Mechanism Investigation On The Anti-hepatocellular Carcinoma Effect Of Sanghuangporus Vaninii Polyphenol Phelliigniarin B

Hepatocellular carcinoma is one of the most serious diseases that endanger human health at present,and its morbidity and mortality are always high.Traditional chemotherapy drugs have several side effects,such as drug resistance,poor prognosis and other shortcomings.Natural products are gradually became a popular direction for anti-cancer research because of their rich sources,small toxic and side effects,as well as multiple pathways for tumor treatment.A large number of studies have shown that Sanghuangporus vaninii(also named as mulberry Sanghuang,SH)has definite anticancer effect in liver cancer,stomach cancer,colorectal cancer and other types of cancer.A new styryl pyranones compound Phelliigniarin B(B12)was isolated from the sporocarps of Phellinus igniarius,the effect of polyphenol Phelliigniarin B(B12)on hepatocarcinoma and its potential molecular mechanism have not been reported.The purpose of this study was to investigate the effect of B12 on liver cancer and the molecular mechanism involved,so as to provide a theoretical basis for the development of antitumor drugs of Sanghuangporus vaninii polyphenols.The main contents and results of this study are as follows:1.Anti-hepatoma activity and mechanism analysis of B12 in vitroThe effect of B12 on human hepatoma HepG2 cytotoxicity,proliferation and migration were evaluated by CCK-8 assay,Colony formation assay and Transwell assay,showed that B12 could significantly inhibit the growth,proliferation and migration of HepG2 cells.B12 induced adverse morphological changes in a dose-dependent manner,with cell morphology characteristic of apoptosis.Flow cytometry results showed that B12 significantly promoted the early apoptosis of HepG2 cell and blocked the cell cycle in S phase.MDC staining and Western blot results showed that B12 promoted apoptosis and activated autophagy.To further explore the anti-hepatocellular carcinoma mechanism of B12,we monitored the gene expression of HepG2 cell treated with 15 μg/m L B12 for 24 h and48h by transcriptomic analysis.Compared with control group,101,46 differential genes were enriched in HepG2 cells after 24 h,48 h B12 treatment.KEGG enrichment showed that differential genes after 24 h B12 treatment were significantly influenced Parathyroid hormone synthesis,secretion and action.Comparatively,48 h B12 treatment significantly affected Ovarian steridogenesis pathway.The analysis and quantitative verification of common genes revealed that B12 inhibited HepG2 cell growth may be related to up-regulated expression of AKR1C1 and down-regulated expression of ID3,TCIM,WFDC1,GPNMB,DOK7 and HAS2,which need to be further verified in the future study.2.Antitumor effect and mechanism analysis of B12 in xenograft tumor model of nude mice with hepatocellular carcinomaThe xenograft tumor model of liver cancer was established in nude mice by orthotopic implantation of HepG2 cells.The mice in B12 group were intragastric administrated with 100 mg/kg B12 once a day for three weeks.The body weight,tumor volume,tissue morphology of hepatocellular carcinoma mice were monitored.B12 significantly reduced the liver tumor volume and attenuate the liver histopathological status of hepatocellular carcinoma mice.To explore the anti-hepatocellular carcinoma effect mechanism of B12,we used transcriptome analysis to explore differentially expressed genes of tumor and adjacent tissues treated with 100 mg/kg B12 for 21 d.As for tumor tissue,54 differential genes were enriched in B12 group(BT)as compared to model group(MT).While as for adjacent tissues,276 differential genes were enriched in B12 group(BC)as compared to model group(MC).KEGG enrichment showed that differential genes among MT vs.BT significantly enriched in Amoebiasis pathways;whereas differential genes among MC vs.BC significantly enriched in Fluid shear stress and atherosclerosis.KEGG enrichment showed that that differential genes in both cellular and individual were significantly influenced Fluid shear stress and atherosclerosis.In this pathway,HMOX1 gene expression was significantly up-regulated at both cellular and individual levels after B12 treatment.It is speculated that the antitumor effect of B12 may be related to HMOX1 gene.These results provided a direction for further investigation of the antitumor mechanism of B12.In summary,as a natural styrene-pyranoid monomeric substance,B12 exhibited anti-hepatocellular carcinoma activity both in vitro and in vivo.In vitro,B12 mainly inhibited hepatoma cell proliferation through promoting cell apoptosis and inducing cell autophagy.The transcriptomic analysis revealed that B12 could up-regulated the expression of AKR1C1,but down-regulated expression of ID3,TCIM,WFDC1,GPNMB,DOK7 and HAS2 to exert anti-tumor effect.In vivo,it can significantly reduce the volume of liver tumor and improve liver tissue lesions.Transcriptomic analysis revealed that B12 perhaps exerted its antitumor effects by upregulating the expression of HMOX1 gene in the fluid shear stress,and atherosclerosis pathway in vivo and in vitro.However,the specific in vitro and in vivo antitumor mechanisms require further investigation.