| Objective1. To study the inhibitory effect of Compound of Phyllanthus Urinaria â…¡ (CPU â…¡) 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 â…¡ in vitro. To observe the regulatory effect of Phyllanthus compound â…¡on 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 â…¡.Exper i mental contents1. Study of the synergistic and toxicity-decreasing effects of CPU â…¡ 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 â…¡, CTX group, high and low dosage group of CPU â…¡ 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 â…¡ 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 â…¡ on anti-hepatoma.2.1 Study of the effect of CPU â…¡ on proliferation inhibitionHuman hepatoma cells were used as a model in vitro, Huh-7 cells proliferation inhibitory effect of CPU â…¡ was observed by testing the OD value with MTT method. It showed that CPU â…¡ 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 â…¡ 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 â…¡ on Cell CycleDetermining the Huh-7 cells which was intervened 48h by CPU â…¡ 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 â…¡ 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 â…¡ in human hepatocel lular carcinoma Huh-7 cells.Determining the Huh-7 cell which was intervened 48h by CPU â…¡, 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 â…¡ were significantly reduced, the differences were statistically significant compared with the control group(P<0.01). It showed that CPUâ…¡ 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 â…¡ 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 â…¡ 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 â…¡ 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. |
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