| Background: Hepatocellular carcinoma(HCC)is an incurable disease with a large population of new cases and deaths,and because of its insidious onset,patients are usually at an advanced stage when symptoms appear.Modern medical treatment options for hepatocellular carcinoma are well established,and in particular the development of molecularly targeted drugs has benefited many HCC patients.Sorafenib is the most important molecularly targeted drug among the first-line hepatocellular carcinoma guidelines.It is a multi-kinase inhibitor and can inhibit the growth of hepatocellular carcinoma mainly through direct inhibition of HCC growth and inhibition of angiogenesis.Due to the heterogeneity of HCC,some patients are not sensitive to sorafenib treatment and some patients are prone to develop drug resistance after applying sorafenib.It is necessary to explore a way to treat HCC with efficacy and also enhance the sensitivity of sorafenib treatment.Chinese medicine has a profound understanding of the treatment of cancer,and accumulation has been recorded in the Yellow Emperor’s Classic of Internal Medicine,where HCC is classified under the categories of "liver accumulation","zhengjia" and "accumulation".Based on the search and collation of ancient prescriptions for liver cancer,it is believed that the basic pathogenesis of HCC in ancient times was blood stasis and Zheng deficiency,and that eliminating blood stasis and eliminating accumulation and supporting Zheng to strengthen the root was the main treatment.In the process,it was found that the Sanlengjiashen Pill,which was boiled in vinegar to treat all recent accumulations in the distant past,was quite consistent with the above-mentioned treatment principle,as it was composed of San Leng,Chuan Xiong and Da Huang,all of which can enter the liver meridian,and has the potential to become the basic formula for the modern treatment of liver cancer.In conjunction with Luo Tianyi’s inheritance of the academic ideas of the Yi Shui school of medicine of "strengthening and protecting the spleen and stomach" and "nourishing the righteousness and eliminating accumulation",the group proposed that ginseng could play a beneficial role in the formula,and therefore added ginseng to Luo Tianyi’s original formula to form a new prescription for the treatment of HCC.Objective: To investigate the rules of ancient medicine used in the treatment of HCC through data mining technology,with a view to obtaining the rules of ancient prescriptions for the treatment of liver cancer,such as common medicines,common pairs of medicines and treatment principles,and to provide a theoretical basis for the modern treatment of HCC,and to compose new prescriptions for the treatment of HCC accordingly;to explore the pharmacological basis of the new prescriptions for the treatment of liver cancer;to investigate the possible effects of the new prescriptions for the treatment of HCC through network pharmacology and molecular docking technology The new prescription was used to investigate the possible mechanisms of action of the new prescription in the treatment of HCC through network pharmacology and molecular docking techniques,and to provide ideas and basis for subsequent experimental studies.Methods:1.The Chinese Medical Dictionary was searched and integrated with the key words "liver accumulation" to construct a "database of prescriptions for HCC in ancient texts".The results of the analysis were analyzed and new prescriptions were prepared.2.To prepare lyophilised powder for the new prescription,and to collect the chemical composition of the new prescription by liquid chromatography-mass spectrometry,and then compare the data with the mz Cloud database to obtain the best chemical composition of the new prescription.3.Network pharmacology and Molecular docking: Effective active ingredients are obtained through screening of Swiss ADME parameters,their Canonical SMILES are imported into Swiss Target Prediction database to predict drug targets,and main compounds are screened according to conditions to obtain main active compounds and corresponding targets;Obtain HCC related targets through various disease databases;Construct a network diagram of "Traditional Chinese Medicine-Compound-Target" for Sanlengjiashen formula;Protein network interactions