The Inhibition Effects Of Ginkgo Biloba Extract On Aflatoxin B1-induced Hepatocarcinogenesis In Wistar Rats

Background and ObjectivePrimary hepatic carcinoma (PHC) is more common malignant tumor in human. The incidence of PHC is the fifth most common cancer and the mortality rate is the third leading cause of cancer in worldwide. PHC is the second cause leading cancer death in China. In any given year20%of total fatalities caused by malignant tumor are PHC in China. The incidence and fatalities of PHC is especially striking in Qidong of Jiangsu and Fusui Country of Guangxi province where PHC is the first most common cancer. The patients of PHC have the characteristics with the difficult of diagnosis and treatment, illness progress fast and poor prognosis. PHC is a terrible disease and this situation is very harmful to people's health.Epidemiological research and experimental study of zoology evidenced that chronic hepatitis B virus (HBV) infection and diet aflatoxins (AFT) pollution are thought to be two largely responsible for the high incidence of PHC in southern Africa and southeast China. However, The two factors can be synergies between, jointly promote the development of PHC. AFT is a metabolic product of aspergillus flavus and Parasitic aspergillus and the chemical is belong to two furan coumarin derivatives. According to the different of its molecular structure, the natural AFT divided into B1, B2, G1and G2. In our country, a lot of food, grain and oil, drugs raw materials, agricultural and sideline products and finished goods are contaminated by AFT, which Aflatoxin B1(Aflatoxin B1, AFB1) of the pollution is the most extensive and toxicological effect is the most strongest. At present, AFB1is one of the strongest carcinogenic substance. Research shows that the positive correlation between the amount of AFB1in food and the occurrence of hepatocellular carcinoma. It has been confirmed by many epidemic research institutions in Asia and Africa.Research shows that, the parent compound of AFB1is without carcinogenicity before metabolic activation. After entering the body, this chemical is biotransformed by cytochrome P450system to the reactive intermediante AFB1-exo-8,9-epoxide (AFBO)exert its gene toxicity and carcinogenicity. Cytochrome P450is the key enzyme when it catalytics the metabolic reactions. cytochrome P450is mainly exist in the liver cells, which the content of cytochrome P4503A4is the most and activity is the highest.AFBO can combined with biological macromolecules such as nucleic acids and proteins to form corresponding the adducts. AFB1and DNA covalent bonding form AFB1-DNA adducts is formed the basis of genetic mutations, also which led to the first step of Primary hepatic carcinoma. AFB1-DNA adducts exist mainly in liver cells. Part of AFB1-DNA adducts are converted into non-toxic substance and eliminated from the body under the action of the body Ⅱ phase metabolic enzymes such as Glutathione S-transferase (GST). Due to the change of the electronic field in molecules, AFB1can also add other DNA spontaneously formed and lead to the DNA damage.AFT is very harmful to human health. In order to prevent or reduce the harm caused by AFT, all kinds of methods were used such as avoid the food pollution and remove toxins measures and so on. Chemoprevention is a pharmacological approach to suppress, delay or reverse cancerous process, and to prevent cancer ways have also increasingly concerned by people. Research shows that some plant chemicals such as chlorophyll, tea polyphenol, flavonoids compounds has a unique anti-cancer effects in the neighborhood of cancer prevention. Through add chemical preparation plants or its content of food is higher in high pollution area to prevent the function of AFT lead to PHC attracted more and more researchers'attention. Food and its composition as chemical prevention agent has the advantages such as safe, easy to accept. It became one of the important fields in conducting chemical preventive research.Gingko (Ginkogo biloba L) for ginkgo secco plant, its leaves and fruit has important medicinal value. Ginkgo biloba leaf extract, Standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, typically contains about24%flavonoid glycosides and about5-7%terpene trilactones that are unique to Ginkgo. Recent research shows that EGb761and its components has the function of