Study On The Safety Of Bear Bile Powder And Its Mechanism Of Preventive Treatment Of Hepatocellular Carcinoma

Liver cancer is the fifth most common cancer and one of the leading causes of cancer death in the world.China’s pet industry is also facing the same challenges.With the improvement of breeding conditions and medical conditions,the lifespan of pets is increasing,and tumor diseases have increasingly become one of the main reasons that threaten the lives of pets.Bear bile powder(BBP)is a natural medicine from animals and is a traditional Chinese medicine approved by the Ministry of Health.Studies have shown that bear bile powder and its main components can inhibit proliferation,promote apoptosis and block cell cycle of human hepatocellular carcinoma cells.Recent studies have also found that BBP has a protective effect on DNA damage.However,their anti-cancer mechanism remains a mystery.Traditional Chinese medicine has the characteristics of small toxic and side effects and slow efficacy.In his important instructions on the work of traditional Chinese medicine in 2019,General Secretary Xi Jinping also pointed out that "give full play to the unique advantages and role of traditional Chinese medicine in disease prevention and treatment,and contribute to building a healthy China and realizing the great rejuvenation of the Chinese nation." Therefore,in order to confirm the preventive and therapeutic effect of BBP and analyze its molecular mechanism,we first evaluated the safety of long-term consumption of BBP,then verified the preventive therapeutic effect of continuous low-dose BBP on liver cancer,and preliminarily analyzed the mechanism.Finally,the protective effect of BBP on DNA damage was confirmed.These results will provide scientific basis for the clinical application of BBP and the development and utilization of anticancer traditional Chinese medicine.The specific experiments are as follows:1.Subchronic toxicity test of bear bile powder by repeated intragastric administration in SD rats for 90 daysIn order to confirm the safety of long-term use of BBP,90 days repeated dose toxicity test of BBP was carried out in SD rats.Six weeks old SPF SD rats were randomly divided into three groups: control group,low-dose BBP group(BBP-L)and high-dose BBP group(BBP-H)according to their body weight,and given 0,40 or 200 mg/kg of BBP for up to 90 days.Clinical signs and general appearances were observed were measured once a week.An autopsy was performed at the end of the experiment.Blood samples were collected from the abdominal aorta under light ether anesthesia after deep anesthesia.At autopsy,weights of liver,spleen,lung,kidney,heart,stomach,intestine,brain,testis(♂)or ovary(♀)were measured.In addition,these organs were fixed in 10% neutral buffered formalin for histopathological examination.During the experiment,there was no significant difference in body weight,food intake and water intake between the BBP group and the control group,neither deaths nor remarkable changes in general appearance were observed in treated groups.In female of the BBP-H group,absolute and relative weights of ovary,the absolute weights of liver and kidney were significantly decreased,as compared to the control group(P < 0.01).In hematological examination,the number of monocytes,the ratio of monocytes and the number of neutrophile granulocyte in male of the BBP-H group were significantly increased,as compared to the Control group(P < 0.05).In addition,significant decrease in the mean corpuscular hemoglobin and a significant increase in the mean corpuscular red blood cell were observed in female of the BBP-H group(P < 0.05).In serum biochemical examinations,a significant increase of the urea in male of the BBP-L group and a significant decrease of the magnesium ion in male of the BBP-L and BBP-H groups were observed.In addition,a significant increase of alkaline phosphatase in female of BBP-H group was observed.Histopathologically,there was no difference between BBP-H group and Control group(P > 0.05).In summary,although some changes were observed in the organ weight,hematology and serum biochemical indexes,but there were no corresponding organ histopathological changes,and they were all within the normal range of rats.These results suggest that there were no toxic changes related to the administration of 200 mg/kg BBP in rats,and the NOAEL(no-observed-adverse effect level)of BBP in SD rats was estimated to be 200 mg/kg or more.2.The prevention therapeutic effect of low-dose bear bile powder on hepatocarcinoma.In