| Hepatocellular carcinoma (HCC) is the one of major health care problem of cancer mortality worldwide. Especially, HCC is more obvious in China and southeast Asian and Africa. And it is the second leading cause of cancer death in China, and the mortality of HCC is the first most common cancer in Guangxi province. And that have a significant rise. The patients of hepatocellular carcinoma have the characteristics with the difficult of diagnosis and treatment, illness progress and development are rapid and poor prognosis. But all of Middle-late stage patients with the prognosis were poor. Even the early, the effective treatments methods of HCC were taken, the rates of 5-years survival also have 50%. So that, the works of HCC risk factors and pathogenesis research, and to develop the tertiary prevention had were particularly important.Yet, we were recognized at now, the three risk factors of HBV infection, aflatoxin (AFB1) intake and people drinking water pollution were considered to be causes HCC. The regions high rates of HCC occurrence were pretty serious by AFB1 pollution. Although launched "mouldproof to poison" education work such as cancer propaganda. For a variety of reasons, the foods were contaminated by high-risk pollution with AFB1 inordinately. The lifes of human in daily were exposured difficult to avoid in the areas of high-risk pollution with AFB1. Therefore, to investigate the significance have been found for prophylaxis and treatment HCC that to find the formation against natural objects or its extract could fight AFB1 induced HCC in these areas. However, the mechanisms of carcinogenic by AFB1-induced were not very clear. But studies have reported, AFB1 within the body have role of oxidative metabolism by the liver enzyme could become biotransformations form active intermediates with carcinogenicity. Because of this epoxy cures with high activity and low stability in the body, and the adduct make AFB1 reduce or lose biological activity. The CYPs and GSTs of drug metabolizing enzymes mainly have CYP3A4 and GST-Pi as well as several subtribes which close-related with drug metabolism that mediated AFB1 metabolic activation and detoxification. The Camellia chrysantha(Hu)Tuyama (CCT) contains many amino acids, tea polyphenol and many kinds of composition. CCT have the role of block cell proliferation through anti-oxidation, anti-angiogenic and regulate gene expression. But so far, the reports have not yet been research about CCT to restrain AFB1causing to hepatocellular carcinoma. This paper is to make it clear that AFB1 cause liver cancer of extracting effective components and analysis by CCT working on AFB1 induced cancer animal models, By immunohistochemical techniques, it observes the effects with dynamically the expression level of related gene with process of AFB1 causing liver cancer, and to investigate the molecular biology mechanism of the blocking hepatocellular carcinoma occurrence and development. Similar research report has not been seen in domestic and foreign.MethodsEstablish the model wistar Rats in Aflatoxin B1-induced hepatic carcinoma, 70 Wistar rats were divided randomly into A, B and C groups. Groups A was AFB1 group with 25 rats. The specific injection to 53w of experiment and dosage about AFB1:200ug/kg weight. Group B was CCT group with 25 rats, from the experimental in the first week began feeding containing 30% concentrate CCT of the stomach every irrigation experimental end of experiment, AFB1 was injected with dosage and intra peritoneal time same to group A; Group C was control group with 20 rats, in the first week, from the experiment began to end feeding normal feed, not giving AFB1 processing end. Animals were sacrificed at the 73rd-week and liver tissues were collected. The each animal are be weight every week once, and according to the amount of weight calculation AFB1 injections. The each animal of liver biopsies were had performed every ten weeks. Animals were sacrificed at the 73rd-week and serums, hepatic tissues and hepatocarcinoma tissues were collected. Using the hepatic microsomal mixed-function oxidase enzyme system dynamically examined the CYP3A4 activity by quantitative fluorescence spectrophotometry and the expressions of GST-Pi, STAT3 protein were examined by immunohistochemical staining with hepatic tissues different poit-times in hepatocarcinogenesis induced by AFB1 in rats.Results1. The datas of rats tumor occurrence,The earliest time that AFB1 evoked rat liver malignant tumor (fibrosarcoma) formation is at 52W, and the first case occurrence of hepatocellular carcinoma is AFB1 