Mechanism Of Resveratrol Inhibition Of DEN Induced Precancerous Injury In Rat Hepatocellular Cancer Based On Metabolomics Research

Objective:To investigate the effect and mechanism of resveratrol（RES）on diethylnitrosamine（DEN）induced hepatocarcinogenesis in rats.Methods:1.The effect of resveratrol on human hepatoma Smmc-7721 cellsHuman hepatocellular carcinoma Smmc-7721 cells were cultured in a medium containing a certain proportion of fetal bovine serum and placed in 5%CO₂ incubator.They were divided into the following 8 groups:negative control group,solvent control group,5μmol/L resveratrol group,10μmol/L resveratrol group,20μmol/L resveratrol group,40μmol/L resveratrol group,80μmol/L resveratrol group and 100μmol/L resveratrol group.MTT assay was used to detect cell viability at 24h,48h and 72h after dosage.2.The effect of resveratrol on inflammatory stage of hepatocellular carcinoma induced by DEN in ratsThirty-two healthy male SPF SD rats were randomly divided into four groups:normal control group,RES treatment group,DEN treatment group and RES-DEN treatment group,with 8 rats in each group.Normal control group rats was intragastrically given normal saline 4 times a week for 8 weeks in the first week.The second week began to increase 0.5%sodium carboxymethyl cellulose solution,once a day,for 7 weeks;RES treatment group At the first week,rats were given normal saline intragastric administration 4 times a week for 8 weeks.From the second week,RES（0.1g/kg,dissolved in 0.5%sodium carboxymethyl cellulose solution）was increased once a day for 7 weeks;DEN treatment group rats was given DEN intragastric administration（0.002g/rat,dissolved in normal saline solution）4 times a week for 8weeks.The second week began to increase 0.5%sodium carboxymethyl cellulose solution,once a day,for 7 weeks;RES-DEN treatment group At the first week,rats were given DEN（0.002g/rat）four times a week for 8 weeks,and at the second week,RES（0.1g/kg）was given once a day for 7 weeks.Pathological changes and collagen fiber changes of liver were observed by HE staining and Masson staining,and alanine aminotransferase（ALT）and aspartate aminotransferase（AST）were detected by enzyme activity to evaluate liver function.Western blot（WB）was used to detect the levels of total PCNA,intracellular PCNA,inflammatory factor IL-6 and fibrosis factor TGF-β1 in liver proliferation index cells of rats.3.The effect of resveratrol on hepatocellular fibrosis stage in rats induced by DENThirty-two healthy male SPF SD rats were randomly divided into normal control group,RES treatment group,DEN treatment group and RES-DEN treatment group,with8 rats in each group.Normal control group rats was intragastrically given normal saline4 times a week for 13 weeks.The 7th week began to increase 0.5%sodium carboxymethyl cellulose solution,once a day,for 7 weeks;RES treatment group At the first week,rats were given normal saline intragastric administration 4 times a week for 13 weeks.At the7th week,RES（0.1g/kg）was given orally once a day for 7 weeks;DEN treatment group rats was given DEN（0.002g/rat）4 times a week for 13 weeks.The 7th week added 0.5%sodium carboxymethyl cellulose solution,once a day,for 7 weeks;RES-DEN treatment group rats was given DEN（0.002g/rat）4 times a week in the first week for 13 weeks.At the 7th week,RES（0.1g/kg）was added by gavage once a day for 7 weeks.Pathological changes of liver and collagen fibers were observed by HE staining and Masson staining,and liver function was evaluated by enzyme activity ALT and AST.Protein immunoblotting was used to detect the level of TGF-β1 in rat liver.4.Metabolomics analysis of the effect of resveratrol on glucose metabolism in liver tissue of rats with precancerous injury of liver cancer induced by DENThe peaks extracted from 8-week precancerous SD rats and QC samples were analyzed.The liver metabolic profiles were analyzed by UHPLC under ESI positive ion and ESI negative ion modes.Firstly,principal component analysis（PCA）was used to analyze the metabolites of the control group（N）,RES treatment group（NB）,DEN treatment group（G）and RES-DEN treatment group（GB）to find out the groups with significant differences.Then,orthogonal partial least squares discriminant analysis（OPLS-DA）was used to further analyze the differential metabolites.the Variable importance for the projection（VIP）and VIP（threshold>1）obtained from the above OPLS-DA model were used in combination with the P value（threshold<0.05）of univariate test to search for differentially expressed metabolites.Then,the selected metabolites were input into KEGG website to determine the metabolite pathway.5.The effect of glucose metabolism reprogramming on resveratrol inhibiting DEN-induced malignant proliferation of rat hepatocytes in precancerous stageThe supernatant was extracted from the liver tissue of 8 weeks with the corresponding extract solution.Enzyme activity tests were performed on Tricarboxylic Acid Cycle（TCA）Isocitrate Dehydrogenase Cytoplasmic（ICDH）and the carbohydrate-limiting enzyme Lactate dehydrogenase（LDH）.Western blot was used to detect the key enzymes in the metabolic pathway of phosphoenolpyruvate-pyruvate-lactate during