| Objective: To investigate the mechanism of Jianpi Yiqi Recipe(JYR)in treating liver cancer,the active components of the Recipe,and their targets were explored.Furthermore,the effect of JYR on the IL-6/STAT3/Vimentin signaling pathway in diethylinitrosamine(DEN)-induced rat hepatocellular carcinoma was studied.Methods: 1.The TCMSP database,CNKI database and Swiss Target Prediction platform were used to search the active components of JYR and their corresponding targets.Hepatocellular carcinoma(HCC)-related genes were acquired by Gene Cards,CTD,and Gene databases.The potential cancer-related drug targets were obtained,and the active component-target network diagram was mapped by Cytoscape software.The protein-protein interaction(PPI)network,GO enrichment and KEGG enrichment were conducted for potential targets.Furthermore,the results were verified by experiments in DEN-induced rat liver cancer model.2.70 healthy male SD rats were randomly divided into seven groups: Control group,Model group,JYR low-,medium-and high-dose groups,STAT3 inhibition group and gp130 inhibition group.All the groups were given intraperitoneal injection of DEN according to the weight of 70 mg/kg once a week for 10 weeks,except the control group which was given normal saline in the same way.After 16 weeks,all the experimental groups were intervened once a day for 4 weeks.The control group and the model group were given 10.5 g/kg normal saline.The JYR low-,medium-and high-dose group were given 5.25 g/kg,10.5g/kg and 21 g/kg JYR,respectively.The STAT3 inhibition group and gp130inhibition group were given 4.5 mg/kg/d C188-9 and SC144 intraperitoneally,respectively.After Hematoxylin-Eosin(HE)staining,the pathological changes in liver tissue were observed by the microscope.The m RNA levels of IL-6,gp130,JAK1,JAK2,STAT3,ZBP-89,Caspase-3,Vimentin,ROCK1,ROCK2 and Aurora B in liver cancer tissue were detected by quantitative reverse transcription PCR(RT-q PCR).The serum level of IL-6 was measured by ELISA,and the activity of Caspase-3 enzyme in liver cancer tissue were determined by colorimetry.The protein levels of gp130,JAK1,p-JAK1,JAK2,p-JAK2,STAT3,p-STAT3,ZBP-89,Caspase-3,Vimentin,ROCK1,ROCK2,Aurora B and p-Vimentin were examined by Western blot.Results: 1.64 active components,146 potential targets and 120 potential HCC-related drug targets were identified.The HCC-related drug targets involved in 2318 biological processes,35 cellular components,156 molecular functions and 167 pathways.STAT3 participated in many biological processes and pathways,and the JAK-STAT signaling pathway is an important one.2.During DEN-induced rat hepatocellular carcinoma,the liver morphology and pathological sections were changed accordingly.After treated with JYR,C188-9 or SC144,the canceration of liver tissue was improved to some extent.3.Compared to the control,the ZBP-89 m RNA level was significantly decreased(P<0.01)in the liver tissue of rat model,but the m RNA levels of IL-6,STAT3,Vimentin,ROCK2 and Aurora B were significantly increased(P<0.01).After the intervention of JYR or inhibitors,the ZBP-89 m RNA level was significantly increased(P<0.01),and the m RNA levels of IL-6,gp130,STAT3,Vimentin,ROCK2 and Aurora B were significantly decreased(P<0.05 or P<0.01),compared to the model group.4.The serum level of IL-6 in the rat model was significantly increased(P < 0.01),and significantly decreased(P<0.01)after the intervention of JYR or inhibitors,compared to the control.5.Compared to the control,the ZBP-89 protein level was significantly decreased(P<0.01),but the protein levels of p-JAK1,p-JAK2,gp130,STAT3,p-STAT3,Caspase-3,Vimentin,ROCK2,Aurora B and p-Vimentin were significantly increased(P<0.01)in the liver tissue of rat model.After the intervention of JYR or inhibitors,the ZBP-89 and Caspase-3protein level,and the enzymatic activity of caspase-3 were significantly increased(P<0.05 or P<0.01),but the protein levels of p-JAK1,p-JAK2,gp130,STAT3,p-STAT3,Vimentin,ROCK2,Aurora B and p-Vimentin were significantly decreased(P<0.05 or P<0.01),compared to the model group.Conclusions: 1.JYR that suppress the IL-6/STAT3 inflammation signal pathway may be essential to treat hepatocellular carcinoma,and STAT3 may be its potential therapeutic target.2.JYR promotes the expression of ZBP-89 and Caspase-3,and reduces the expression of ROCK2 and Aurora B by inhibiting IL-6/STAT3 signaling,which further leads to down-regulating the level of Vimentin m RNA and protein expression and its phosphorylation level. |
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