Mechanism of 17-hydroxy-rockcaprolactone B in inducing apoptosis in human hepatocellular carcinoma HepG2 cells through the mitochondrial apoptosis pathway mediated by the PI3K-Akt pathway based on network pharmacology and experimental verification
Objective To investigate the effects of 17-hydroxyjolkinolide B(HJB)on apoptosis of human hepatocellular carcinoma cells based on network pharmacology and to analyze the possible mechanisms.Methods Network pharmacology was used to screen the targets of HJB action,construct target networks and protein-protein interactions(PPI)networks,and predict the potential targets of HJB anti-hepatocellular carcinoma action and related pathways.HepG2 cells were treated with 0(blank control),2.5,5,10,20,40 and 80 µmol/L of HJB for 24,48 and 72 h,and the proliferative activities of the cells in each group were detected by CCK-8 assay.After HepG2 cells were treated with 0(blank control),5,10 and 20 µmol/L of HJB for 48 h,apoptosis of cells in each group was detected by Annexin V-FITC/PI double staining,mitochondrial membrane potential changes of the cells were observed by JC-1 staining,and reactive oxygen species levels of the cells were observed by DCFH-DA staining.Western blot assay was performed to detect mitochondrial apoptosis of cells and the proliferative activity of cells in each group.Western blotting assay was used to detect the mitochondrial apoptosis and protein expression changes related to PI3K-Akt signaling pathway in each group.Results The network pharmacological prediction yielded 291 common targets between HJB and hepatocellular carcinoma;KEGG enrichment analysis showed that the results of HJB treatment for hepatocellular carcinoma were mainly in the signaling pathways of cance r pathway,proteoglycans in cancer,lipids and atherosclerosis,and PI3K-Akt signaling pathway and the time-dose related significant inhibition of HepG2 cell proliferation by HJB.Compared with the blank control group,after the action of HJB,the cellular r eactive oxygen species level,apoptosis rate,Bax,Cyt C,cleaved caspase-9,cleaved caspase-3 protein levels were increased in a concentration-dependent manner(P<0.05),and the mitochondria's membrane potential,Bcl-2,p-PI3K,and p-Akt protein expression were significantly reduced(P<0.05).Conclusion The study showed that HJB inhibited the value-added of hepatocellular carcinoma HepG2 cells and promoted apoptosis of HepG2 cells,and the mechanism may be related to the PI3K-Akt pathway mediating the mitochondrial apoptotic pathway.
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