Effect And Mechanism Of Babaodan On Primary Hepatocellular Carcinoma And Fatty Liver

Hepatic carcinoma is a fatal malignant tumor which causes a large number of deaths.In recent years,its high incidence and high mortality rate has become a serious challenge to China's medical and health undertakings.The recurrence rate of HCC patients was still high after surgical resection.The chemotherapy success rate and overall survival rate of HCC is very low.The urgent problem need to address is to reduce the incidence of hepatic carcinoma and inhibit the development of liver cancer.Many chronic liver diseases can cause chronic liver injury,such as hepatitis B,hepatitis C,chronic alcohol abuse or metabolic syndrome.Chronic liver injury can further develop into a chronic inflammation of the liver.During the development of inflammation,the liver tissue and hepatocytes are necrotic,and the harsh environment caused by inflammation makes the normal liver cells unable to adapt and then die,while the adapted and survived hepatocytes are malignantly transformed into liver cancer cells.What is the source of this part of malignant transformed cells and what causes them to further develop.Hepatitis progenitor cell(HPC)which has self-renewal,proliferative capacity and multiple differentiation ability can be found in the liver.Liver injury caused by disease results in the activation ofHPCs,which participate in the restoration of the damaged liver tissue.HPC connect with hepatic carcinoma and can cause occur of HCC.Some studies have found that HCC has the characteristics of HPCs which express HPC markers,such as CK19 protein.Many studies have also shown that HPC is the origin of CSCs.Because HPCs are long-lived cells that have long enough time to accumulate DNA damage and increase the risk of mutations.Liver injury,liver microenvironment changes,a large number of inflammatory factors in the liver,leading to the process of signal pathway disorders,so that HPC turn into liver cancer stem cells(CSCs),leading to liver cancer.We also found that lipopolysaccharide can inhibit the normal differentiation of HPCs,and can promote the expression of CD133 liver precursor cells.Therefore,HPC is the initiation of hepatocellular carcinoma,the activation of which is closely related to the inflammatory microenvironment.Macrophages(Kupffer cells)are the important inflammatory cells in thehumanbody,which play an important immune function,such as the removal of damaged tissue and pathogens.Studies have suggested that macrophages play a link between tumor progression and inflammation.Macrophages are highly plastic,and their phenotype can change inresponse to a variety of microenvironment.Macrophages can be polarized into M1 phenotype and M2 phenotype.The two differentphenotypes have different biological function.The main function of M2 is tissue remodeling and Th2 immune response.M2 macrophages are not capable of efficient antigen presentation.In liver cancer microenvironment M1 macrophages are involved in tumor proliferation,angiogenesis,ECM remodeling,and help tumor cells invasion and metastasis.Thesurface molecules and biological functions of tumor-associated macrophages(TAM)and M2 macrophages are similar,and all can promote the development of hepatocellular carcinoma.The deeper TAM infiltrated in the tumor,the worse the prognosis of the patient.In addition,studies have shown that T lymphocytes and B lymphocytes can be indirectly through macrophages and other effects on tumor cells,which can promote the occurrence and development of tumors.Therefore,macrophages play an important role in the development of hepatic carcinoma.Toll-like receptors in immune cellsare important immune response receptors,which participate in the regulation of both innate and adaptive immune responses.TLR4 is one of the Toll-like receptor(TLR)and overexpress in HPC cells.TLR4 can specifically respond to LPS.Therefore,inhibition of TLR4 expression may be one of the strategies to inhibit the development of HCC.Obesity is a worldwide epidemic with 2.1 billion people overweight globally.NAFLD(Nonalcoholic fatty liverdisease)starts with aberrant accumulation of triglyceride in the liver,which causes steatosis.Steatosis presented with hepatocyte injury,inflammatory infiltrate,and/or fibrosis is referred to as nonalcoholic steatohepatitis(NASH),which can progress to cirrhosis.Individuals with cirrhosis can ultimately progress to hepatocellular carcinoma.It is suggested that effective treat obesity can significantly reduce the risk of NAFLD to a certain extent.Therefore early control of obesity can prevent the incidence of hepatic carcinoma.Babaodan(BBD)is a kind of Chinese patent medicine,was widely and clinically used in protecting liver,amending the alimentary canal symptoms,relieving pain and enhancing immunity.Our previous studies have shown that Babaodan can inhibit the expression of TLR4 in liver tissue induced by LPS.Babadan may be able to protect the liver by regulating the inflammatory microenvironment and inhibiting the occurrence of liver cancer and fatty liver.In order to evaluate the role and the possible mechanism of Babaoda on hepatic carcinoma and fatty liver,our study would be carry out from the following three parts:Part 1.The role and mechanism ofBabaodan on the occurrence of hepatic carcinomaIn this part,we used DEN-induced rat and LPS-induced WB-F344 cells as in vivo and in