Effect Of Jian Pi Xiao Ji Recipe Aqueous Extract On Autophagy Of Human Hepatocellular Carcinoma Cells And Its Mechanism

Objective:To investigate the effect of Jianpi xiaoji recipe aqueous extract on SMMC-7721 cell autophagy.Using the whole genome expression spectrum chip technology and Systematic pharmacology to find out the target gene of jianpi xiaoji recipe inhibiting the proliferation of liver cancer cells,and to select the key monomer of Jianpi xiaoji recipe aqueous extract regulating the autophagy of liver cancer and reveal the treatment mechanism of the disease.Methods:Using CellTiter GLO spleen method to detect different conc entration Jianpi xiaoji recipe aqueous extract SMMC-7721 human liver ca ncer cell inhibition rate,calculate the half inhibitory concentration(half-inhibitory concentration,IC50);Different concentrations(1200ug/ml,1500ug/ml)of Jianpi xiaoji recipe aqueous extract were used to act on the SMC C-7721 cells.The SMMC-7721 cells cultured in the same amount of unt reated medium were used as the control group.After 48 hours of action,CellTiter GLO cell cloning and cell formation experiment was conducted t o detect the effect of Jianpi xiaoji recipe aqueous extract on the proliferat ion and apoptosis of the SMCC-7721 cells.The SMMC-7721 cells were tr eated with 1500ug/ml Jianpi xiaoji recipe aqueous extract.The SMMC-7721 cultured in the same amount of untreated medium were used as the c ontrol group:Using western blot test the influence of autophagy related pr otein P62?LC3A/B expression,The SensGFP-StubRFP-LC3 fusion protein is expressed in the cells by the lentivirus,autophagy body formation and autophagy body with the lysosome fusion process are monitored.Experim ent group are as follows:The normal target cells,and cells treated with Jianpi xiaoji recipe aqueous extract.The whole RNA of SMMC-7721 cells of human hepatocellular carcinoma was extracted after intervention with Jianpi xiaoji recipe aqueous extract.Using high content screening(HCS)e xperiment to choose cell proliferation inhibition significant genes,ultimatel y determine the key genes.Through IPA pathway-based analysis,the prog nosis of the present Jianpi xiaoji recipe aqueous extract and the changes i n the classical pathway were observed,and the effect of key gene shRNA lentivirus on SMMC-7721 cells was verified.On the basis of TCMSP C MSP(http://lsp.nwsuaf.edu.cn/tcmspsearch.php)?TCMID(http://119.3.41.228/tcmid/)?ETCM(http://www.nrc.ac.cn:9090/ETCM)database,the network pharmacological analysis of jianpi xiaoji prescription was carried out.We b uilt traditional Chinese medicine composition database for the composition s of Jianpi xiaoji recipe.based on ADME(Metabolism of Absorption Abs orption,Distribution,Metabolism,Excretion discharge).Based on ADME(M etabolism of Absorption Absorption,Distribution,Metabolism and Excretio n discharge),the active ingredient selection target selection,screening acti ve compounds through data mining screen to eliminate repetition and rem ove with cancer or tumor clearly not related compound.Results:(1)The IC50 of SMMC-7721 cells was 1500ug/ml(2)After 48 hours of intervention with different concentrations(1200ug/ml,1500ug/ml),compared with the control group,the proliferation of liver cancer cells in the intervention group was significantly inhibited,the number of clones was decreased,and the apoptosis rate was increased.(3)Autophagy related protein detection and autophagy flow monitoring showed that,compared with the control group,the ratio of autophagy marker protein LC3II/LC3I increased in the 1500ug/ml Jianpi xiaoji recipe aqueous extract,and the expression of P62 was up-regulated,and autophagy flow was inhibited.(4)The results of the gene chip showed that,compared with the control group,the gene data analysis of the Jianpi xiaoji recipe aqueous extract intervention group showed that 430 differemtial genes expression,among them 265 gene were up-regulated and 165 gene were down-regulated.Selecting the gene the high abundance of expression and reported for the first associated with liver cancer to do HCS,according to the results,HIST1H2BK is the positive proliferation related genes,interfere with human liver cancer SMMC-7721 cells HIST1H2BK gene,according to the results of HIST1H2BK genes affect cell proliferation apoptosis.Based on IPA pathway analysis,ERK/MAPK pathway is significantly inhibited in the pathways affecting autophagy.According to the TCMSP database and Genecards database,Ingredients targets for liver cancer targets do take intersection map,select 45 compounds,Metascape analysis results show that the active targets have multiple targets in different biological processes and pathways of enrichment,is one of the most significant biological processes such as calcium salt pathways in cancer cell migration,acacetin?kaempferol?beta-sitostero?luteolin and quercetin are the main compounds regulating the liver cancer cell proliferation in the spleen and negative side played a key role in the prevention and treatment of liver cancer.MAPK?PI3K-Akt autophagy related pathways have higher levels of significance and gene enrichment.Conclusions:1.The Jianpi xiaoji recipe aqueous extract has inhibitory effect on the proliferation of SMMC-7721 cells2.Jianpi xiaoji recipe aqueous extract can regulate autophagy process in SMMC-7721 cells by inhibiting the process of autophagy body to autophagy-lysosome.3.HIST1H2BK gene is the key gene for jianpi xiaoji recipe to regulate the proliferation and inhibition process of liver cancer cells.The mechanism of action may be that MAPK pathway plays a regulatory role in autophagy by inhibiting the proliferation and inducing apoptosis of liver cancer cells4.Acacetin?Kaempferol?beta-sitostero?luteolin and quercetin are the main compounds are the key compound in Jianpi xiaoji recipe.The mechanism of action may be that ERK/MAPK pathway plays a regulatory role in autophagy by inhibiting the proliferation of liver cancer cells and inducing their apoptosis.