Substances And Mechanism Of Jiedu Granule In Patients With Hepatocellular Carcinoma

Primary liver cancer is one of the most common tumors in the world,and more than 90%of them are hepatocellular carcinoma,which has become the fourth most common cause of cancer-related deaths worldwide and the third most common cause of cancer-related deaths in China.Because of the insidious onset of hepatocellular carcinoma,the outcome of patients with hepatocellular carcinoma is poor,which poses a great challenge to the current various tumor treatment methods.Jiedu Granule is a traditional Chinese medicine compound consisting of root of Valvate Actinidia,root of Valvate Actinidia,Salvia Chinensis,and membranes of chicken gizzards.It has achieved good results in clinical treatment of hepatocellular carcinoma,but currently the efficacy of Jiedu Granule in the treatment of hepatocellular carcinoma is still lacking.The support of large-scale clinical research,and the material basis and mechanism of Jiedu Granule remain unclear.Our study aims to study the clinical efficacy of Jiedu Granule in the treatment of early and intermediate-stage hepatocellular carcinoma,and to clarify the pharmacodynamic substance and the molecular mechanism of Jiedu Granules,so as to provide a basis for clinical application and secondary development.This study is divided into three main sections.1.UHPLC-LTQ-Orbitrap combined ultra-high-pressure liquid phase and linear ion trap orbital ion trap combined high-resolution mass spectrometry was used to establish detoxification particles as a research carrier to establish a rapid screening and identification method for Jiedu Granule.Consult the original literature and database,establish a chemical composition database of Jiedu Granule,and quickly identify the compound composition of Jiedu granules by extracting ion chromatograms and secondary mass spectra.The liquid phase conditions were:the column was Acquity UPLC HSS T3(1.8 ?m× 2.1 mm),and the mobile phase consisted of 4 mmol/L ammonium acetate 0.08%formic acid aqueous solution(phase A)and methanol(phase B).The elution gradient was:0-8 min:5-40%B,8-16 min:40-50%B,16-24 min:50-95%B,24-26 min:95%B,26.01-30 min:85.01-90%B.The flow rate was 0.1 mL/min.The mass spectrometry conditions are:the electrospray ion source(H-ESI)is connected to the liquid phase,and the positive and negative ion full-scan mode is used.The scanning range is m/z 50-1500,and the gas used by the ion source is nitrogen.The results showed that the main chemical components of Jiedu Granules were analyzed,and a total of 65 components were identified or speculated,including 9 phenylpropanoids,14 triterpenes,7 phenolic acids,7 flavonoids,etc.UHPLC-LTQ-Orbitrap combined technology was used,with Jiedu granules as the research carrier,and a rapid screening and identification method for related chemical components was established to study the blood-injection components of rats after taking the preparation.The liquid phase conditions were as follows:The chromatographic column was a Platisil ODS C18 column(5 ?m × 4.6 mm).The mobile phase consisted of 4 mmol/L ammonium acetate 0.08%formic acid aqueous solution(phase A)and methanol(phase B).The elution gradient was:0-20 min:5-40%B;20-40 min:40-50%B;40-80 min:50-95%B;80-85 min:95%B;40-80 min:85.01-90%B.The flow rate was 0.1 mL/min.The mass spectrometry conditions are:the H-ESI is connected to the liquid phase,and the positive and negative ion full-scan mode is used.The scanning range is m/z 50-1500,and the gas used by the ion source is nitrogen.The blood-injection components of Jiedu Granules were analyzed,and about 15 components such as caffeic acid,danshensu,and asiatic acid were identified in the plasma samples.Through network pharmacology,31 representative compounds of Jiedu Granule were screened in the PubChem database to obtain 1,720 targets,and 566 targets related to liver cancer were screened in the OncoDB.HCC and Liverome databases.A total of 62 potential targets for liver cancer were obtained.The drug-component-target network diagram was obtained by Cytoscape software,the protein interaction network was constructed by the String database,and the key functions and pathways related to the treatment of liver cancer by Jiedu Granule were obtained