Experimental Study On Anti-hepatoma Effect And Mechanism Of Kangairuanjian Decoction

ObjectiveTo evaluate the anti-cancer effects of Kangairuanjian decoction(KQRGD)on hepatoma 22 bearing mice models in vivo.To observe the cells growth inhibitory effects in the human hepatoma Huh7 cells by KQRGD,and study the regulating effects on aberrant network consisting of IL-6 signaling pathway and MicroRNA let-7a,inqure the mechanism of anti-hepatoma with KQRGD.Experimental contents1.Inhibitory effects of Kangairuanjian detoction(KQRGD)on mice in bearing H22 tumour32 NIH mice were established for transplanted tumour model of H22,then they were randomly divided into four groups:model group(group A),high and low dosage group of KQRGD(group B,group C),and cyclophosphamidum as the positive control(group D).Mice in group B and group C were administrated orally with KQRGD at dosage of 49.0g/kg and 24.5 g/kg daily respectively.Mice in group D were injected with cyclophosphamidum intraperitoneal at dosage of 20mg/kg daily.After treatment for 8 days,the tumour-inhibition rate were calculated according to the tumour mass weight in mice.It showed that tumour mass weight in group B and group C were obviously lower to that of group A(P<0.01).The tumour-inhibition rate in group B and group C were 41.4%and 32.8%resepectively.The thymus index and spleen index in group B and group C were all significantly higher than those of the group D(P<0.01),but no significanf difference comparing with group A(P>0.05).it showed that KQRGD has good effect of suppressing transplanted tumour growth on H22 tumour-bearing mice.2.Study of the effect and mechanism of KQRGD on anti-hepatoma2.1 Inhibitory effect of KQRGD on growth and proliferation in human hepatoma Huh7 cellsStudy of Inhibitory effect on growth and proliferation in human hepatoma Huh7 cells with KQRGD by MTT assay.Results showed that KQRGD can significantly inhibit the growth and proliferation in human hepatoma Huh7cells,and there was a significant dose-effect relationship.After treatment of Huh7cells for 48h,the concentration which caused 50%inhibition of cell growth(IC50)was 55.46mg/ml.Compared with the control group,high and low dose groups of KQRGD(48.0 mg/ml and 24.Omg/ml respectively)all showed significantly inhibitory effect on growth and proliferation in human hepatoma Huh7 cells at the treatment time point of 24h,48h and 72h(P<0.05,P<0.01).2.2 Study of the effect of KQRGD on regulating the abnormal network consisting ofIL-6 signaling pathway and microRNA let-7a in human hepatoma Huh7cells Testing the 0D value with MTT assay and detecting amounts of IL-6 with RIA in the conditioned medium after treatment Huh7cells for 48h.It showed that OD value and IL-6 level in high and low dose groups of KQRGD were all remarkable reduced Compared with the control group(P<0.05,P<0.01).Comparing with the control group,in both groups of KQRGD,the expression level of microRNA let-7a elevated and miR-21 decreased(P<0.01),Gene mRNA expression level of NF-?B/P65?STAT3?AKT2?K-ras?Bcl-2 and c-myc obviously decreased(P<0.01),but PTEN increased(P<0.01).it showed dose-dependent response.Exposure Huh7cells with 48.0 mg/ml(high dose group)and 24.0 mg/ml(low dose group)of KQRGD for 48h,it showed that protein expression level of PTEN elevated,STAT3 and AKT2 reduced in both groups of KQRGD Comparing with the control group(P<0.01)?Conclusion1.KQRGD has obvious effect of suppressing transplanted tumour growth on H22 tumour-bearing mice in vivo.2.KQRGD can significantly inhibit the proliferation of human hepatoma Huh7cells in vitro,its anti-tumor effects may be attributed,at least in part,to interfere with an autocrine/paracrine loop based on the production of IL-6,and then inhibit the activation of IL-6/STAT3 signaling pathway,modulate the abnormal network consisting ofIL-6 signaling pathway and microRNA let-7a,then depress the expression of its downstream signaling molecules NF-?B/P65?STAT3?AKT2?K-ras?Bcl-2 and c-myc,and up-regulated the expression of PTEN,leads to the proliferation of human hepatoma Huh7cells restrained,it showed the whole treatment and regulation virtue of traditional Chinese medicine.3.MicroRNA are a class of noncoding RNAs that be connected with cancer initiation,they located at the up-stream of oncogene or tumour suppressor gene,have the function of controlling the expression of coding genes related to cancer.Aberrant expression of microRNA involved in hepatocarcinogenesis.it provide a new clue for study the molecular mechanism of hepatocarcinoma,microRNA has become new target for medical therapy.IL-6 has been confirmed as the downstream target gene of let-7a,miR-21 regulate the expression of PTEN.In the study,after treatment of human hepatoma Huh7 cells with KQRGD,expression level of microRNA let-7a was up-regulated,the mRNA and protein expression level of IL-6 decreased.Meanwhile,expression level of miR-21 was down-regulated,the mRNA and protein expression level of PTEN increased.It suggested the anti-tumor effects of KQRGD are connected with regulating the expression of tumor-related microRNA,which enrich the scientific connotation of the therapy,and provide a new evidence for its clinical treatment.