order to confirm the prevention therapeutic effect of low-dose BBP on hepatocarcinoma,we established a two-stage carcinogenic model by using Ndiethylnitrosamine(DEN)and N-nitrosomorphine(NMOR).And,we carried out the drug efficacy test of BBP on hepatocarcinoma.Forty-five six weeks old male SD rats were randomly divided into three groups: Control group,DEN+NMOR group and DEN+NMOR+BBP group.Firstly,all animals in the DEN+NMOR+BBP group received a daily intragastric administration of 10 mg/kg BBP for 15 weeks.At the 13 th week of the experiment,all animals in the DEN+NMOR+BBP and DEN+NMOR groups received an intraperitoneal injection of DEN at a dose of 200mg/kg body weight,and given in drinking water containing 80 ppm NMOR for 10 weeks from the 15 th week.At the end of the experiment,rats were euthanized by ether anesthesia,and livers and lung were excised and weighed.As a result,body weight gains were suppressed in the NMOR-treated rats from 13 weeks after intraperitoneal injection of DEN to the end of the experiment.However,there were no significant difference in final body weight,relative and absolute liver and lung weight,and survival did not change between the DEN+NMOR and the DEN+NMOR+BBP groups.In histopathological examination,the incidence of hepatocarcinoma and adenoma did not significantly different between the DEN+NMOR group and DEN+NMOR+BBP group(P > 0.05).However,the number and area of placental glutathione S-transferase placental form(GST-P)positive lesions tended to decrease in the DEN+NMOR+BBP group compared with the DEN+NMOR group.In addition,the Ki67 positive cell ratio in DEN+NMOR+BBP group was significantly decreased than that in DEN+NMOR group(P < 0.01).RT-qPCR analysis showed that BBP treatment significantly increased the mRNA expression of apoptosis-related genes Caspase3 and Caspase9.These results suggest that low-dose BBP treatment has a certain prevention therapeutic effect on hepatocarcinoma induced by DEN+NMOR,and its mechanism is mainly by inducing the activation of apoptosis pathway Caspase-9/3 and inhibiting cell proliferation.Thereby,BBP inhibiting the occurrence and development of preneoplastic lesions on the liver.3.Effect of bear bile powder on the expression of DNA repair genes in different stages of hepatocarcinoma.In the drug efficacy test of BBP pre-treatment on hepatocarcinoma,we found that BBP inhibiting the occurrence and development of preneoplastic lesions on the liver.In addition,in the experiment of BBP treatment effect at promotion stage of hepatocarcinoma,BBP significantly inhibited the preneoplastic lesions on the liver.Accordingly,in order to detect the effect of BBP on the expression of DNA repair genes in different stages of hepatocarcinoma,the expression of DNA repair gene APE1,XRCC1,JWA in liver tissue treated with BBP at different stage of hepatocarcinoma were analyzed by RT-qPCR assay.The results showed that BBP up-regulated the expression of JWA mRNA in the BBP pre-treatment experiment.Especially,the expression of JWA significantly increased compared with the DEN+NMOR and the control groups(P < 0.01).Based on the above results,we also detected the expression of JWA in liver tissue from the experiment of BBP treatment effect at promotion and medium or end stage of hepatocarcinoma.As results,the expression of JWA mRNA in the liver tissue treated by BBP significantly increased compared with model groups.Especially,the expression of JWA mRNA in the experiment of BBP treatment effect at promotion stage also significantly increased compared with control group.Therefore,only pre-treatment or early treatment of BBP can effectively inhibit the occurrence and development of preneoplastic lesions of hepatocarcinoma.These results suggest that BBP enhances the DNA repair ability by up-regulating the expression of environmental response gene JWA,and inhibiting the occurrence of preneoplastic lesions in early hepatocarcinoma.Overall,long-term treatment with low-dose BBP could not only induce toxic effect,but also enhance the ability DNA repair ability by increase of JWA expression.In addition,it can inhibit cell proliferation and induce apoptosis by regulate the expression of apoptosis-related genes Caspase3 and Caspase9.Thereby,BBP reduce liver injury induced by DEN and NMOR,and inhibit the development of hepatocarcinoma.Farther,the high expression of JWA may be the key for BBP inhibit the occurrence and development of early hepatocarcinoma,but the prevention therapeutic cancer mechanism of BBP mediated JWA needs to be further explored.