group rats in the 55W.They are all AFB1 group rats. Microscopically dynamic observation biopsy liver histopathology shows the rat liver AFB1 induced cancers process can appear different degrees of liver cell degeneration,and all so it have Significant differences and different damage degrees each other about three groups. Using CCT intervention rats induced cancer in various stages of liver damage is lighter than AFB1, and happened proliferative lesion also later than AFB1 group, average survival time, rats and final hepatocellular carcinoma incidence significantly lower than AFB1 group. The control group was without the changes.The end of experiment at 73W, the rats of occurred malignant tumor in group AFB1 and CCT were 76.5 percent (13/17) and 25.0 percent (4/16), the rats of hepatocellular carcinoma in group AFB1 and CCT were 58.8 percent (10/17) and 18.8 percent (3/16). In group AFB1, the rats of occurred malignant tumor and hepatocellular carcinoma are significantly higher than the CCT groups (P = 0.0053 and P = 0.0324). The control groups have no rat tumor occurrence.2. The changes of CYP3A4 activity in rat hepatic tissues metabolic enzymesIn the whole experiment process, The CYP3A4 metabolic enzymes activity show increased gradually with peak value at the 23rd-week and then decreased gradually, in each groups with dynamic observation. And the CYP3A4 activity increased again at the 53rd-week, forming double peaks. CYP3A4 activities decreased significantly during53rd- and 63rd-week in AFB1 and CCT group compared to control group (P<0.05). The level of CYP3A4 activity in control group was decreased at the 33rd-week and 63rd-week, and no change significantly in other stage.3. The expression of GST -Pi in rat hepatic tissuesGST-Pi positive dyeing located in cell plasma, positive cells is multifocal dyeing or flake distribution. In the whole experiment process, the expression of protein were gradually rise heighten with the time extend induced cancer, especially obvious in AFB1 group. The rates of GST-Pi in AFB1 group were 73%, 86.7%, 93.3% and in CCT group were 66.7%, 80.0%, 80.0% at 33W, 53W and 73W. The rates of GST-Pi in AFB1 group are corresponding higher than in CCT group. And that the control group was 10% at the 73W, and the expression was negative in other time. The same time is compared among different groups, the expression of GST-Pi in AFB1 groups and CCT groups are significantly higher than the control groups (P < 0.05) from 33W to 73W, the AFB1 groups are no significantly higher than and CCT groups from 33W to 63W. AFB1 group is not only significantly higher than CCT group, but also higher than those in the control groups (P = 0.000) in the 73W. The GST-Pi protein expressions were no significant difference (P > 0.05) at different time-points between groups or between group rats.4. The expression of STAT3 in rat hepatic tissuesSTAT3 positive dyeing located in cell hepatic tissues plasma, positive cells in scattered upon or dyeing focal distribution. Induced cancer process, In group AFB1 and group CCT , the rates expressions of STAT3 protein were gradually rise heighten with the time extend induced cancer, The rates of STAT3 in AFB1 group were 20.0%, 53.3%, 86.7% and in CCT group was 13.3%, 40.0%, 60.0% at 33W, 53W and 73W. And that the control group was 10% at the 53W and 73W, and the expression was negative in other time. The same time is compared among different groups, the expression of STAT3 in CCT groups are significantly higher than the control groups (P < 0.05) from 53W to 73W, the AFB1 groups are no significantly higher than and CCT groups from 53W to 73W (P > 0.05). Conclusions1. CCT concentrate had inhibited the rates of hepatocellular carcinoma incidence in AFB1 induced. The effects of hepatocellular carcinoma have prevented, that likely to become human hepatocellular carcinoma with AFB1 correlation of potential chemoprevention agent.2. The CYP3A4 activity was inhibited during hepatocarcinogenesis in CCT groups. This may be due to decrease of carcinogens metabolic and that reduce carcinogenicity and hepatic injury of chemical to attain the role of protect liver.3. In the carcinogenic process, the expression of GST - Pi and STAT3 were inhibited during hepatocarcinogenesis in CCT groups, and so it could prevent cell division and malign hyperplasia and delay the development of tumor. This may be due to the methylation about promotor of CpG-island and activation of STAT3 downstream gene, and concerned those interdict activity of some important anti-tumor genes. |
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