| Background and ObjectiveHCC (Hepatocellular Carcinoma) is the sixth most prevalent cancer and the third most frequent cause of cancer-related death. The dominant risk factor of HCC is chronic infection with hepatitis B virus (HBV), together with exposure to aflatoxin B1 in developing countries. In China, about 80% of HCC patients are infected with HBV. It is well known that the death and continuous proliferation of liver cells, the release of massive inflammatory and pro-fibrotic cytokines along with the change of microenvironment caused by the infiltration of inflammatory corpuscles play a significant role in canceration. As hepatitis, cirrhosis process inside the liver, dysplastic nodules with initial chromosomal changes are often observed. These dysplastic nodules can progress to the early stages of HCC. In recent years, due to the anti-viral therapy for HBV and HCV, tremendous progresses have been made in the treatment of clive irrhosis so that the incidence of HCC is reduced to certain extent. Although progresses have been made in HCC research, including improved survival rate, the treatment effect is still not satisfactory compared with other malignancies. Therefore, to finding effective antifibrotic drugs to block the process of hepatitis, cirrhosis, liver cancer is still a hot topic and the focus of domestic and international research.Recent epidemiological studies found that the use of statin drugs can reduce the incidence of liver cancer. A study in diabetic population also found that the use of statins was significantly associated with lower the incidence of hepatocellular carcinoma. According a study in Taiwan China, statin use may reduce liver cancer risk in patients infected with hepatitis B virus. Statins are HMG coenzyme A (HMG-CoA) reductase inhibitors and are widely used as a class of lipid-lowering drugs in clinics. Its mechanism is that, through the competitive inhibition of endogenous cholesterol synthesis rate-limiting enzyme (HMG-CoA reductase), the mevalonate pathways within cells are blocked, resulting in a reduction of the intracellular synthesis of cholesterol, which thereby as a feedback stimulates the liver cell surface such that the Low density lipoprotein (LDL) receptor is enhanced in activity and the clearance of serum cholesterol is raised.It is generally acknowledged that inhibition of 3-hydroxy-3methylglutaryl-coenzyme A reductase by statins interferes with the ratelimiting step of the mevalonate pathway, leading to reduced levels of mevalonate and its downstream products; thus, statins may reduce tumor initiation, growth, and metastasis. In recently years along with the wide use and intensive study on statins, it has been found that statins can improve endothelial functions, reduce inflammatory reactions, and have anticoagulation and antiplatelet effects. The multi-effect of statins provides us with new thoughts on the anti-cancer research.Earlier research reported that the inhibition of HMG-CoA reductase by statins impedes proliferation and protein synthesis of fibroblasts, and reduces profibrotic cytokine expression in extrahepatic tissues. Different groups have previously investigated the effect of statins on hepatic inflammation, fibrosis, and also cirrhosis with portal hypertension. Since the incidence and development of cancer are closely related with microenvironment, we believe statins play a role against liver cancer through its anti-inflammatory and anti-fibrosis effects.In this study, we use the DEN-induced hepatitis-cirrhosis-liver cancer model in rats, and orally administer atorvastatin intervention to study the effect of atorvastatin in this process and find its mechanism. This research could offer a precise theoretical foundation for atorvastatin used as anti-cirrhosis therapy and adjuvant therapy for the prevention and treatment of liver cancer.Methods1. Establish DEN induced hepatitis-cirrhosis-liver cancer model. The incidence of cancer is almost 100%.2. Liver specimens, liver and the body mass ratio, blood tests, HE staining were observed in hepatitis period (4 weeks), cirrhosis period (9 weeks).3. Through 15 weeks of DEN-induced cancer (hepatocellular period) each rat tumor incidence, tumor volume and the number of tumors, liver and body mass ratio, blood chemistry testing and HE staining