to foam,and a foam cell model is constructed.The lipid deposits in the cells are observed under a microscope with the oil red O staining method Happening.4.After successful cell modeling,flow cytometry was used to detect the apoptosis of the model group,10%blank serum control group,10%drug-containing serum low-dose group,and 10%drug-containing serum high-dose group to determine the most The best medicated serum concentration.5.To study the effect of Gualou Xiebai Banxia Decoction on foam cell apoptosis,divide the cells into blank control group,model group,blank serum control group and drug-containing serum group,and use Western Blot method to detect apoptosis-related proteins in each group The expression levels of SIRT1,FOXO1,P53,and P21;qRT-PCR was used to detect the expression levels of apoptosis-related genes SIRT1,FOXO1,P53,and P21 in each group.Results:1.The active ingredients of the three drugs in the compound of Gualou Xiebai Banxia Decoction have been screened through multiple screenings to obtain a total of 29 main compounds that play a role,including quercetin,naringin,hesperetin,?-sitosterol,etc.,which are used to treat crowns.There are 54 core targets for heart disease,including AKT1,MAPK3,TNF,MMP9,IL6,PTGS2,SIRT1,etc.According to GO function enrichment analysis,there are 203 biological processes(BP)and 29 cell components(CC).There are 52 items,molecular functions(MF),mainly related to the process of nitric oxide biosynthesis,inflammation,and apoptosis.KEGG pathway enrichment analysis results showed that according to P<0.05 as the screening criteria,a total of 71 related pathways were collected,mainly related to estrogen signaling pathway,arachidonic acid metabolism pathway,PI3K-Akt signaling pathway,FOXO signaling pathway,etc.2.The oil red O staining results showed that compared with the blank control group,the lipid deposition in the cells of the ox-LDL model was obvious,indicating that the model was successful.3.The results of cell apoptosis detection by flow cytometry showed that compared with the model group,the apoptosis rate of the drug-containing serum group was significantly reduced(P<0.01),and the apoptosis rate of the 10%drug-containing serum high-dose group was even more reduced.Significantly(P<0.01);compared with the blank serum control group,the 10%drug-containing serum high-dose group had a more significant decrease in apoptosis rate(P<0.01).4.The results of qRT-PCR detection of mRNA expression in each group showed that compared with the blank control group,the expression level of SIRT1 mRNA in the model group was significantly reduced(P<0.01),and the expression level of FOXO1,P53,P21 mRNA was significantly increased(P<0.01)or P<0.05);Compared with the model group,the SIRT1 mRNA expression level in the drug-containing serum group was significantly increased(P<0.01),and the FOXO1,P53,P21 mRNA expression levels were significantly reduced(P<0.01 or P<0.05);5.The results of Western blot detection of cell protein expression in each group showed that compared with the blank control group,the expression level of SIRT1 protein in the model group was significantly reduced(P<0.05),and the protein expression level of FOXO1,P53,and P21 was significantly increased(P<0.01 or P<0.05);Compared with the model group,the SIRT1 protein expression level in the drug-containing serum group was significantly increased(P<0.01),and the FOXO1,P53,and P21 protein expression levels were significantly reduced(P<0.01 or P<0.05).Conclusions:1.By constructing a compound-target-pathway network relationship diagram,it is further clarified that Gualou Xiebai Banxia Decoction intervenes in the development of coronary heart disease through multiple pathways and multiple targets,and SIRT1 is one of the main core targets.2.Gualou Xiebai Banxia Tang's regulation of foam cell apoptosis may be related to the up-regulation of SIRT1 gene and protein expression and then down-regulation of FOXO1,P53,P21 gene and protein expression. |
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