through the GO and KEGG pathway enrichment analysis,providing a basis for subsequent cell experiments.2.Human gene expression profiling chip was used to detect the difference genes between the normal SMMC-7721 cell control group and Jiedu granules.The functional targets and Ingenuity Pathway Analysis analysis were used to screen the key targets of Jiedu granules.The analysis of TCGA,HCCDB,GEPIA,HPA databases revealed that the expression of SLC39A10 in liver cancer tissues was significantly higher than in normal tissues,and the expression of advanced liver cancer was significantly higher than that of early liver cancers.The survival rate of patients with low expression of SLC39A10 was higher than that of SLC39A10.In patients with high expression,SLC39A10 overexpression is associated with poor prognosis in patients with HCC.Multivariate analysis confirmed the predictive significance of SLC39A10 expression level for the prognosis of liver cancer.Western Blot experiments showed that the relative expression of SLC39A10 in HCCLM3 cells was higher,and the relative expression of SLC39A10 in MHCC97-L cells was lower.Therefore,the expression of SLC39A10 was interfered in HCCLM3 cells,and SLC39A10 was overexpressed in MHCC97-L cells.Morphological changes,scratch experiments,and Transwell chamber migration and invasion experiments found that the ability of HCCLM3 cells to invade and migrate after SLC39A10 interference was significantly reduced,while that of MHCC97-L overexpressed SLC39A10 significantly improved the ability to invade and migrate.Western Blot experiments showed that transfection of a plasmid over-expressing SLC39A10 can down-regulate the expression of E-cadherin and promote the expression of N-cadherin and Vimentin.Conversely,when SLC39A10 is disturbed,the expression of E-cadherin increases and N-cadherin and Vimentin increase.Expression decreased.The active ingredients of Jiedu granules were selected for intervention,protocatechuic acid and Madecassic acid.Through cell morphological changes,scratch experiments,Transwell cell migration and invasion experiments,it was found that the invasion and migration ability of over-expressing SLC39A10 cells was significantly reduced,Western Blot experiments It showed that the addition of protocatechuic acid or asiatic acid can antagonize the decline of E-cadherin expression after over-expression of SLC39A10,and antagonize the expression of N-cadherin,Vimentin,MMP-9,MMP-2,Snail,Wnt2.Mice were inoculated with H22 hepatoma cells to establish ascites H22 transplanted tumor model,then divided randomly into 4 groups:low dose of Jie-du granule preparation treatment group(JL),high dose of Jie-du granule preparation treatment group(JD),Sorafenib treatment group(SF)and negative control group(NA).All drugs were administrated separately for 14 days,the tumor volume was monitored.After treatment for 15 days,6 mice in each group were killed to identify the weight pathological changes of tumors,and the effects of treatment on livers and kidneys.After treatment,compared with NA,the tumor volume of SF and JD groups were significantly decreased(P<0.01).Compared with those in NA group,the tumorous cellular density,atypia and mitotic counts in tumors of JH group and SF group were significantly decreased and the tumor necrosis was increased(P<0.01),the effect of JH group was better than that of JL group in the tumorous cellular density and atypia(P<0.01).These findings indicate that Jiedu Granule preparation has antitumor activity in the H22 tumor-bearing mice,and seems to be a safe and effective supplementary agent for the treatment of HCC.Therefore,we can draw the following conclusions:1.The main components of Jiedu Granule include 9 phenylpropanoid components,14 triterpenes,7 phenolic acids,7 flavonoids,etc.The serum transfer components of Jiedu granules include coffee acid,salviansis,snow oxalic acid and 17 components.Furthermore,based on the network pharmacology,the main mechanism and active components of Jiedu Granule in the treatment of liver cancer were discussed.2.SLC39A10 promotes the invasion and migration of HCC by activating JAK2/STAT3 and PI3K/AKT signaling pathway,which can be inhibited by the active components of Jiedu Granule,madecassic acid and protocatechuic acid.