vitromodels to investigate the role and mechanism of Babaodan on the occurrence of hepatic carcinoma.The results indicated that Babaodan could prevent or postpone the initiation of hepatocellular carcinoma formation.Babaodan could protect hepatocytes from DEN-injury,inhibited inflammatory infiltration and inflammatory cytokines expression,and ameliorated liver fibrosis.DEN could obviously induced activation of HPCs,while Babaodan ameliorated it by inhibit the expression of the CK-19 and SOX-9.We found that LPS could induce WB-F344 cells neoplastic transformation with high colony-formation,sphere-formation and proliferation,and Babaodan obviously decreased the ability of colony-formation,sphere-formationand proliferation.According to further studies,we found that Baobaodan could inhibit the expression of TLR4 induced by DEN or LPS in the in vivo and in vitro cell experiments,and found that Baobaodan can inhibit the expression of signal molecules MyD88 and TRAF6 downstream of TLR4.The results suggest that Babaodan can inhibit expression of TLR4 and its downstream signaling molecules MyD88 and TRAF6,and relieving the inflammatory response,improve liver function,reduce the secretion of inflammatory factors and activation of HPCs.Thus,Babaodan play a role in inhibiting the initiation of hepatic carcinoma.Part 2.The role and mechanism of Babaodan on the development of hepatic carcinomaIn this part,we still use DEN-induced rat and LPS-induced WB-F344 cells as in vivo and in vitro models,but the intervention time of the babaodan was set up after the tumor formation.We would observethe effect of Babaodan on tumor development.AfterDEN-induced tumor formation in rat,we treated the rats with Babaodan.The result show thatthe number and size of tumor were significantly decreasedand the survival rate of rats was significantly longer than that of non-intervention group.Inflammatory cellsinfiltration of the liver after tumor formation can also be significantly redsuced.We also observed that Babaodan could inhibition of tumor progression.We further study the transformation of macrophages in liver tissue after BBD interventipon,and we found that DEN can cause increased macrophages in the liver,which is consistent with the results of that can cause inflammation,but Babaodan intervention can decline the total amount of macrophages.Then,we observed macrophage differentiation showed that the expression of M2 in tumor tissue was significantly increased,but the expression of M2 was significantly decreased after treatment with Babaodan.Similarly,weobserved LPS induce could promote macrophages to transformationto M2.After treated with Babaodan,the amount of macrophages transformation of M2 was significant decline.We further studied the possible mechanism and found that the expression of TLR4 in macrophages induced by DEN-induced liver tissue,and after treated with Babaodan,the expression of TLR4 in liver tissue was significantly decreased.As the same result,after LPS induction,TLR4 and its downstream signaling molecules MyD88 and TRAF6 in macrophages were also significantly increased.However,TLR4 and its downstream signaling molecules MyD88 and TRAF6 were also significantly decreased after treatment with Babaodan.The above results suggest that we may be use BBD torelief liver tumor progressionthrough inhibiting of TLR4 expression and its downstream signaling molecules MyD88 and TRAF6 and reducing the liver macrophages transformation to M2 type.Part 3.The effect and mechanism of Babaodan on nonalcoholic fatty liverHere we investigated the effect Babaodan on obesity and the mechanism behind the observation.Our results showed that BBD could significantly decrease body weight gain by losing of stored body fat because treatment with Babaodan significantly decreased the fat weight and adipocyte size compared with HFD mice.These results suggest that Babaodan administration suppresses HFD-induced adipose tissue mass and body weight gain and inhibits lipid accumulation.Additionally,the supplement of Babaodan decreased both the plasma levels of TG,TC and liver TG,TC in Babaodan group.The expression levels of IL-6 and TNF-?,both serum level and liver expression,were shown increased in HFD mice.And BBD administration could suppress the expression level of IL-6 and TNF-?.Additionally,the favorable effect of Babaodan also reflected by significantly decreased activities of AST and ALT.Our data suggested Babaodan could improve the lipid metabolism by inhibiting the down regulation of AMPK induced by high fat diet andreducing the synthesis of lipid.In summary,Babaodan supplementation protects against high fat diet induced fat accumulation,hepatic fat accumulation,inflammation and fibrosis by regulating genes involved in hepatic lipid metabolism.Based on the above study,we get the following conclusions:1,Babaodan can inhibit TLR4 and its downstream signaling molecules MyD88 and TRAF6 expression,thereby inhibiting the inflammatory response,improve liver function,reduce inflammatory and HPCs activation,thus inhibiting the initiation of hepatic carcinoma.2,Babaodan inhibited the expression of TLR4 to reduce the transformation of macrophages M2,thereby relief hepatic carcinoma progression.3,Babaodan supplementation protects against high fat diet induced fat accumulation,hepatic fat accumulation,inflammation and fibrosis by regulating genes involved in hepatic lipid metabolism.