antitumor through anti-oxidation, antiangiogenic and gene regulation. So, the study of EGb761on the application of cardiovascular system disease and cerebrovascular disorder has been expanded to antitumor territory.The experimental study was confirmed that EGb761has Inhibition on Aflatoxin B1-induced Hepatocarcinogenesis in wistar Rats. However, the specific mechanism was not completely clear. In recent years, the studies found that the expression of related genes in hepatocellular carcinoma of experimental rat such as IGF-II, NADE, P16INK4a, GST-Pi, UGT etc were significantly greater than normal liver, and the relationship of P53, AKR7A, P75NTR, COX-2with the tumor are very outstanding. Therefore, we investigated dynamic the influence of EGb761on liver related metabolic enzymes and tumor gene expression on Aflatoxin B1-induced Hepatocarcinogenesis in wistar Rats. We investigated the molecular biology mechanism of the blocking hepatocellular carcinoma occurrence and development. For the drug of prevention liver cancer, it can provide more experimental basis. There will be a great economic and social benefits.MethodsThe model of aflatoxin B1-induced Primary hepatic carcinoma was established in wistar Rats. Among the totale of70, four-weeks old male wistar Rats were divided randomly into three groups:AFB1, EGb761+AFB1and normal control groups. AFB1group:25rats were exposed to AFB1by intraperitoneally injection from the fourth week experimental and feeding normal feed in the first week until the end of experiment. AFB1+EGb761group:25rats were exposed to AFB1done as AFB1group and were fed feedstuff containing2g/kg EGb761from the experiment in the first week until the end of experiment. Control groups,20rats were not exposed to AFB1and were fed normal feed in the first week from the beganing until the end of experiment. During hepatocarcinogenesis, liver biopsies were performed on all of animals at13thW,23thW,33thW,43thW,53thW and63thW of the experiment. All survivals animals were sacrificed by cervical dislocation at73rd-week and liver tissues and tumor were collected. The Liver tissue samples were separated into three pieces. Microsomal and cytosolic fractions were prepared from fresh liver tissue and applied to detect the hepatic drug-metabolizing enzymes CYP3A4activity and GSH. One section of liver tissue samples snap-frozen in liquid N2and stored at-80℃for investigated related gene expression of the tumor. Some liver tissues were immerged in10%formalin fixation and paraffin embedded for HE dyeing. The relative quantitative analysis technology Real time-PCR and Western Blotting were used to investigate the genes expression situation in the tumors such as IGF-II, p16Ink4a, P53, NADE and p75NTR etc. The correlation of the protein expression were analysed by statistics.Results1. The data of rats tumor occurrence:The effective numbers of animals and tumorigenesis effective at the end of experiment (73W)as follows:the effective numbers of animals in AFB1group was17, occurred malignant tumor13cases (76.5%), including hepatocellular carcinoma (HCC)10cases, bile duct carcinoma two cases, fibrosarcoma one case, PHC incidence was70.6%(12/17); EGb761+AFB1group was17, occurred malignant tumor6cases (35.3%), including hepatocellular carcinoma5cases, bile duct carcinoma one case, PHC incidence was35.3%(6/17); The control group was16, no tumor was occurrenced. The incidence of PHC in EGb761+AFB1group was significantly lower than that in AFB1group (P<0.01).2. The morphology change of rat liver tissue:The earliest time AFB1-induced rat liver malignant tumors(including fibrosarcoma) formation was at52thW. The first case of Primary hepatic carcinoma was occured in AFB1group rats at the55thW.Microscopically dynamic observation of liver biopsy histopathology shows that AFB1-induced rats liver cancerous process can be divided into three stages: The first stage (13thW-33thW), there were some different degrees of liver cell denaturation appear in rats liver. The second stage (34thW-53thW), the abnormal liver cell hyperplasia and focal hyperplasia nodules, mainly for basophilic nodules were developed. The third stage (54thW-73thW), liver disease become accentuate further, gradually visible carcinoma nodules. In the date using EGb761intervention the liver damages in different stage were lighter than AFB1group at the same stage. The proliferative