observation, analysis of atorvastatin DEN induced liver cancer.4. Respectively, the liver tissues of NC, DEN, DEN+Ato three groups (9 weeks) were RNA-seq sequenced by Illumina HiseqTM2000, and pathway analysis followed.5. To detect accumulation of collagen fibers in liver tissues by Sirius red staining to analyze the effect of atorvastatin on DEN-induced liver fibrosis.6. To detect Ki67, TGF-beta, a-SMA protein expression in liver tissues (4 weeks,9 weeks,15 weeks) by immunohistochemistry assay.7. To detect the expression of VEGFa, protein in paraneoplastic tissues and cancer tissues by immunohistochemistry methods.8. To detect the anti-proliferation of atorvastatin on HSC-T6 (Rat hepatic stellate cells line) by MTT assay in vitro.9. To detect pro-apoptotic of atorvastatin on HSC-T6 by flow cytometry in vitro.10. To detect gene expression level of Ki67, TGF-beta, PDGFb, a-SMA in atorvastatin intervene ethanol stimulated HSC-T6 by RT-PCR.11. To detect expression of TGF-beta, PDGFb, a-SMA protein in atorvastatin intervene ethanol stimulated HSC-T6 by Western Blot assay.Results1. This study successfully established DEN induced hepatitis-cirrhosis-liver cancer model in rats.2. Atorvastatin significantly reduced the DEN-induced liver cell edema, fatty degeneration, inflammatory cell infiltration.3. Atorvastatin significantly reduced the quantity and burthen of DEN-induced liver cancer in rats.4. RNA-seq sequencing results show that atorvastatin significantly reduced expression of PCNA, Mki67, PDGFb, TGF-beta, CTGF, a-SMA in liver tissues. Pathway analysis results show TGF-beta signaling pathway plays an important role in the protective effect of atorvastatin in DEN-induced rat cirrhosis model.5. Atorvastatin could continuously inhibit DEN-induced liver extracellular collagen fiber in rats, thereby preventing liver cirrhosis. It is noteworthy that the atorvastatin can reduce tumor capsule.6. Atorvastatin could significantly improve liver function in cirrhotic rats and still decreased serum GGT levels in the liver cancer rats.7. Atorvastatin could inhibit proliferation of liver cells in DEN-induced hepatitis, cirrhotic rat.8. Atorvastatin could inhibit TGF-beta protein expression in liver tissue of DEN induced hepatitis, cirrhotic rat.9. Atorvastatin could inhibit the expression of TGF-beta, ?-SMA in para-carcinoma tissues. There is almost no TGF-beta,?-SMA protein expressions in hepatic tissue of the DEN+Ato (Tumor Free) rat.10. Atorvastatin could significantly reduce the expression of VEGFa in carcinoma paraneoplastic tissue.11. In group DEN+Ato compared with group DEN, the expression of Ki67 protein did not show a significant difference in carcinoma paraneoplastic tissue.12. Atorvastatin could significantly inhibit proliferation of HSC-T6 cell in vitro.13. Atorvastatin could significantly induce apoptosis of HSC-T6 cell in vitro.14. Atorvastatin could significantly inhibit the expression of TGF-beta in rat hepatic stellate cells of ethanol stimulation..ConclusionThis study successfully established DEN induced hepatitis-cirrhosis-liver cancer model in rats. We studied the effect of atorvastatin in various pathological stages in DEN-induced liver cancer by a variety of experimental methods. We found that the protective effect of atorvastatin on liver cells is limited in hepatitis period. Atorvastatin has significant anti-fibrotic effect and reduces the quantity and burthen of DEN-induced liver cancer in rats. The anti-inflammatory and anti-fibrotic effects of atorvastatin are mainly through the inhibition of stellate cell activation and proliferation, to induce apoptosis, which thereby results in sustained suppression of liver tissues TGF-beta level. Atorvastatin reduced tumor development and progression by regulating stromal microenvironment. Meanwhile, atorvastatin may also exert anti-tumor effect by inhibiting the proliferation of liver cells and reducing cancer and paracancerous tissues expression VEGFa inhibit tumor angiogenesis. Meanwhile, atorvastatin may also inhibit hepatocyte proliferation and VEGFa expression in cancer and adjacent tissues. The results provide an experimental basis for the atorvastatin clinical application of anti-fibrotic and the prevention and treatment of liver cancer adjuvant therapy. |
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