Uncovering The Main Effective Components And Mechanisms Of Compound Kushen Injection Against Hepatocellular Carcinoma Based On A Novel Network Pharmacology Strategy

Background: Hepatocellular carcinoma(HCC)is one of the 10 most prevalent malignancies in the world,and the mortality is ranked second in digestive system malignancies.The incidence of HCC is increasing,and the new cases in China account for more than half of the cases in the world every year.The current treatment methods for HCC mainly focus on surgical resection,radiotherapy and chemotherapy,but the 5-year survival rate of patients is less than 30%.Therefore,exploration of the pathogenesis of HCC and the mechanisms of drugs for prevention and treatment of HCC is an urgent need to improve the survival rate and solve the problem of clinical medication.Compound Kushen Injection(CKI)has been proverbially applied in China for more than 15 years to treat various types of solid tumors,especially carcinomatous pain and digestive system tumors including hepatocellular carcinoma(HCC).Clinical researches show that CKI can effectively alleviate the symptoms of patients with advanced HCC from following aspects,such as assisted double interventional therapy,combined arterial perfusion embolization,combined chemotherapy,and other methods.Experimental pharmacological studies show that CKI has anti-tumor effect on H22 hepatoma-bearing mice,which can markedly inhibit the proliferation of HCC cells and regulate the cell cycle.However,does CKI have inhibitory effect on other animal models of HCC? Can CKI inhibit the metastasis of HCC? Besides regulating apoptosis and cell cycle-related signaling pathways,what are the specific mechanisms of CKI for inhibition of proliferation and metastasis of HCC cells? What is the material basis of CKI against HCC? These key scientific problems are still unclear and need to be studied deeply and systematically.In this study,the anti-HCC effect of CKI was clarified in vitro and in vivo.The effective components group was predicted by utilizing computational systems pharmacology model.The quantitative component-oriented network pharmacology approach was used to predict and verify the mechanism of CKI against HCC.The results will provide new scientific basis for the clinical use of CKI against HCC,provide material basis for the development of new drugs,and provide new strategies for the research of other traditional Chinese medicine injections in preventing and treating tumors.Objective:(1)To clarify the pharmacological effects of CKI on HCC.(2)To explore the effective components group of CKI against HCC by constructing the computational system pharmacological model.(3)To explain the anti-HCC mechanism of CKI based on quantitative component-oriented network pharmacology approach.Methods:(1)The effects of CKI on human hepatoma SMMC-7721 cells were determined by cell viability,adhesion,wound healing assay,invasion assay.The therapeutic effects of CKI on HCC were clarified in DEN-induced HCC rats by using histopathological evaluation and biochemical analysis of livers.(2)The effective components group of CKI against HCC was identified by utilizing computational systems pharmacology model called Dijkstra program to imitate the propagation mode of therapeutic effect of drug on diseases by integrating metabolomics analysis,data collection,network algorithm,and bioinformatics tools.(3)The high-content components were used to screen anti-HCC activities and the components with high content and strong activity were focused.The key targets and pathways of CKI against HCC were predicted by network pharmacology.The key targets and Wnt/β-catenin signaling pathway were validated by western blotting in vitro and in vivo,and the anti-HCC mechanism of CKI in regulating Wnt/β-catenin signaling pathway was confirmed.Metabolomics was used to detect the differential metabolites regulated by CKI for interpreting the mechanism of CKI in regulating metabolic disorder.Finally,the anti-HCC mechanism of CKI was integrated and analyzed.Results:(1)2 mg/mL CKI markedly inhibited the proliferation and clone formation of SMMC-7721 cells,1.5 and 3 mL/kg CKI could alleviate the injure of cellular structure of diethylnitrosamine-induced HCC rats,and regulate the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(AKP)and gamma-glutamyl transpeptidase(γ-GT),indicating that CKI could inhibit the growth of HCC.1 and 2 mg/mL CKI significantly inhibited the migration,invasion and chemotactic movement ability of SMMC-7721 cells,and could significantly enhance cell-cell adhesion and alleviate cell-matrix adhesion,indicating that CKI could inhibit the invasion and metastasis of hepatoma cells.