lesion was also later than AFB1group and the control group was not changes alone.3. Effect of EGb761on hepatic tissues metabolic enzymes3.1The changes of CYP3A4activity in rat hepatic tissuesDuring the whole experimental process, the activity of CYP3A4in AFB1group and EGb761+AFB1group had different levels of changes. The activity of CYP3A4were formed double peaks at23thW and53thW. The activity of CYP3A4in AFB1group and EGb761+AFB1group were higher than that in control group except13thW. The activity of CYP3A4increased significantly during53thW and63thW in AFB1group compared to EGb761+AFB1group (P＜O.05). The activity of CYP3A4in control group was decreased at the33thW and63thW. There are no change significantly in other stage.3.2The changes of GSH conten in rat hepatic tissuesThe content of GSH in rat hepatic tissues is lower in AFB1group and EGb761+AFB1group compared to control group. Along with the progress of the experiment, the content of GSH decreased gradually in AFB1groups and EGb761+AFB1group. The content of GSH decreased significantly during43W and53W in AFB1group compared to EGb761+AFB1group (P＜0.05). The change of GSH in normal control group was not obvious during each period of the whole process of experiment.4. Effect of EGb761on the expression of related cncer genes4.1Effect of EGb761on the expression of p16Ink4a mRNA and corresponding protein in rat hepatic tissuesThe expression of p16Ink4a mRNA is not significantly higher or lower in each group at13thW and33thW(P>0.05), however, in AFB1group and EGb761+AFB1group were significantly lower than that in the control groups during53thW and73thW(P<0.05). The expression of p16Ink4a mRNA in AFB1group was significantly lower than that in the EGb761+AFB1group from53thW to73thW (P＜0.05).The p16Ink4a protein expressions were not significant difference in each group during13thW and33thW(P>0.05), however, in AFB1group and EGb761+AFB1group were significantly lower than that in the control groups during53thW and73thW(P<0.05). The p16Ink4a protein expression in EGb761+AFB1group was significantly higher than that in the AFB1groups (P＜0.05).4.2Effect of EGb761on the expression of P53mRNA in rat hepatic tissuesThe expression of P53mRNA was low expression and no significant difference in each group during13thW. The expression of P53mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control group during33thW,53thW and73thW(P<0.01). The expression of P53mRNA in EGb761+AFB1group was significantly higher than that in the AFB1group (P＜0.01). 4.3Effect of EGb761on the expression of IGF-II mRNA and corresponding protein in rat hepatic tissuesThe expression of IGF-II mRNA was not significant difference in each group during13thW and33thWCP>0.05). The expression of IGF-II mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control groups during53thW and73thW. The expression of IGF-II mRNA in EGb761+AFB1group was significantly lower than that in the AFB1group (P＜0.01).The IGF-II protein expressions were low expression or not expression in each group during13thW and33thW. There was not significant difference in each group during13thW. Along with the progress of the experiment, the IGF-II protein expressions increased gradually. The IGF-II protein expressions in AFB1group and EGb761+AFB1group were significantly higher than that in the control group during53thW and73thW. The IGF-II protein expressions in EGb761+AFB1group was significantly lower than that in AFB1group during53thW and73thW (P<0.05).4.4Effect of EGb761on the expression of NADE mRNA and corresponding protein in rat hepatic tissuesThe expression of NADE mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control group from33thW to73thW and the difference have statistical significance (P<0.01). The expression of NADE mRNA in EGb761+AFB1group was higher than that in the AFB1group. However, there was not statistical significance on the differences between AFB1andEGb761+AFB1group(P>0.05). The NADE protein expression in AFB1group and EGb761+AFB1group were significantly higher than that in the control group from33thW to73thW (P<0.05). The NADE protein expression in EGb761+AFB1group was slight higher than that in the AFB1group, however, there was not statistical significance on the difference among them (P>0.05).4.5Effect of EGb761on the expression of p75NTR mRNA and corresponding protein in rat hepatic tissuesThe