Meta-analysis Of Replenishing Qi,strengthening Spleen And Removing Toxicity In The Treatment Of Liver Cancer And Study On The Mechanism Of Qizhu Xiaozheng Formula

The morbidity and mortality of primary liver cancer(PLC,hereinafter referred to as liver cancer)are still high,and most patients with liver cancer lose the opportunity of radical cure when they are diagnosed,and the overall survival rate of liver cancer patients is low.Liver cancer has high metastasis and recurrence rates,and the pathogenesis of liver cancer is complex.Also,the efficacy of modern medicine is often difficult to last.Traditional Chinese medicine(TCM)has the outstanding advantage of increasing efficiency and reducing toxicity in the treatment of cancer,comprehensive treatment of western medicine combined with TCM has become a better choice.The basic pathogenesis of liver cancer in TCM can be summarized as "deficiency of spleen qi and carcinomatous toxin",and "replenishing qi,strengthening spleen and removing toxicity" is an important method for the treatment of liver cancer.TCM unique prescription is a specific means to embody the method of treatment,and many studies have reported that TCM unique prescription of "replenishing qi,strengthening spleen and removing toxicity" in the treatment of liver cancer could improve the clinical effect.However,the clinical efficacy of "replenishing qi,strengthening spleen and removing toxicity" in treating liver cancer is still lack of evidence-based medical evidence,and its related mechanism remains to be further studied.It has a broad prospect to make an in-depth study of this kind of prescriptions,to clarify its mechanism,and to excavate its core target for the pathogenesis of liver cancer.Based on our previous research and the clinical experience of our team,we have summarized the TCM unique prescription-Qizhu Xiaozheng formula for the treatment of liver cancer,which contains several Chinese materia medicas such as Astmgali Radix and Phyllanthus.It has the effect of "replenishing qi,strengthening spleen and removing toxicity",which is consistent with the TCM pathogenesis of liver cancer.Taking the clinical treatment as the starting point,this paper evaluates the effect of the method of "replenishing qi,strengthening spleen and removing toxicity" on the clinical observation indicators of patients with liver cancer,which can enrich the evidence-based basis of this TCM method for the treatment of liver cancer;the potential action mechanism of Qizhu Xiaozheng formula,which is the representative prescription of "replenishing qi,strengthening spleen and removing toxicity",was excavated using network pharmacology;based on the results of network pharmacology,the scientific and practical nature of Qizhu Xiaozheng formula in the treatment of liver cancer was explored using experimental study,so as to provide a reference for its clinical application.Objective1.The relevant TCM literature on liver cancer was collated to clarify the main etiology and pathogenesis of liver cancer from the perspective of TCM and the theoretical basis of"replenishing qi,strengthening spleen and removing toxicity" and its representative prescription-Qizhu Xiaozheng formula in the treatment of liver cancer.2.Based on Meta-analysis of randomized controlled studies(RCTs),the clinical efficacy of all TCM unique prescriptions in the treatment of liver cancer was determined,and then the clinical effect of the TCM unique prescriptions including "replenishing qi,strengthening spleen and removing toxicity" was evaluated,and prescription rules of the TCM unique prescriptions were also analyzed to verify the clinical practicability of"replenishing qi,strengthening spleen and removing toxicity".3.The potential action target and signaling pathway of Qizhu Xiaozheng formula(the representative prescription of"replenishing qi,strengthening spleen and removing toxicity")in the treatment of liver cancer were analyzed by network pharmacology,which predicted its molecular mechanism of anti-liver cancer.4.Based on the results of network pharmacology,the experimental study was utilized to explore the mechanism of Qizhu Xiaozheng formula on anti-liver cancer through PI3K/Akt pathway and cancer-related genes.5.Network pharmacology indicates that quercetin(QCT),the bioactive ingredient of this prescription,exists in several Chinese materia medicas with the effect of "replenishing qi,strengthening spleen and removing toxicity",which can reflect the prescription rules of Qizhu Xiaozheng formula,and its number of corresponding targets is the