(2)Twenty-one components were identified in CKI based on UPLC-MS approach.The targets of 21 chemical components of CKI were predicted based on multiple databases,and the component-target network of CKI was constructed.Then,277 genes of CKI for regulation of metabolic disorder of HCC were integrated as disease genes,and a comprehensive component-target-interacting protein-disease gene network of CKI was constructed based on protein-protein interaction data.17,412 shortest distance chains,which diffused from targets of components to disease molecular network,were calculated by using Dijkstra algorithm.According to the propagation coefficient,11 pharmacodynamic components were identified,including sophoridine,9β-hydroxylamprolobine,matrine,sophoranol,isomatrine,(-)-acetylbaptifoline,N-methylcyticine,oxysophoridine,oxymatrine,oxysophoranol and(-)-oxylehmannine.The accuracy and reliability of the effective components were verified by the coverage of pathogenic genes and functional pathways.(3)Five components with high content were used for screening of anti-HCC activities.Matrine,oxymatrine,sophoridine and N-methylcytisine with remarkable anti-HCC activities were analyzed by network pharmacology.The targets-interacted proteins network of CKI was constructed for predicting the key targets.Some of the key targets predicted by network pharmacology were experimentally validated in HCC cells and rats.The results showed that CKI significantly increased the expression of CASP3 and significantly inhibited the expression of MMP2,MYC,and REG1 A in HCC cells and rats.Furthermore,CKI suppressed the EMT process by down-regulating the expression of Vimentin,increasing the expression of E-cadherin in HCC cells and rats.The pivotal proteins β-catenin and GSK-3β and downstream protein COX2 in Wnt signaling pathway were significantly regulated by CKI.Meanwhile,CKI treatment can significantly reduce the expression of β-catenin and COX2,and increase the expression of GSK3β in LiCl-treated cells.CKI can inhibit the EMT,migration and invasion of HCC cells induced by LiCl.The above results suggested that Wnt/β-catenin signaling pathway might be involved in the anti-invasion and anti-metastasis effect of CKI.Through the pathway enrichment analysis of the targets predicted by network pharmacology,it was found that the anti-HCC effect of CKI was mainly related to metabolic pathway.The metabolomics results of serum and liver shown that CKI significantly regulate the content of differential metabolites citrate and lactate in hepatoma rats,and improve the metabolic disorder of hepatoma.The mechanism may involve key metabolites and metabolic enzymes in glycolysis process,and c-Myc,the key downstream target of Wnt/β-catenin signaling pathway,may mediate the inhibitory effect of CKI on glycolysis.The integrated analysis of the anti-HCC mechanism of CKI showed that CKI might play an anti-HCC role by regulating β-catenin/c-Myc signaling pathway,and then interfering with metabolic reprogramming of HCC and inhibiting EMT process.Conclusion:(1)CKI could inhibit hepatoma cells and diethylnitrosamine-induced hepatoma rats,and inhibit invasion and metastasis of hepatoma cells.(2)The main effective components of CKI against HCC may be sophoridine,matrine,oxymatrine,N-methylcytisine,while the secondary effective components may be sophoriol,isomatrine,(-)-acetoindigine,sophoridine oxide,sophoriol oxide,9β-hydroxylamprolobine and(-)-oxylehmannine.(3)CKI might play an anti-HCC role by regulating β-catenin/c-Myc signaling pathway,and then interfering with metabolic reprogramming of HCC and inhibiting EMT process.(4)The computational systems pharmacology model can be used to study the material basis of traditional Chinese medicine formulas.The quantitative component-oriented network pharmacology method proposed in this study can be used to predict the mechanism of action of traditional Chinese medicine formulas with clear component content.