based on intersecting targets;Conduct GO and KEGG enrichment analysis;The binding ability of core compounds to key target proteins are verified by molecular docking.4.The effects of Trigonella plus Ginseng on human HCC cells were observed by CCK-8 assay and two cell lines were selected for subsequent experiments;the effects of Trigonella plus Ginseng on human normal hepatocytes were observed;the effects of Trigonella plus Ginseng on human umbilical vein endothelial cells were observed by combining Trigonella plus Ginseng with sorafenib compared to Chinese medicine alone or western medicine alone.The effect of the combination of Sanlengjiashen formula and sorafenib on human umbilical vein endothelial cells was observed to verify the effect of Sanlengjiashen formula in inhibiting the angiogenesis of hepatocellular carcinoma cells;the expression levels of angiogenesis-related proteins in the hepatocellular carcinoma cell lines of Sanlengjiashen formula and the combination of Sanlengjiashen formula and sorafenib were detected by WB assay.5.To construct a nude rat HCC model in situ and assess the success rate of the model by MRI of small animals;to assess the efficacy of drug intervention by HE staining of livers after drug intervention;to assess the toxic effects of the new prescription on major organs by taking the heart,lung,spleen and kidney of experimental animals;to stain liver sections for proliferation and vascular neovascularization-related proteins by immunohistochemical assay;to detect by WB assay The expression of angiogenesis-related proteins in HCC in situ tissues of nude mice after different drug interventions was detected by WB assay;the effects of Sanlengjiashen formula and the combination with sorafenib on hepatocellular carcinoma angiogenesis were verified by chicken embryo allantoic membrane assay.Results:1 Results of data mining for the Sanlengjiashen formula:1.1 Status of all included prescriptions: A total of 342 Chinese herbal prescriptions were included,involving 103 ancient texts.The prescriptions were mainly for internal use,including pills,dispersants and tonics;among the pills,honey pills,vinegar pills and wine pills were most common,and were usually taken smoothly with date soup,wine or white soup;the frequency of administration was mostly 2-3 times a day;the time of administration was mainly on an empty stomach,after eating and before eating;the decoction of tonics was usually cooked with ginger or with ginger and dates;the degree of administration was usually to facilitate downward movement.1.2 Overall frequency analysis results: 385 Chinese herbal medicines were involved,with a cumulative frequency of 3637 occurrences,of which Mu Xiang,San Leng,Ginseng and Da Huang had the highest frequency of occurrence.In terms of nature and flavour,the majority of drugs were warm,flat,cold,pungent,bitter and sweet,and most of them belonged to the spleen,stomach and liver meridians.In terms of drug efficacy,those with the highest proportion were tonifying drugs,qi-regulating drugs and blood-stasis-activating drugs.1.3 Overall factor analysis results: 31 components were obtained by factor analysis of high-frequency drugs in all the formulas included.Eleven components were included when the eigenvalue was 1.Among them,San Leng,Da Huang and Ginseng were in the same component in component 4,indicating that the use of the three drugs together could represent to a certain extent the action of the drugs used in the treatment of HCC in ancient times.1.4 The results of frequency analysis by era: the frequency of occurrence of rhubarb,ginseng and white peony was high in the Tang and pre-Tang periods;the frequency of occurrence of Mu Xiang,rhubarb and trigon was high in the(Five Dynasties)Song,Jin and Yuan periods;the frequency of occurrence of several drugs such as trigon,Mu Xiang,Chen Pi,rhubarb,green peel and ginseng was high in the Ming dynasty;the frequency of occurrence of fu ling,ginseng and atractylodes was high in the Qing dynasty.1.5 The results of the aptitude and ascription of drugs in various eras: drugs used in the treatment of HCC in the Tang and pre-Tang dynasties were mainly cold,warm and flat;(Five Dynasties)the Song,Jin,Yuan and Ming dynasties were mainly warm,cold and flat;and the Qing dynasty was mainly warm,flat and