expression of p75NTR mRNA was not significantly difference in each group during13thW and33thW(P>0.05). The expression of p75NTR mRNA in AFB1group and EGb761+AFB1group were significantly lower than that in the control group during53thWand73thW (P＜0.05). The expression of p75NTR mRNA in EGb761+AFB1group was higher than that in the AFB1group (P＜0.01).The p75NTR protein expression in AFB1group and EGb761+AFB1group were significantly lower than that in the control group from55thW to73thW(P<0.01). The p75NTR protein expression in AFB1group was significantly lower than that in the EGb761+AFBl group from55thW to73thW (P＜0.05).4.6Effect of EGb761on the expression of COX-2mRNA in rat hepatic tissuesThe expression of COX-2mRNA were low expression in AFB1group and EGb761+AFB1group during13thW and33thW. The expression of COX-2mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control group during53thW and73thW (P<0.01). There was not significantly difference between AFB1group and EGb761+AFB1group(P>0.05). The expression of COX-2mRNA was low or no expression in control group from13thW to73thW.4.7Effect of EGb761on the expression of UGT mRNA in rat hepatic tissuesThe expression of UGT mRNA in AFB1group and EGb761+AFB1group are significantly higher than that in the control group from13thW to73thW and the difference have statistical significance (P<0.01). The expression of UGT mRNA in EGb761+AFB1group are significantly higher than that in the AFB1group during whole of the experimental process and the difference have statistical significance (P<0.01).4.8Effect of EGb761on the expression of AKR7A mRNA in rat hepatic tissuesThe expression of AKR7A mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control group from13thW to73thW and the difference have statistical significance (P＜0.01). The expression of AKR7A mRNA in EGb761+AFB1group are significantly higher than that in the AFB1group(P<0.01).4.9Effect of EGb761on the expression of GST-Pi mRNA in rat hepatic tissuesThe expression of GST-Pi mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control group from13thW to73thW and the difference have statistical significance (P<0.01). The AFB1group are not significantly higher than that in AFB1+EGb761group during33thW and53thW. The expression of GST-Pi mRNA in AFB1group was not only significantly higher than that in the control group, but also higher than those in the EGb761+AFB1group during73thW(P<0.01).5.0Effect of EGb761on the mutual relations of p16Ink4a, IGF-II, NADE and p75NTR protein expressionThe protein expression between p16Ink4a and p75NTR in in AFB1group and EGb761+AFB1group was positive correlation during73thW,55thW and33thW(P <0.01).Conclusions1. EGb761can reduce the incidence of PHC induced by AFB1. It indicated that EGb761has the effects of anti-cancer development and may be used at the chemoprevention for human PHC related with AFB1.2. EGb761can restrain the liver enzymes I phase CYP3A4activity, reduced the AFBO formation, and weaken the carcinogenicity of AFB1.3. EGb761can improve the content of rat liver GSH level, so as to improve the body's anti-oxidation ability, reduced lipid peroxidation damage caused by free radicals in the carcinogenesis induced by AFBl, which is one of the mechanism of EGb761anti-cancer effects.4. In the carcinogenesis AFB1-induced, EGb761can upregulate the expressive level of tumor-suppressor genes such as P16Ink4a, p53and P75NTR, downregulate the expressive level of IGF-II related proliferation which is one of the foundation of molecular biology of anti-cancer effects.5. In order to enhance the detoxify ability against the mycotoxin's for organism itself, EGb761can greatly improve the expressive level of metabolic enzymes related detoxification in the liver tissue such as UGT and AKR7A, during hepatocarcinogenesis aflatoxin B1-induced in Wistar Rats. 6. EGb761can cut the expression level of GST-Pi during hepatocarcinogenesis aflatoxin Bl-induced in Wistar Rats.This may be due to the methylation about promotor of CpG-island.7. There was a positive correlation between the protein expression of p16Ink4a and p75NTR during hepatocarcinogenesis aflatoxin B1-induced in Wistar Rats. This phenomenon was indicated that EGb761can adjust different genes that block the cell cycle, and induced apoptosis of corresponding of tumor cells through the meanwhile, this could be one of its anti-cancer mechanism.