most.Therefore,in this study,QCT was selected as a representative bioactive ingredient to further verify the reliability of network pharmacology prediction results.This study is helpful to elucidate the scientific connotation of Qizhu Xiaozheng formula in the prevention and treatment of liver cancer and lay a foundation for the development of new drug against liver cancer.MethodsThis paper makes a systematic study based on the outline of "literature researchsummary of clinical effect-research on action mechanism".1.First of all,this paper discusses the TCM etiology,pathogenesis,principle of treatment,and method of treatment of liver cancer,and summarizes the theoretical basis of"replenishing qi,strengthening spleen and removing toxicity" and Qizhu Xiaozheng formula against liver cancer.2.Meta-analysis of RCTs.In order to comprehensively collect the RCTs associated with the method of"replenishing qi,strengthening spleen and removing toxicity",we searched the databases of CNKI,Wanfang data,Weipu(VIP),SinoMed,Cochrane Library,PubMed,EMBASE,Web of Science,and selected RCTs of TCM unique prescriptions first,and further analysis was made on those who contained"replenishing qi,strengthening spleen and removing toxicity".Through the Systematic Review and Meta-analysis of the response evaluation criteria in solid tumors,survival rate,Kamofsky score(quality of life),immune function,and adverse events,the efficacy and safety of TCM unique prescriptions and prescriptions of "replenishing qi,strengthening spleen and removing toxicity" in the treatment of liver cancer were evaluated.The prescription rules of all included TCM unique prescriptions were summarized,and the core drug combination was excavated.3.Network pharmacology research.Network pharmacology was utilized to predict the mechanism of Qizhu Xiaozheng formula for liver cancer.In this study,hepatocellular carcinoma(HCC),the most common type of liver cancer,was selected as the disease model.TCMSP,SymMap,TCMIP,BATMAN-TCM,HERB,and MalaCards databases were used to screen the target genes related to disease(HCC)and the bioactive ingredients of Qizhu Xiaozheng formula.Then the "bioactive ingredients-common targets" network of Qizhu Xiaozheng formula was constructed,and the pathway analysis for common targets of disease and Qizhu Xiaozheng formula were analyzed using David database.Protein and protein interaction network was established by the STRING databank and Cytoscape(version 3.7.1),and Hub genes were identified.Besides,the expressions of mRNA and protein and prognostic value of Hub genes were analyzed by bioinformatic methods using UALCAN,HPA,and Kaplan-Meier databases.Finally,protein receptors of MAPK3,RAC1,RHOA,and TP53 were docked with their corresponding bioactive ingredients using molecular docking method.4.Experimental research on Qizhu Xiaozheng formula.①Experimental study on inhibition of proliferation and induction of apoptosis of liver cancer cells by Qizhu Xiaozheng formula-containing serum.The Qizhu Xiaozheng formulacontaining serum was prepared using SPF grade Sprague-Dawley(SD)rats,and the effects of Qizhu Xiaozheng formula-containing serum on the growth activity(proliferation)and apoptosis of HepG2 cells were observed by CCK8 assay and Annexin V-FITC/PI respectively.②Identification of bioactive ingredients of Qizhu Xiaozheng formula(freeze-dried powder).The freeze-dryer was used to get the freeze-dried powder of Qizhu Xiaozheng formula.Based on the results of network pharmacology and our previous research,the bioactive ingredients in the Qizhu Xiaozheng formula were detected by HPLC-Q-TOFMS/MS.③Experimental study on the effects of Qizhu Xiaozheng formula(freeze-dried powder)on the proliferation,migration,invasion,and apoptosis of liver cancer cells.MTT assay,apoptosis-related experiments,plate clone formation assay,scratch,and transwell experiments were used to observe the effects of Qizhu Xiaozheng formula on the proliferation,apoptosis,clone formation,migration,invasion of HepG2 and Hep3B.④Verification of the molecular mechanism of Qizhu Xiaozheng formula(freeze-dried powder)against liver cancer cells.Based on the prediction results of network pharmacology,real-time quantitative PCR(RT-qPCR)and western blot were performed to observe the effects of Qizhu Xiaozheng formula on the expression of critical genes(TP53,MAPK3,RHOA,RAC1,and β-catenin)as well as PI3K/Akt pathway-related proteins(Akt,PI3K,pAkt,p-PI3K),which is helpful to clarify the molecular biological mechanism of its anti-liver cancer.5.Experimental research on quercetin(QCT),the bioactive ingredient of Qizhu Xiaozheng formula.