slightly cold.The treatment of HCC before the Tang Dynasty and the Tang Dynasty was mainly bitter,pungent and sweet in order of the five flavours;(Five Dynasties)Song,Jin,Yuan and Ming Dynasties and the Qing Dynasty were mainly pungent,bitter and sweet in order of the five flavours.In the Tang and pre-Tang dynasties,medicines for HCC were mainly classified as spleen,lung and liver meridians;in the(Five Dynasties)Song,Jin,Yuan and Ming dynasties,they were mainly classified as spleen,liver and stomach meridians.Drugs for treating HCC in the Tang and pre-Tang dynasties were mainly for invigorating blood stasis,tonifying and clearing heat;(Five Dynasties)Song,Jin,Yuan and Ming dynasties and the Qing dynasty were mainly for tonifying,regulating qi and invigorating blood stasis.1.6 Results of association rules by era: Tang and Tang dynasties included 55 pairs of association rules,and the pairs with a high degree of association included: Da Huang-Ginseng,Da Huang-Bai Shao;(Five Dynasties)Song,Jin and Yuan dynasties included 9pairs of association rules,and the pairs with a high degree of association included: San Leng-Mu Xiang,Da Huang-Mu Xiang;Ming dynasty included 60 pairs of association rules,and the pairs with a high degree of association included: Chen Pi-San Leng,Chen Pi-Ginseng,San Leng-In the Qing dynasty,33 pairs of association rules were included,and the pairs with a high degree of association included: Fu Ling-Ginseng and Bai Zhu-Ginseng.It can be seen that rhubarb,ginseng and trigon appear more frequently in the pairs with a higher degree of association.1.7 Results of cluster analysis by dynasties: the drugs in the ancient texts of each dynasty for the treatment of HCC prescriptions were clustered into three categories,respectively,in which the main drugs in this study: San Leng,rhubarb and ginseng appeared in all three clusters in each dynasty.1.8 Rationing results of the Sanlengjiashen formula: The ratios of the four drugs in the Sanlengjiashen formula in the included formulas,when occurring at least two by two,were analysed to explore the dose ratios of the Sanlengjiashen formula groups.It was found that the four herbs appeared together only once in all the included formulas,in the ratio of1:1:1:1.2,while San Leng-Chuan Xiong-Rhubarb appeared together three times and Chuan Xiong-Ginseng-San Leng appeared together two times.The rest appear as pairs,with the1:1 ratio being the most common.As three herbs in the Sanlengjiashen formula are from the ancient formula of San Leng Pills boiled in vinegar,the three herbs in the new prescription of Sanlengjiashen formula follow their dosage ratios: San Leng:Chuan Xiong:Da Huang =8:4:1.Combined with result 8,it is suggested that ginseng should be used as the ruling herb together with San Leng in the new prescription,with San Leng:Ginseng = 1:1.2.2 Results of the substance basis of the Sanlengjiashen formulaBy using high-performance liquid chromatography mass spectrometry technology to analyze the composition of compounds in Sanlengjiashen formula,a total of 812 possible compounds were obtained.According to the conditions,132 compounds were selected from the compound.3 Network pharmacology and molecular docking results of Sanlengjiashen formula3.1 Network pharmacology results: The 132 compounds obtained in the second part were screened through Swiss ADME parameters to obtain 109 effective active ingredients,and their Canonical SMILES were imported into the Swiss Target Prediction database to predict drug targets.There are a total of 99 compounds predicted to target,and 25 effective active ingredients were obtained by screening with a Probability≥0.3 as the screening condition.After removing duplicate values,79 drug targets were finally obtained;By searching the Gene Cards database,OMIM database,and TTD database for "Hepatocellular Carcinoma","HCC",and "liver cancer",11155,59,and 47 disease targets were obtained,respectively.After union and deduplication,a total of 11188 targets were obtained.The intersection of traditional Chinese medicine compound targets and disease targets resulted in a total of 58 common targets for drugs and diseases.The "Chinese medicine compound target" network diagram was constructed by Cytoscape 3.7.2 software.According to the Degree value,the core effective compounds were obtained: