Effects And Mechanism Of Astragalus Injection On Different Stages Hepatocarcinogenesis In Sd Rats

With the rapid development of China’s pet industry,neoplastic diseases have become one of the main reasons that threaten the lives of pets.As a frequently-occurring disease,tumor has a rising incidence.60%of the carcinogens that humans and animals are exposed to in their daily life can cause liver cancer.Most carcinogens play carcinogenicity by inducing DNA damage and promoting the proliferation of tumor cells.Therefore,inhibiting cancer cell proliferation is the focus of cancer treatment.Based on traditional Chinese medicine,astragalus has the effect of tonifying Qi and lifting Yang,strengthening the exterior and reducing sweat,as well as promoting the secretion of saliva or body fluids.Astragalus comprises flavonoids,saponins,polysaccharides,various amino acids and trace elements.Previous studies have shown that astragalus increases telomerase activity and has antioxidant,anti-inflammatory,immune-regulatory,anticancer,hypolipidaemic,antihyperglycaemic,hepatoprotective,expectorant,and diuretic effects.Astragalus injection（AI）is purified from the 18 active ingredients extracted from astragalus,and mainly used in patients with myocarditis,cardiac insufficiency,hepatitis and other diseases.The previous study has shown that astragalus and its main components inhibit cell proliferation and induce apoptosis in various cancer cells.However,whether astragalus injection has the same effects on liver cancer in vivo as not been confirmed.Especially the mechanism of action on the proliferation activity of hepatocarcinogenesis has not been systematically studied.In this study,we established early and medium-term stage hepatocarcinoma models using the genotoxic carcinogen N-diethylnitrosamine（DEN）as an initiator,piperonyl butoxide（PBO）or N-nitrosomorpholine（NMOR）as a promoter,and conducted the anticancer efficacy test of astragalus injection.First,we confirmed the inhibitory effect of astragalus injection on different stages hepatocarcinogenesis.Secondly,from the two aspects of cell cycle and apoptosis-related signaling pathway,it is detected the effects of astragalus injection to the proliferation of hepatocytes on mRNA and protein levels,and analyzed its anti-cancer molecular mechanism.The specific experimental results are as follows:1.Effects and mechanism of astragalus injection on different stages of early hepatocarcinogenesis in a two-stage hepatocarcinogenesis model using rats.To clarify the suppressive effects of astragalus injection（AI）on different stages of early hepatocarcinogenesis induced by weak promotion,SD rats initiated with a single intraperitoneal（i.p.）injection of N-diethylnitrosamine（DEN）at 200 mg/kg body weight and promoted with 0.5%piperonyl butoxide（PBO）in diet were repeatedly administered AI at 5 mL/kg body weight/day in the early postinitiation（EPI）or late postinitiation（LPI）period for 2 or 8 weeks,respectively.The number and area of glutathione S-transferase placental form（GST-P）-immunoreactive^（+）foci tended to increase in DEN+PBO group compared to DEN-alone group.Among PBO-promoted groups,number and area of GST-P⁺foci did not apparently change in DEN+PBO+AI-EPI group compared to DEN+PBO group.In contrast,number and area of GST-P⁺foci tended to decrease in DEN+PBO+AI-LPI group compared to DEN+PBO group.Number of Ki67⁺cells was increased in DEN+PBO group compared to DEN-alone group,and was decreased in both AI-administered groups compared to DEN+PBO group.Gene expression analysis revealed that DEN+PBO+AI-LPI group increased transcript level of Ccne1,Cdkn1b,Rb1,Bax,Bcl2,Casp3 and Casp9 compared to DEN+PBO group;however,DEN+PBO+AI-EPI group did not change the transcript level of any genes examined compared to DEN+PBO.These results suggest that AI administration during LPI period caused a weak suppression of hepatocarcinogenesis under a weak promotion with low PBO dose by the mechanism involving facilitation of cell cycle suppression causing G1/S arrest and apoptosis through mitochondrial pathway.In addition,AI administration during EPI period has no effect on weakly promoted hepatocarcinogenesis.2.Therapeutic effect and mechanism of astragalus injection on medium-term and end stage hepatocarcinoma in a two-stage hepatocarcinogenesis model using rats.The medium-term and end stage hepatocarcinoma model of SD rats was established using DEN and NMOR,and the modeled animals were divided into DEN+NMOR group（the model group）and DEN+NMOR+AI group（the administration group）.In addition,a control group that was not treated was established.At the beginning of the experiment,all rats in the model group and the administration group were intraperitoneally injected with 200 mg/kg DEN and then intragastrically administered with 80 ppm NMOR for 17weeks.Rats in the administration group were intraperitoneally injected with 5 mL/kg/day of astragalus injection for 6 weeks from the 18^th week after treatment NMOR,and the model group was not treated.At the end of the experiment,all living animals were sacrificed and grossly dissected to weigh the heart,liver,lungs,spleen,and kidneys.The experimental results showed that the survival rates of the model group and the administration group at the end of the experiment were 55%and 73%,respectively.Both the body weight of the model group and administration group were significantly lower than that of the control group,and the body weight of the administration was significantly lower than that the model group.Both the relative lung weights of the model group and the administration group were significantly increased as compared with the control group,and the absolute kidney weights and absolute heart weights were significantly decreased.The relative lung weights of the administration group were significantly increased compared to the model group,and the absolute weights of the kidney and heart was significantly decreased.Pathological examination showed that the incidence of hepatocarcinoma was 91%in both the model group and the administration group,and the incidence of hepatocellular adenoma was 63%and 54%,respectively,but there was no statistical difference.The metastasis of hepatocarcinoma was mainly occurred in the lungs,and there was no significant difference in rate of metastatic between the two groups.In addition,in the histopathological examination of the two groups,it was also found pathological changes such as hepatic bile duct hyperplasia,lung adenoma,extramedullary hematopoiesis of spleen.The results of immunohistological examination showed that the Ki67⁺cell rate in the administration group was significantly decreased than that in the model group.The number and area of GST-P⁺lesions and the positive cell rate of apoptosis staining were not significantly different between the model group and the administeration group.The results of gene expression analysis showed that the mRNA expression levels of Casp3,Rb1,p21 and Cdkn1b which were significantly increased in the administration group as compared with the model.These results indicate that astragalus injection can prolong the lifespan of rats by inducing cell cycle arrest and promoting the activation of apoptotic signaling pathways,but it cannot inhibit the metastasis of hepatocarcinoma.In summary,although astragalus injection can’t effectively inhibit the metastasis of hepatocarcinoma,it has a certain degree of inhibition of cell proliferation activity on hepatocarcinoma at different stages,and this inhibition contributes to the anti-cancer effect of astragalus injection.Especially in the promotion period of early hepatocarcinogenesis is more obvious.In addition,the action mechanism of astragalus injection to inhibit cell proliferation is attributed to cell cycle arrest and regulation of apoptotic signaling pathways.Specifically,astragalus injection induces apoptosis through G1/S phase arrest and caspase-9/3 pathway activation,thereby inhibiting the development of hepatocarcinoma.In this experiment,in order to simulate the clinical situation,a heptocarcinogenesis animal model was established using carcinogen induction method,and carry out an anti-cancer experiment.These results not only provide an important scientific basis for cancer treatment of animals and humans,but also provide an important theoretical basis for the research and development of anticancer mechanisms of natural anticancer drugs.