The Experimental Study Of Prevention And Treatment With Aitongxiao Prescription On The Induced Hepatic Carcinoma In Rat Based On The Preventive Treatment Of Disease Thought

OBJECTIVE:1. To observe the cancer rate and survival time of rats and confirm the optimal concentration of DEN to induce cancer, after liver cancer in rats was induced by free-drinking with different concentrations of two diethylnitrosamine (DEN) water solutions.2. To identify protogene related to hepatocarcinogenesis by observing expression of Bax, Bcl-2, Survivin, Fas, FasL and Caspase-3mRNA induced by64ppm DEN in rats at different time.3. To discuss the mechanism of prevention and treatment of primary hepatic carcinoma from the pathological changes of liver tissue of rats induced by ATXP (Aitongxiao major Prescription) intervention at different time, expressional changes of relative cell apoptosis genes (gene and protein), such as Bax, Bcl-2, Survivin, Fas, FasL, Caspase-3and the in-situ cell apoptosis.METHODS:1.96SPF male Wistar rats were randomly divided into4groups (n=24). Each group was fed freely with water containing DEN40ppm,64ppm,80ppm and purified water respectively for16weeks. In the16th week of the experiment,12rats in each group were randomly killed to take out livers to make specimens with HE staining, whose pathology and cancer-generating rate were observed under the optical microscope. The rest rats in each group were used to observe survival time.2.60male Wistar rats were randomly divided into model group (n=40) and control group (n=20). The model group was fed freely with running water containing DEN64ppm for16weeks and then running water while the control group just with running water.4rats in model group and2in control group in the4th,8th,12th,16th and18th week respectively were randomly decapitated to take out liver tissues to observe the pathology and to detect gene expression of Bcl-2, Bax, Caspase-3, Survivin and Fas/FasL through RT-PCR.3.40male Wistar rats, randomly divided into prevention group and model control group on average, were fed with drinking water containing DEN64ppm for16weeks and then with running water. From the cancer-induced day, prevention group was irrigated with ATXP (0.2g/100g), one time per day, continuous administration for6days in a week with a course of18weeks.4rats in the4th,8th,12th,16th and18th week respectively were randomly decapitated to take out liver tissues (passive part). The expressions of Bax, Bcl-2, Survivin, Fas, FasL, Caspase-3mRNA and the protein in liver tissues were detected through HE staining pathological observation, RT-PCR and SABC methods. The condition of liver cell apoptosis was inspected through TUNNEL method.RESULTS:The cancer induction rate in the18th week was91.16%in the80ppm group,83.33%in the60ppm group and0in the40ppm group; in the80ppm group, death occurred in the22th week (4rats died) and at the26th week the death rate was100%. While in the64ppm group, rats began to die in the25th week and mortality was100%in the30th week; in the40ppm group, fatality rate was16.67%in the30th week. In terms of cancer rate, there was no obvious difference (P>0.05) between the80ppm group and the64ppm group. However, in the aspect of survival time, the former group was significantly longer than the later (p<0.05).2. Pathological analysis of the rats indicated hepatic damage occurred all the time. The pathological damage involved inflammation of the liver, fat lesions, hepatic fibrosis, liver cirrhosis and liver cancer, the same as the pathological mechanism of HBV infection. Over-expression of Bax, Bcl-2existed in the DEN carcinogenesis rats all the time, while Survivin, FAS just occurred in the8th week; over-expression of Fasl appeared in the8th,12th and18th week; Casepase slightly expressed or did not express.Results of RT-PCR:The intervention process of ATX can lower the expression of survivin and Bcl-2, up-regulate the expression of casepase all the time, up-regulate Bax only at the12th week and Fas, Fasl at the4th week, with no obvious up-regulation in the rest of the time.4.1mmunohistochemical results:protein and gene expressions of survivin, casepase-3, Fas/Fasl, Bax, Bcl-2are consistent; Survivin expressed clearly in16w,18w and Casepase-3proteins less expressed in the whole process of cancer inducing in control group; The Bcl-2protein expressed in the early period of inducing cancer, present continuous high expression in process induced carcino-ma,18w to spike; While Bax, Fas/Fasl protein expression in8w quantity highest; Prevention grou-p of survivin apoptosis gene and the Bcl-2during the whole observation time point expressed lower than control group, P<0.05; Casepase-3and Bax protein expression were significantly higher during the whole observation time point, P<0.05; Fas, Fasl protein expression increased more significantly than those of control group in the4w, P<0.01).5. TUNEL results:negative control group did not see apoptotic cells in each time point; model group in the4w and8w,12w no apoptotic cells,16w,18w visible small amounts of fluorescence, sugge-sting a small amount of apoptosis cells; Prevention group in4w no fluorescence,8w,12w a small amount of fluorescent,16w,18w fluorescence intensity increased, cell apoptosis quantity become more, the highest cell apoptosis index,16w,18w apoptosis significantly compared with model group, with significant difference(P<0.01).CONCLUSION:1. DEN carcinogenesis with a concentration of64ppm is an ideal hepatocellular carcinoma model in rats which is used for drug effectiveness evaluation.2. The whole process of DEN carcinogenesis prompts the activeness of cell proliferation and the apoptosis reduction through the expression of apoptosis gene inhibition and the expression of apoptosis promoting gene during the carcinogenesis process.3. ATXP prevention and treatment of liver cancer promotes cell apoptosis to fulfill its antitumous effect by means of down-regulating the Bcl-2and survivin and up-regulating the expression of casepase-3.