glycolysis,M2 pyruvate kinase（PKM2）and human hypoxia inducible factor-1α（HIF-1α）and lactate dehydrogenase（LDHA）Results:1.After treating human hepatocellular carcinoma Smmc-7721 cells for 24h,48h and72h,high concentration of RES inhibited hepatocellular malignant proliferation in a time-dependent manner.2.Inflammatory stage of liver cancer:HE staining showed that liver plate thickening was improved in the RES-DEN treatment group compared with the DEN treatment group.Masson staining showed that RES-DEN improved the proliferation of collagen fibers compared with DEN treatment group.The results of serum ALT and AST levels showed that liver injury was significantly reduced in the RES-DEN treatment group compared with the DEN treatment group.The expression levels of IL-6,TGF-β1 and PCNA showed that the expression levels of IL-6 in the RES-DEN treated group were lower than those in the DEN treated group,and the levels of TGF-β1 in the RES-DEN treated group were not significantly different.The total PCNA protein level and intracellular PCNA protein level of rat hepatocytes in the RES-DEN treated group were significantly decreased.These results all indicated that resveratrol inhibited DEN-induced hepatitis injury in rats and inhibited malignant proliferation of hepatocytes.3.Hepatocellular fibrosis stage:HE staining results showed that compared with DEN treatment group,the cell morphology of rats in the RES-DEN group was complete and no obvious cell atypia occurred.Masson staining showed that collagen fiber deposition was significantly improved in RES-DEN treatment group compared with DEN treatment group.The results of serum ALT and AST levels showed that the liver injury in the RES-DEN group was significantly reduced compared with that in the DEN group.The expression level of TGF-β1 in the RES-DEN treatment group was significantly lower than that in the DEN treatment group.These results all indicate that resveratrol inhibits the occurrence of DEN-induced liver fibrosis in rats.4.The results of non-targeted metabolomics and metabolic pathway enrichment analysis showed that,compared with the control group,the levels of 5-phosphate ribose and ribose were inhibited,and the conversion level from pentose phosphate pathway to glycolysis was increased in the RES-DEN treated group.The level of conversion from pentose phosphate pathway to glycolysis in RES-DEN treated rats was higher than that in DEN treated rats,suggesting that RES could regulate the flux of pentose phosphate pathway and inhibit cell proliferation.TCA circulation levels were reduced in DEN treated rats.The results showed that DEN induced the conversion of glucose metabolism from oxidative phosphorylation to aerobic glycolysis.Compared with the control group,there was no significant change in TCA cycle level in the RES-DEN treatment group,indicating that RES weakened the inhibition effect of DEN on TCA cycle to a certain extent.In addition,lactic acid levels in the RES-DEN treated group were still higher than those in the control group,but similar to those in the DEN treated group.These results indicated that although the transformation of pentose phosphate pathway to glycolysis pathway was enhanced in liver cells of RES-DEN treated rats,the glycolysis level was not significantly improved compared with that of DEN group rats,suggesting that the metabolic pathway of phosphoenolpyruvate-pyruvate-lactic acid was inhibited.5.To further investigate the effect of phosphoenolpyruvate-pyruvate-lactic acid,a key metabolic pathway enzyme in the sugar metabolic reprogramming,on resveratrol’s inhibition of DEN induced precancerous liver cancer injury in rats.The results showed that,compared with the control group,the levels of key enzymes PKM2,LDHA and related protein HIF-1αin the DEN treated group were significantly increased.However,it decreased significantly after RES treatment.It is suggested that DEN may induce PKM2 to migrate into the nucleus and promote HIF-1αand LDHA transcription in the precancerous stage.However,levels of LDHA,HIF-1α,and PCNA were significantly reduced in RES-DEN treated rats,suggesting that RES may block PKM2 migration to the nucleus.Conclusion:In vitro experiments have demonstrated that high concentration of RES can inhibit the malignant proliferation of human hepatoma Smmc-7721 cells in a time-dependent manner;In vivo experiments showed that resveratrol could inhibit the development of hepatocellular carcinoma inflammatory stage and hepatocellular carcinoma fibrosis stage in rats induced by DEN;Through untargeted metabolomics analysis,resveratrol can inhibit the reprogramming of glucose metabolism in DEN induced rat liver tissue,suggesting that the phosphoenolpyruvate-pyruvate-lactic acid metabolic pathway is inhibited during glycolysis;Further verification revealed that the expression levels of key enzymes PKM2 and LDHA and related protein HIF-1αin this metabolic pathway were inhibited.RES can inhibit DEN induced excessive proliferation of rat hepatocytes in the precancerous stage of liver cancer by regulating glucose metabolism reprogramming,which provides experimental basis for the prevention of liver cancer.