①Experimental study on the effects of QCT on the proliferation,migration,and apoptosis of liver cancer cells.The effects of QCT on the proliferation,colony formation,apoptosis,and migration of HepG2 cells were determined by MTT assay,clone formation test,flow cytometry,and scratch test.②Verification of the molecular mechanism of QCT against liver cancer cells.The effect of QCT on the expression of PI3K/Akt pathway-associated proteins(Akt,PI3K,p-Akt,pPI3K)in HepG2 cells was detected using western blot method.Results1.The result of theoretical research showed that there were many related discussions on liver cancer in literature and ancient books of TCM,and the TCM pathogenesis of liver cancer is characterized by "deficiency of spleen qi and carcinomatous toxin".The"replenishing qi,strengthening spleen and removing toxicity" is an important method for the treatment of liver cancer.Qizhu Xiaozheng formula has the effect of replenishing qi,strengthening spleen,and removing carcinomatous toxin,which demonstrated that Qizhu Xiaozheng formula conforms to the TCM pathogenesis characteristics of liver cancer.2.In Meta-analysis,57 RCTs related to TCM unique prescriptions(24 RCTs including the method of "replenishing qi,strengthening spleen and removing toxicity")were obtained through retrieval and screening,which included 4485 patients(1709 for "replenishing qi,strengthening spleen and removing toxicity")with liver cancer.16 RCTs(7 for"replenishing qi,strengthening spleen and removing toxicity")described specific randomized approaches,all 57 studies did not describe allocation concealment and blind method,11 RCTs(3 for"replenishing qi,strengthening spleen and removing toxicity")mentioned the patients who were lost to follow-up,and 12 RCTs(5 for "replenishing qi,strengthening spleen and removing toxicity")had the possibility of report bias.The result of Meta-analysis demonstrated that,compared with hepatic artery interventional therapy alone,TCM unique prescriptions combined with interventional therapy could improve the Kamofsky score,improvement rate of Karnofsky score,the effective rate of evaluation criteria for solid tumors(WHO or RECIST),and the survival rate of 6 months(half a year),1 year,2 years and 3 years of patients with liver cancer.After confirming that the TCM unique prescriptions can effectively treat liver cancer,RCTs with prescriptions of"replenishing qi,strengthening spleen and removing toxicity" were analyzed separately,and results indicated that the prescriptions of "replenishing qi,strengthening spleen and removing toxicity" could improve the effective rate by WHO criteria[OR=1.80,95%CI(1.41,2.29),P<0.0001],the effective rate by the RECIST criteria[OR=1.52,95%CI(1.06,2.18),P=0.02],improvement rate of Karnofsky score(quality of life)[OR=3.90,95%CI(2.55,5.97),P<0.0001],Karnofsky score[MD=8.77,95%CI(5.27,12.26),P<0.0001],the survival rate of 6 months[OR=2.59,95%CI(1.54,4.36),P=0.0004],the survival rate of 1 year[OR=2.87,95%CI(1.98,4.15),P<0.0001].Descriptive analysis indicated that TCM unique prescriptions or prescriptions of"replenishing qi,strengthening spleen,and removing toxicity" could improve the patients’immune function.Safety evaluation showed that TCM unique prescriptions or prescriptions of "replenishing qi,strengthening spleen and removing toxicity" are safe.The funnel plots of some observation indicators indicated that our study might have a slight publication bias.The analysis of prescription rules demonstrated that TCM unique prescriptions for the treatment of liver cancer often include Chinese materia medicas for replenishing qi and strengthening the spleen,and clearing heat and removing toxicity.3.In the analysis of network pharmacology,the network of bioactive ingredients(Qizhu Xiaozheng formula)-common targets showed that QCT,wogonin,luteolin,and baicalein were the important bioactive ingredients of Qizhu Xiaozheng formula for treating liver cancer.Signaling pathway analysis showed that the anti-liver cancer effect of Qizhu Xiaozheng formula may be correlated with pathways in cancer,hepatitis B,PI3K-Akt,MAPK,Ras signaling pathways,etc.Fifteen Hub genes were found via the protein and protein interaction network,and the protein and mRNA expressions of 7 Hub genes(TP53,MAPK1,MAPK3,PIK3CA,MAPK8,RHOA,and RAC1)in HCC were higher than those in normal liver tissues.Survival analysis demonstrated that nine genes,including TP53,STAT3,MAPK3,SRC,PIK3R1,HRAS,EGFR,RHOA,and RAC 1 were significantly correlated with the survival time of patients with HCC.The data of molecular docking revealed that the protein receptors of MAPK3,RAC1,RHOA,and TP53 were well docked with the corresponding ingredients.4.Experimental results of Qizhu Xiaozheng formula:①The data of CCK8 and Annexin V-FITC/PI assays indicated Qizhu Xiaozheng formula-containing serum could effectively inhibit the proliferation and promote the apoptosis of HepG2 cells(statistically different from control,P<0.05).