| BackgroundTraditional Chinese medicine(TCM)is an important treatment strategy for middle and advanced hepatic cancer,and seeking and exploring the advantageous populations and critical pointcuts of TCM treatment has become the focus of the recent studies.The supervisor’s topic group has long devoted to the clinical study of TCM for hepatic cancer,and has successfully developed the therapeutic protocols for soothing liver and removing stasis and toxicity and Decoction for Soothing Liver and Removing Stasis and Toxicity.It has been proved through summarizing the clinical cases that the decoction can effectively prolong the patients’ survival time and improve their quality of life.However,what is the action mechanism of Decoction for Soothing Liver and Removing Stasis and Toxicity in the treatment of middle and advanced hepatic and whether it is associated with the prognosis of hepatic cancer are issues urgently to be resolved.This study might provide novel methods for inhibiting the progression of hepatic cancer and theoretical basis for further study of TCM in intervening the prognosis of hepatic cancer.ObjectiveThe syndrome factor diagnostic scale for hepatic cancer was preliminarily established through literature review and retrospective study,and the traditional Chinese medicine(TCM)formulas for the treatment of middle and advanced hepatic cancer prescribed by Professor Lin Li-zhu based on syndrome differentiation were collected to analyze and extract the clinical evidence of Decoction for Soothing Liver and Removing Stasis and Toxicity in the treatment of hepatic cancer.Network pharmacology,molecular docking and molecular dynamic techniques were adopted to disclose the action targets of Decoction for Soothing Liver and Removing Stasis and Toxicityin the treatment of hepatic cancer,and prognostic model was established based on bioinformatics to disclose the specific molecular mechanisms referring to cell assays,aiming to provide experimental basis for the clinical application of Decoction for Soothing Liver and Removing Stasis and Toxicity.Methods1 Clinical study on patients with hepatocellular carcinoma(HCC)from outpatient clinic of Professor Lin Li-zhu based on syndrome factors(1)A total of 304 eligible patients with hepatocellular carcinoma(HCC)treated by Professor based on syndrome differentiation and treatment in outpatient clinic from January2015 to December 2018 were selected as the study subjects.The information acquired from the 4 diagnostic methods was screened and included into Wenfeng III auxiliary diagnostic and therapeutic system syndrome differentiation software to screen standardized information acquired from the 4 diagnostic methods and identify the syndrome factors.(2)Traditional Chinese Medicine Inheritance Auxiliary Platform 3.0 software was used for the statistical analysis of the frequency of the information acquired from the 4diagnostic methodsand syndrome factors,with 26 syndrome factor-related analyses obtained.2 Study on the rule of formulas for the treatment of middle and advanced hepatic cancer by Professor Lin Li-zhu based on syndrome differentiation in the outpatient clinicThe 347 formulas prescribed by Professor in the outpatient clinic were retrospectively analyzed and the Traditional Chinese Medicine Inheritance Auxiliary Platform 3.0 software was used for frequency summarization of the medications,effects,properties,tastes and belonged meridians and the correlation rule analysis of the formulas,so as to summary the rules for specifying and prescribing the formulas and extract the core formulas.And some cases with long-term survival was taken as an example.3 Molecular target identification and mechanism study on Decoction for Soothing Liver and Removing Stasis and Toxicityin the treatment of hepatic cancer based on network pharmacology,molecular docking and molecular dynamicsThe molecular targets and mechanisms of Decoction for Soothing Liver and Removing Stasis and Toxicityin the treatment of hepatic cancer were identified based on on-line TCM database concomitant with R language,and molecular target network was established to screen the possibly active core targets and compounds.And Autodocksoftware and Pymol software were further used to simulate the molecular docking and dynamics of the core compounds and targets to verify the reliability of the results.4 Establishment of prognostic model and nomogram of Decoction for Soothing Liver and Removing Stasis and Toxicity in the treatment of hepatocellular carcinoma(HCC)based on bioinformaticsThe genes closely related with the occurrence and development of HCC were identified using bioinformatics,the targets of Decoction for Soothing Liver and Removing Stasis and Toxicity in the treatment of hepatic cancer were sued to establish the prognostic model,and AUC of the ROC was adopted to assess the reliability of the model.The nomogram was established referring to the clinical characteristics of HCC patients to assess their prognoses.5 Study on molecular mechanisms of Decoction for Soothing Liver and Removing Stasis and Toxicityin the treatment of hepatic cancerThe concentrated juice and drug containing serum of Decoction for Soothing Liver and Removing Stasis and Toxicity were prepared,and human hepatic cancercell Hep G2 and human normal livercell LO2 were cultured for cell generation.Cytotoxicity test,DAPI staining test,Annexin V-FITC/PI double-staining test,immunofluorescence assay,and Western blotassay were completed