Study On The Mechanism Of Xihuang Pill In Promoting Pyroptosis Of Liver Cancer SMMC-7721 Cells

Objective: Screen the targets of Xihuang Pills through network pharmacology,and explore the mechanism of Xihuang Pills in promoting the pyroptosis of SMMC-7721 cells in liver cancer.Method:1.Use network pharmacological analysis related platforms and methods to screen out the active ingredients and targets of Xihuang Pills,and screen out potential signal pathways and targets.2.Taking liver cancer SMMC-7721 cells as the research object,using cell proliferation-toxicity(CCK-8)to detect the effect of different concentrations of Xihuang Pill on the inhibition rate of liver cancer cell lines.3.Use caspase-3 fluorescent probe and TUNEL to detect the apoptosis-promoting effect of Xihuang Pills on SMMC-7721 cells of liver cancer.4.The Western-Blot method was used to verify the regulation of Xihuang Pills on the expression of caspase-3,GSDME,cleave-GSDME,NF-?B,NLRP3,and TNF-? protein in SMMC-7721 cells of liver cancer effect.Result:1.A total of 27 biologically active ingredients were obtained from Xihuang Pills,and 21 common targets of Xihuang Pills and cell pyroptosis were obtained through screening and intersection.The more frequent occurrences are CASP3,RELA,VEGFA,BCL2,CASP9,CASP8,EGFR,IL6,etc.,these targets are mainly involved in the regulation of apoptosis signaling pathways,cysteine endopeptidase activity is involved in the process of apoptosis,death receptor binding and other biological processes,and are mainly concentrated in apoptosis,NF-?B signaling pathway,TNF signaling pathway and other signaling pathways.2.Cell proliferation-toxicity(CCK-8)test results suggest that the inhibitory effect of Xihuang Pills on SMMC-7221 cells of liver cancer is concentration and time dependent.The IC50 of Xihuang Pills extract is250?g/ml.3.Apoptosis detection results suggest:Compared with the blank group,the fluorescence intensity of caspase-3 in the Xihuang Pills group is higher(P< 0.05).Under the microscope,the SMMC-7221 cells in the Xihuang Pills group showed classic apoptotic vacuoles.Observed under an optical microscope,SMMC-7221 cells in the Xihuang Pills group showed classic apoptosis vacuoles.Observed under a fluorescence microscope,SMMC-7221 cells in the Xihuang Pills group showed unclear nuclei,irregular shapes,darker coloration,and dark blue,accompanied by pyknosis of the nucleus and apoptotic bodies.4.The results of Western-Blot method suggest:(1)Xihuang Pills has no obvious effect on the protein of caspase-3,but has a protein up-regulation effect on the active spliceosome caspase-3(P<0.01).(2)Xihuang Pills can up-regulate GSDME and spliced GSDME protein(P<0.05),and adding caspase-3 inhibitor on the basis of Xihuang Pills can down-regulate the expression of spliced GSDME protein(P<0.01).(3)Xihuang Pills has an up-regulation effect on NF-?B,NLRP3 and TNF-? protein(P < 0.01),and adding caspase-3 inhibitor on the basis of Xihuang Pills has an effect on NF-?B,NLRP3 and TNF-? protein.Down-regulate the effect of protein(P<0.05).Conclusion:Network pharmacology analyzed the relationship between Xihuang Pills and pyroptosis,and predicted that the apoptosis pathway and cytokines are closely related.The caspase-3 gene and related NF-?B pathway and TNF-? inflammatory pathway are Xihuang Pills have important potential mechanisms of action in the treatment of liver cancer.From the cellular level,the research shows that Xihuang Pills can play an anti-liver cancer effect by promoting the pyroptosis of SMMC-7721 cells of liver cancer.