Experimental Study On Anti-hepatoma Effect And Mechanism Of Compound Of Phyllanthus

Objective1. To study the inhibitory effect of Compound of Phyllanthus Urinaria Ⅱ (CPU Ⅱ) on H22 hepatoma mice, measure mice peripheral hemogram and hepatic and renal function, also calculate spleen index and thymus index, and evaluate its synergistic and toxicity-reduced effects in chemotherapy.2. To observe the inhibitory effect on the multiplication and the cycle of human hepatoma Huh-7 cells with Phyllanthus compound Ⅱ in vitro. To observe the regulatory effect of Phyllanthus compound Ⅱon the activation of PI3K/AKT/mTOR signal path by modulating the expression of miR-122, decreasing its target gene expression level of mRNA and the protein of IGF-1R, Exploring the molecular mechanism of anti-hepatoma with CPU Ⅱ.Exper i mental contents1. Study of the synergistic and toxicity-decreasing effects of CPU Ⅱ on H22-bearing mice with chemotherapy.Hepatoma 22 bearing mice models were established and then divided randomly into six groups:model group, high and low dosage group of CPU Ⅱ, CTX group, high and low dosage group of CPU Ⅱ combined with CTX group. Continuous administration of 8 days, the tumour-inhibition rate were calculated according to the tumour mass weight in mice, the serum levels of ALT\AST> CREA and UREA were determined, index of spleen and thymus were observed. Results showed that tumor weight in group of high, low dosage of CPU Ⅱ combined with cyclophosphamide were significantly lower than those in model group respectively (P<0.01); Tumour-inhibition rate in group of high, low dosage of CPU II combined with cyclophosphamide were 74.3% and 66.9% respectively, obviously higher than that of CTX group, also higher than two drug combination theory additive effect value of (Fa)1-2 72.2% and 64.8% respectively. The results suggest that the two drugs used in combination show noticeable synergism in tumour-inhibition. The thymus index, spleen index of CPU II high, low dose group were significantly higher than that of cyclophosphamide group, the difference is significant (P<0.05);The spleen index of CPU II high dose cyclophosphamide group was highter than that of cyclophosphamide group (P<0.05), but no significant differences were observed in thymus index (P>0.05). The mouse serum levels of ALT, AST, CRE and UREA were significantly higher than that of cyclophosphamide group (P<0.05). It shows that CPU II can not only improve the non-specific immunity of mice, but also can effectively improve the immune organ atrophy induced by cyclophosphamide to a certain extent, and can effectively improve the function of liver and kidney induced by chemotherapeutic drugs damage.2. Study of the effect and mechanism of CPU Ⅱ on anti-hepatoma.2.1 Study of the effect of CPU Ⅱ on proliferation inhibitionHuman hepatoma cells were used as a model in vitro, Huh-7 cells proliferation inhibitory effect of CPU Ⅱ was observed by testing the OD value with MTT method. It showed that CPU Ⅱ can significantly inhibit the proliferation of human hepatoma Huh-7 cells,and there was a significant dose-effect relationship in it.1C50 of the 48h was 58.6mg/ml. at the time point of 24h,48h and 72h, high and low dose groups of CPU Ⅱ all could significantly inhibit the proliferation of hepatoma Huh-7 cells compared with the control group(P<0.05, P<0.01).2.2 Study of the effect of CPU Ⅱ on Cell CycleDetermining the Huh-7 cells which was intervened 48h by CPU Ⅱ through flow cytometry assay Annexin V-FITC/PI. Compared with the control group, the G0/G1 cell ratio in high and low dose groups of CPU Ⅱ increased significantly, and the S, S+G2/M ratio were all significantly decreased, the differences were statistically significant (P<0.01).2.3 Influence on the expression of miR-122, IGF-1RmRNA and the protein, PI3KmRNA, AKT2mRNA, mTORmRNA, cyclinD1mRNA,P-AKT protein by CPU Ⅱ in human hepatocel lular carcinoma Huh-7 cells.Determining the Huh-7 cell which was intervened 48h by CPU Ⅱ, compared with the control group, the expression of miR-122 increased significantly, and the expression of IGF-1RmRNA was significantly decreased(P<0.01).The expression of PI3KmRNA, AKT2mRNA, mTORmRNA, cyclinDlmRNA and P-AKt protein in high and low dose groups of CPU Ⅱ were significantly reduced, the differences were statistically significant compared with the control group(P<0.01). It showed that CPUⅡ has strong the effects of inhibiting the activation of PI3K/AKT/mTOR signal path by regulating the expression of miR-122, then reduced its target gene IGF-1R mRNA and the protein exoression, and leaded to the expression of cell cycle protein cyclinDl lowed, so that inhibited the proliferation of hepatocel lular carcinoma Huh-7 cellsConclusion1. CPU Ⅱ have obvious effect on tumour-inhibition to hepatoma 22 bearing mi ce models. Combined with chemotherapy drugs, can enhance the tumor inhibiting effect,and also can attenuate the toxic side effects of chemotherapeutic drugs.2. CPU Ⅱ can significantly inhibit the proliferation of human hepatoma Huh-7cells, can significantly block Huh-7 cell cycle progression, so that the cells are mostly arrested in G0/G1 phase, can not enter S phase to DNA synthesi ultimately cells proliferation was Inhibited. At the same time, CPU Ⅱ can significantly improve the expression of miR-122 and decreased the expression of target gene IGF-1R mRNA and the protein, leading to the expression of downstream cascade reaction of PI3K, AKT, P-AKt and mTOR was inhibited. Therefore the effect on the activation of PI3K/AKT/mTOR signal path was also inhibited, leading to the expression of cyclinDl was reduced, the proliferation of hepatocellular carcinoma cells was inhibited.