cis Resveratrol,Mefenamic acid,19 norandrostenedione,tangerine,Ferulic acid.3.2 Results of protein interactions and KEGG enrichment analysis: The common target was imported into the STRING database,and after two screenings,four core target genes were ultimately selected,namely PTGS2,CYP3A4,PGR,and ESR1.KEGG enrichment analysis shows that the effect of Sanlengjiashen formula on liver cancer may be related to PPAR pathway and NF-κB pathway and VEGF pathway are related.3.3 Molecular docking results: key target proteins were Macromolecular docking with core effective compounds,and the average binding efficiency was 7.44 kcal/mol;The core effective compounds of Sparganum fortunei plus ginseng have good binding efficiency with VEGF,the main target of Sorafenib.4 Results of in vitro experiments4.1 Inhibition of hepatocellular carcinoma cells by Trigonella plus ginseng formula:Trigonella plus ginseng formula could inhibit the growth of five types of hepatocellular carcinoma cells in a concentration-dependent manner.among the five types of hepatocellular carcinoma cell lines,Trigonella plus ginseng formula had the strongest inhibitory effect on Huh7 and the weakest inhibitory effect on Hep3 B.to prevent bias in the results of subsequent experiments,the above two cell lines were not selected.Since SMMC-7721 is a cell contaminated with Hela cells,HCCLM3 and Hep G2 were chosen as the cell lines for the subsequent experiments in this study.4.2 Safety of Sanlengjiashen formula: Sanlengjiashen formula has no toxic side effects on human normal hepatocytes.4.3 Effect of Sanlengjiashen formula on the inhibitory effect of sorafenib on hepatocellular carcinoma cells: The effect of Sanlengjiashen formula and sorafenib alone and in combination on hepatocellular carcinoma cell lines was examined by CCK-8 assay.This indicates that the Sanlengjiashen formula can enhance the inhibitory effect of sorafenib on the proliferation of hepatocellular carcinoma cells.4.4 Effect of Sanlengjiashen formula combined with sorafenib on human umbilical vein endothelial cells: The effect of Sanlengjiashen formula and sorafenib on human umbilical vein endothelial cells was examined by CCK-8 assay,and the viability of human umbilical vein endothelial cells was significantly inhibited after the three drug interventions compared with the control group.This suggests that Sanlengjiashen formula may inhibit the growth of hepatocellular carcinoma by suppressing the expression of vascular endothelial cells,and may enhance the effect of sorafenib in inhibiting the viability of vascular endothelial cells.4.5 Effect of Sanlengjiashen formula combined with sorafenib on the expression of angiogenesis-related indicators: Western Blot assay was performed to verify whether Sanlengjiashen formula inhibited hepatocellular carcinoma cells through the angiogenesis phenotype at the protein level.The expression of these five proteins was reduced in all concentrations of Sanlengjiashen formula,Sorafenib group and the combination group compared with the control group,indicating that Sanlengjiashen formula may inhibit the growth of hepatocellular carcinoma cells through the VEGF pathway,a major pathway of vascular neogenesis,and that Sanlengjiashen formula can enhance the effect of Sorafenib in inhibiting vascular The effect of sorafenib on the inhibition of vascular neoplasia-related targets was enhanced.5 In vivo experimental results5.1 Results of in situ hepatocellular carcinoma modeling in nude mice: according to the results of small animal MRI,30 nude mice could see tumour formation in the imaging results,with a modelling success rate of 85.71% and an average tumour area of 2.55±0.5mm in width and 2.9±0.6 mm in length.5.2 The results of HE staining of the main organs of the animals: compared with the blank group,there were no obvious pathological changes in the heart,lung,spleen and kidney in each dose group,sorafenib group and combined group of Sanlengjiashen formula,indicating that oral administration of Sanlengjiashen formula is safe for mice.5.3 The results of HE and immunohistochemical staining of HCC tissues of the animals:HE: Compared with the model group,the tumor foci of different concentrations of Sanlengjiashen formula were reduced in size,and the tumor foci of the combined group were the