respectively.Results1 Clinical study on patients with hepatocellular carcinoma(HCC)from the outpatient clinic of Professor Lin Li-zhu based on syndrome factorsA total of 304 clinical records were obtained ultimately,and standard information acquired from the 4 diagnostic methods was acquired,involving 221 syndromes and 2359 frequency.The top 5 syndromes were “superficial sleep”,“fatigue and weakness”,“thirst and profuse drinking”,“insomnia” and “bitter mouth”,and the common tongue manifestations were light red or dark red tongue with thin-white,yellow or greasy coat and thread pulse.The syndromes were successively extracted,and 38 syndrome factors were obtained,with 1223 frequency in total.The top 10 syndrome factors were dampness,heat,liver,brain,yin deficiency,qi deficiency,qi stagnation,stomach,spleen,and gallbladder.The top 5disease location syndrome factors were liver,mind,stomach,spleen and gallbladder,the disease nature syndrome factors were dampness,heat,yin deficiency,qi deficiency and qi stagnation,and the top 5 concomitant syndrome factors for correlation analysis were dampness + heat,brain + yin deficiency,dampness + liver,heat + liver,and heat + yin deficiency.2 Study on the rules of formulas for the treatment of middle and advanced hepatic cancer by Professor Lin Li-zhu based on syndrome differentiation in the outpatient clinic.Of the 347 formulas collected,148 TCM drugs were used in total,with frequency of5706.The frequently used drugs included Gancao(glycyrrhiza),Taoren(Semen Pruni Persicae),Tubiechong(Eupolyphaga seu Steleophaga),Longkui(Solanum nigrum),Shancigu(pseudobulbus cremastrae seu pleiones),Zhongjiefeng(Glabrous Sarcandra Herb)and Banzhilian(Sculellaria barbata)and so on.The most common TCM types are drugs for tonifying the deficiency,drugs for clearing away heat,drugs for activating blood to remove stasis,and so on.The natures of the drugs were analyzed,which showed that drugs cold,neutral and warm in nature were used in majority;as to the taste,drugs bitter,pungent and sweat in taste were mostly applied.As to the meridians,most drugs belonged to Liver and Spleen meridians.The drugs with highly supported by formula correlation rule analysis were Gancao,Taoren and Tubiechong.3 Molecular target identification and mechanism identification and study on Decoction for Soothing Liver and Removing Stasis and Toxicityin the treatment of hepatic cancer based on network pharmacology,molecular docking and molecular dynamicsGO enrichment analysis demonstrated that the clinical effect was achieved through response to stimulation,the response of cells to stimulation and biological regulation.KEGG enrichment analysis indicated that the clinical effect was possibly associated with PI3K-Akt signal pathway,HTLV-I infection and Ras signal pathway.Meanwhile,AKT1,IL6,MAPK1 and TP53 were the primary core targets for the action while Quercetin Glycoside might target above four targets.The further molecular docking and molecular dynamic simulation analysis revealed that Quercetin Glycoside might be a potential core compound and target since it less bound to AKT1 and MAPK1.4 Establishment of prognostic model and nomogram for Decoction for Soothing Liver and Removing Stasis and Toxicity in the treatment of HCC based on bioinformaticsUnivariate cox analysis of the 10 targets for Decoction for Soothing Liver and Removing Stasis and Toxicity in the treatment of HCC and LASSO regression were performed to establish the prognostic model.The results showed that the median survival time was 5.8 months in low risk group and 3.7 months in high risk group,and there was significant difference(p=0.00189).The AUC of ROC of the prognostic model was 0.74 at year 1,0.679 at year 3 and 0.635 at year 5,indicating that this model had good effect in predicting the patients’ survival.Referring to the cox analysis,CCNB1 and TNM stages were independent risk factors for the prognosis.Nomogram was further established to estimate the relationships between the expression of CCNB1 and TNM stage and the patients’ overall survival(OS),and the results suggested that C-indexwas 0.69(0.635-1)(p<0.001),suggesting that this nomogram had moderate accuracy in assessing the patients’ OS.5 Study on molecular mechanism of Decoction for Soothing Liver and Removing Stasis and Toxicityin the treatment of hepatic cancerDecoction for Soothing Liver and Removing Stasis and Toxicity had dual-regulation effects to Ras/MAPK signal pathway,and early activation of Ras/MAPK signal pathway could induce the autophagy of Hep G2 without significantly affecting the cell apoptosis,whereas late inhibition of Ras/MAPK signal pathway could induce the apoptosis of Hep G2 cells without significantly affecting the autophagy.ConclusionDecoction for Soothing Liver and Removing Stasis and Toxicity has been modified by summarizing and analyzing the rules of medications for the treatment of middle and advanced hepatic cancer by Professor Lin Li-zhu.It is verified through the network pharmacologyand molecular docking,molecular dynamic prediction that Decoction for Soothing Liver and Removing Stasis and Toxicity has dual-regulation effect to Ras/MAPK signal pathway and early activation of Ras/MAPK signal pathway can induce the autophagy of Hep G2 while late inhibition of Ras/MAPK signal pathway can induce the apoptosis of Hep G2 cells,which may also effectively inhibit the drug resistance during the treatment of hepatic cancer. |
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