②The mass spectrometry data indicated that Qizhu Xiaozheng formula(freeze-dried powder)contains QCT,wogonin,luteolin,baicalein,and so on.③The data of MTT assay showed that Qizhu Xiaozheng formula(freeze-dried powder)could remarkably suppress the proliferation of HepG2 and Hep3B cells compared with the control group(P<0.05).The clone formation experiment suggested that low concentrations of Qizhu Xiaozheng formula(freeze-dried powder)could inhibit the clone formation of the two cell lines.The result of Annexin V-FITC/PI assay showed that Qizhu Xiaozheng formula(freeze-dried powder)could promote the apoptosis of Hep3B cells and induce cell cycle arrest in S phase compared with the control group(P<0.05).Also,apoptosis was confirmed by observing the nuclear by using Hoechst 33342 staining in liver cancer cells.Scratch and Transwell experiments revealed that Qizhu Xiaozheng formula(freeze-dried powder)could noticeably inhibit the migration and invasion of HepG2 and Hep3B cells compared with the control group(P<0.05).④The result of PCR showed that Qizhu Xiaozheng formula(freeze-dried powder)could significantly reduce the mRNA expressions of MAPK3,RHOA,and β-catenin in HepG2 cells and β-catenin in Hep3B cells(versus control,P<0.05).Western blotting data suggested that the relative expressions of p-Akt,and p-PI3K decreased after the treatment with Qizhu Xiaozheng formula(versus control,P<0.05).5.Experimental results of QCT:①The data of MTT and colony formation assays revealed that QCT could noticeably inhibit the proliferation(P<0.05)and colony formation of HepG2 cells compared with the control group.Annexin V-FITC/PI double-staining indicated that QCT promoted apoptosis in HepG2 cells compared with the control group(P<0.05),and scratch experiment showed that QCT could remarkably inhibit the migration of HepG2 cells in comparison to control group(P<0.05).②Western blot analysis demonstrated that QCT could noticeably reduce the protein expression levels of p-PI3K and p-Akt in comparison to control group(P<0.05).Conclusion1.The basic pathogenesis of liver cancer is "healthy qi deficiency and pathogen excessiveness"-deficiency of spleen qi and carcinomatous toxin,which are often transformed into each other.Qizhu Xiaozheng formula has the effect of"replenishing qi,strengthening spleen and removing toxicity",which is in line with the pathogenesis of liver cancer.2.Through Meta analysis,on the basis of clarifying the clinical effects of TCM unique prescriptions,it was found that the prescriptions of "replenishing qi,strengthening spleen and removing toxicity" combined with interventional therapy could improve the effective rate of evaluation criteria for solid tumors,survival rate,quality of life,and immune function(index of immune cells)of liver cancer patients.Combination therapy had a good safety profile.Due to the low quality of the methodology of RCTs,the clinical basis provided by this study needs to be further verified.Treatment method of core drugs in TCM unique prescriptions could be summarized as"replenishing qi,strengthening spleen and removing toxicity" and "dispersing stagnated liver qi and nourishing blood",which was consistent with the TCM efficacy of Qizhu Xiaozheng formula(representative prescription of "replenishing qi,strengthening spleen and removing toxicity").3.Qizhu Xiaozheng formula may treat liver cancer through multiple bioactive ingredients,multi-targets,and multi-pathways.Furthermore,Hepatitis B,PI3K-Akt,MAPK,Ras,and other pathways related to tumorigenesis and cancer progression were the main signaling pathways regulated by Qizhu Xiaozheng formula.4.Qizhu Xiaozheng formula could effectively inhibit the proliferation,colony formation,migration,and invasion of HCC cells,and facilitate HCC cells apoptosis,whose molecular mechanism could be associated with MAPK3,RHOA,and β-catenin and PI3K/Akt pathway.5.Quercetin(QCT),the bioactive ingredient of Qizhu Xiaozheng formula,could remarkably suppress the proliferation,colony formation,and migration of HCC cells,and promote cell apoptosis in liver cancer cell lines.Its mechanism may be correlated with PI3K/Akt pathway.