smallest,indicating that the tumor foci of in situ carcinoma in mice were inhibited in size after drug intervention,and the inhibition of tumor foci in the combined group of Sanlengjiashen formula and sorafenib was the most obvious.Immunohistochemistry: Compared with the control group,the expression levels of Ki67,VEGFB and VEGFR2 decreased in all concentrations of San Leng Plus Ginseng,Sorafenib and the combination group,especially in the Sorafenib and the combination group,indicating that Sanlengjiashen formula could inhibit the growth of in situ Hepatocellular Carcinoma masses in nude mice and suppress the expression of tumor foci proliferation and angiogenesis-related indexes.5.4 The expression of vascular neovascularization-related proteins in liver tissues was measured by WB experiment: compared with the blank group,the expression of vascular neovascularization-related proteins in the model group was significantly up-regulated;compared with the model group,the expression of vascular neovascularization-related proteins in all concentrations of Sanlengjiashen formula,sorafenib and the combination group were significantly reduced,especially VEGFR2 and VEGFB.The expression of VEGF-related proteins in the combination group was most significantly reduced,indicating that Sanlengjiashen formula may inhibit the growth of hepatocellular carcinoma through VEGF-related proteins,the main pathway of vascular neogenesis,and that Sanlengjiashen formula can strengthen the effect of sorafenib in inhibiting the expression of VEGF-related proteins.5.5 Results of chicken embryo allantoic membrane experiment: The mechanism of the treatment of hepatocellular carcinoma with Trigonella plus ginseng formula and the effect of the combination with sorafenib were again verified by the chicken embryo allantoic membrane experiment: compared with the blank group,the model group showed a significant increase in neovascularization with a disorganized distribution and a significant increase in the development of small blood vessels;compared with the model group,the small blood vessels in different concentrations of Trigonella plus ginseng formula,sorafenib and the combination group were significantly reduced,and some of the large blood vessels showed disruption and insufficient blood flow,indicating that the phenomenon of neovascularization was reduced after drug intervention.Conclusions:1.Sanlengjiashen formula can inhibit the growth of hepatocellular carcinoma cells and suppress the number of neovascularization caused by hepatocellular carcinoma cells in animals,and can enhance the effect of sorafenib in the treatment of hepatocellular carcinoma.2.The main effects of drugs in ancient prescriptions for HCC were to tonify,regulate Qi,invigorate blood circulation and remove blood stasis.The commonly used drugs were Mu Xiang,San Leng,Ginseng and Da Huang,of which Da Huang-Ginseng was the commonly used pair.Based on the data mining results,combined with ancient famous medical prescriptions and the clinical experience of the supervisor in the treatment of HCC,a new prescription for the treatment of HCC can be formed: Sanlengjiashen formula,with the ratio of San Leng-Ginseng-Chuanxiong-Rhubarb as 8:8:4:1.3.The chemical composition of Sanlengjiashen formula was analysed by high performance liquid chromatography-mass spectrometry(HPLC/MS),which is the material basis for the pharmacological efficacy of Sanlengjiashen formula in the treatment of HCC and provides a modern pharmacological basis for the treatment of HCC with Sanlengjiashen formula.4.The core compounds in Sanlengjiashen formula may be used to treat hepatocellular carcinoma through multiple targets,including key proteins in the VEGF pathway,which is related to vascular renewal.5.The mechanism of action of sorafenib may be related to the inhibition of the tumour vascular neovascularisation pathway,as sorafenib can enhance the inhibition of hepatocellular carcinoma cell proliferation and vascular endothelial cell growth.6.San Leng plus ginseng can inhibit the growth of hepatocellular carcinoma cells and the number of neovascularization induced by hepatocellular carcinoma cells in animals,which can enhance the effect of Sorafenib in the treatment of hepatocellular carcinoma. |
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