Preliminary Discussion Of Its Mechanism And Effects Of Bear Bile Powder On Different Stages Hepatocarcinogenesis In SD Rats

The causes of liver cancer are complex and diverse.Although some progress has been made in the early diagnosis and treatment of liver cancer,the mortality rate of liver cancer is still high.Bear bile power(BBP)is a traditional Chinese medicine approved by the Ministry of health in China,which has rich pharmacological effects and remarkable effects.BBP can inhibit the development of liver cancer through inhibit cell proliferation and induce the apoptosis of cancer cells.However,further experiments are necessary to elucidate the suppression mechanism and to confirm the effects in in vivo.In order to confirm the modification effect of BBP on different stages hepatocarcinogenesis in SD rats,we established early and advanced stage hepatocarcinoma models by two-stage carcinogenesis method,and carried out the anticancer efficacy tests of BBP.In addition,we screened the effective methods for establishing advanced liver cancer models.The details are as follows:1.Study on the effect and mechanism of bear bile powder on early liver cancer in ratsTo clarify the modification effects of the BBP on different periods of hepatocarcinoma formation in SD rats,total of 40 male rats were divided into the following 4 groups,each comprising 10 animals:DEN-alone,DEN+PBO,DEN+PBO+BBP-L,and DEN+PBO+BBP-H,randomly.All animals received an intraperitoneal(i.p.)injection of DEN at a dose of 200 mg/kg body weight and all animals in the DEN+PBO,DEN+PBO+BBP-L,and DEN+PBO+BBP-H groups were fed a diet containing 0.5%PBO for 8 weeks,starting 2 weeks after DEN initiation.To enhance the hepatocellular proliferation,all animals were subjected to two-thirds partial hepatectomy after 1-week of PBO administration.Other,to investigate the suppressive effects of BBP on early hepatocarcinogenesis in rats,animals of DEN+PBO+BBP-L and DEN+PBO+BBP-H groups were received a daily intragastric administration of 200 or 400 mg/kg BBP for 8 weeks with PBO treatment.As results,final body weight did not change between the DEN-alone and DEN+PBO groups.Regarding the liver weights,the absolute and relative liver weights in PBO-promoted groups were significantly increased compared with those of the DEN-alone group.However,no significant difference was observed between the DEN+PBO and each of the BBP-treated groups.The number and area of GST-P~+foci in the DEN+PBO group significantly increased compared with those in the DEN-alone group.In contrast,number and area of GST-P~+foci tended to decrease in the DEN+PBO+BBP-L group compared with the DEN+PBO group.In addition,the Ki67 positive cell ratio obviously increased by DEN and PBO treatment was significantly decreased in the BBP-treated groups.Inreal-time RT-PCR analyasis,expression levels of Ccne1 and Cdkn1b in the DEN+PBO+BBP-H group were significantly increased compared to the DEN+PBO group.Therefore,mRNA expression levels of casp8 and casp9 in the DEN+PBO+BBP-H group was significantly higher than those of the DEN+PBO group.These results suggest that the low dose of BBP in the early period of hepatocarcinoma caused weak suppression of hepatocarcinogenesis by the mechanism involving facilitation of cell cycle suppression causing G1/S arrest and apoptosis via the mitochondrial pathway.2.Screening for establishing methods of advanced liver cancer model in SD ratIn order to develop animal models with short experimental period,low mortality and low modeling cost,which are suitable for various cancer research,our research group has optimized and improved the existing modeling methods based on the DEN+PH method of Ito,et al.In this part,we compared the methods of liver cancer model established by DEN combined with N-nitrosomorpholine(NMOR)in the early stage of the laboratory research group,and explored and screened a new method suitable for the efficacy test of middle and late stage liver cancer.In the first scheme,the model of early stage liver cancer with variant cell focus can be established within20 weeks with 200 mg/kg DEN combined with 40 ppm NMOR,but the model of middle and late stage liver cancer with adenoma or cancer can not be established,and the survival rate is 100%.The second scheme,200 mg/kg DEN combined with 40 or80ppm NMOR intermittent drinking water for 10 weeks and feeding for 17 weeks,can establish 35%of the animal model of adenoma and cancer in the middle and late stage of liver cancer.Third,200mg/kg DEN combined with 0.8mg/kg NMOR was administered continuously for 17 weeks and feeding for 24 weeks.Partial hepatectomy was performed to establish a liver adenoma with an incidence rate of100%,a final survival rate of 55%,and a lung metastasis rate of 55%.It can be seen that the first scheme is not suitable for the establishment of middle and late stage liver cancer.The third scheme,although the formation rate of middle and late stage liver cancer is higher,but the later mortality is higher,which will affect the later treatment time of BBP.In the second scheme,although the formation rate of middle and late stage liver cancer is lower than that of the third scheme,the experimental cycle is shorter and the late survival rate is higher,which can win time for BBP treatment,and is suitable for the efficacy test of middle and late stage liver cancer.3.The effect and mechanism of bear bile powder on middle and late stage hepatocarcinomaA total of 46 male rats were randomly divided into 3 groups:Cont(n=6);DEN+NMOR(n=20);DEN+NMOR+BBP-L(n=20).All animals received an intraperitoneal(i.p.)injection of DEN at a dose of 200 mg/kg body weight,and all animals in the group of DEN+NMOR and DEN+NMOR+BBP-L were given drinking water containing 80 ppm NMOR for 7 weeks.During the experiment,the dose of NMOR(40 ppm)was reduced in the 3 to 4 weeks and NMOR stopped in the second week.To investigate the suppressive effects of BBP on late period of hepatocarcinoma formation in rats,animals of DEN+PBO+BBP-L group were received a daily intragastric administration of 200 mg/kg BBP for 7 weeks after DEN and NMOR treatment.Dosage of BBP is the dose that significantly inhibits preneoplastic lesions in ExperimentⅠ.As results,final body weights in the NMOR-promoted groups were significantly decreased compared to the control group.In addition,the relative liver weights in NMOR-promoted groups were significantly increased compared with those of the control group.However,no significant difference was observed between the DEN+NMOR and DEN+NMOR+BBP-L groups.The absolute weight of lung in the DEN+NMOR was significantly reduced in the control group,but the absolute and relative weight of lung in the DEN+NMOR+BBP-L group was significantly increased compared with those in the DEN+NMOR group.In histopathological examination,the incidence of altered hepatocellular foci or tumor and the number and area of GST-P~+foci in the liver were significantly increased in the NMOR-treated groups compared with the control group,but there was no change between the DEN+NMOR and DEN+NMOR+BBP-L groups.However,the Ki67~+cell ratio in the DEN+NMOR+BBP-L group were significantly decreased compared with the DEN+NMOR group.In the TUNEL assay,no significant differences were found between each group.In real-time RT-PCR analysis,there was no significant change in the expression level of all genes between each group.In conclusion,the overall data suggested that low dose of BBP can inhibits the development of preneoplastic lesion,and this suppression effect are only in early period of hepatocarcinoma formation,but not in late period.In addition,treatment with BBP during the late period of hepatocarcinoma formation can inhibit cell proliferation but cannot suppress the development of hepatocarcinoma.Further studies are necessary to clarify the potential suppression mechanism of BBP in early period of hepatocarcinoma formation.Moreover,it is also necessary to clarify the certain toxicity of low dose BBP which can inhibit preneoplastic lesion.