Bicyclic Alcohol Research, Experimental Non-alcoholic Fatty Liver Protective Effect And Mechanism

Nonalcoholic Fatty Liver Disease (NAFLD) is a syndrome with liver histopathologic findings, which resembles alcoholic hepatitis in patients who lack a history of significant alcohol comsumption (<140 g ethanol/week). The spectrum of histologic abnormalities of NAFLD ranges over simple steatosis, nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and even hepatocellular carcinoma or liver failure. The prevalence of NAFLD in the general population is estimated to be 14% and 24% and has markedly increased recently. To date, the only effective treatment of NAFLD is caloric restriction, which is difficult to achieve for most NAFLD patients, it is of great necessity to find the effective treatment of NAFLD, including drug therapy.Bicyclol is a novel anti-hepatitis drug for the treatment of chronic viral hepatitis. It has been shown to improve the clinical symptoms and inhibit virus replication in HBV patients, featured by low rebounding rate, few adverse effects and convenient usage. Previous pharmacological studies indicated that bicyclol had hepatoprotective action against acute and chronic experimental liver injury caused by certain toxins and alcohol. The hepatoprotective mechanisms of bicyclol were related with the clearance of reactive oxygen species, regulation of mitochondrial dysfunction, etc.Previous study in our laboratory has also demostrated its role in protecting experimental animals from steatosis (induced by high fat diet, tetracycline, carbon tetrachloride and ethionine), however, in-depth investigation on related mechanisms are still needed. Therefore, acute and chronic experimental steatosis models were established in the present study to observe the protective effect of bicyclol and related mechanisms, so as to provide experimental evidences for its clinical application.Part I:Protective effect of bicyclol on tetracycline-induced fatty liver in mice and related mechanismsSingle dose of tetracycline injection caused hepatic steatosis as evidenced by the elevation of serum ALT and AST, accumulation of hepatic TG and CHO, and morphologic changes (small lipid droplets and hydropic degeneration of hepatocytes), which reflected early alterations of NAFLD in mice. Bicyclol treatment significantly protected against tetracycline-induced fatty liver by reducing elevated serum ALT&AST levels and alleviating hepatic lipid accumulation biochemically and pathologically.The oxidation of fatty acids occurs in three subcellular organelles. Mitochondria and peroxisome degrade fatty acid byβ-oxidation, while smooth endoplasmic reticulum metabolizes fatty acid viaω-oxidation by CYP2E1 and CYP4A subfamily. The mitochondrialβ-oxidation was found to be inhibited by 34% after tetracycline injection. In addition, tetracycline also reduced the activities of enzymes responsible for fatty acid oxidation in peroxisome (AOX) and microsome (CYP2E1, CYP4A) were also inhibited by 40%,58% and 30%, respectively in mice. Furthermore, mRNA levels of LCAD, CYP4A10 and CYP4A12 also decreased. Administration with bicyclol ameliorated the decrease of fatty acid oxidation at both enzyme activity and mRNA levels.Peroxisome proliferators-activated receptor a (PPARa) is a kind of transcription factor regulating a serial of enzymes responsible for fatty acid oxidation. The mRNA expression of hepatic PPARa was inhibited after tetracycline injection. Bicyclol (300mg/kg) showed protective effect against tetracycline induced decrease of PPARa mRNA expression.Mitochondrial dysfunction, particularly mitochondrial respiratory chain (MRC) deficiency, plays a key role in the physiopathology of NAFLD. Hepatic MRC I andⅣwere significantly inhibited by 42% and 31% respectively after tetracycline injection. Pretreatment of bicyclol (300mg/kg) reversed the hepatic MRC complexI&IV activity to normal levels. Besides, the isolated mitochondria had a rapid onset of MPT (membrane permeability transition, MPT) by decreased uptake of cationic dye Rodamine 123 and lowered sensitivity to Ca2+after tetracycline injection. Bicyclol remarkably improved tetracycline-induced alteration mentioned above. ATPase is a kind of marker reflecting the condition of mitochondrial energy metabolism. Pretreatment with bicyclol (300mg/kg) significantly reversed the injured activity of ATPase to normal level.Mitochondrial dysfunction accelerates the production of ROS and induced hepatic oxidative injury. Oxidative stress was another pathogenic factor in initiating and maintaining of NASH. Hepatic MDA content was elevated 24h after tetracycline injection. GSH content and SOD activity were also found increased compensably. Pretreatment with bicyclol (300mg/kg) significantly inhibited the elevation of MDA, decreased GSH content and SOD activity.Sterol regulatory element-binding protein (SREBP)-lc is a key transcription factor regulating lipid synthesis. No obvious alteration of mRNA expression of SREBP-1c and its target genes SCD, FAS was observed after tetracycline injection.In conclusion, obvious hepatic steatosis was observed after a single dose of tetracycline in mice. The mechanism was found to be partly due to the inhibition of PPARa and its regulated genes, and mitochondrial dysfunction. Bicyclol showed significant protective effect on hepatic steatosis induced by tetracycline through the modulation of PPARa pathway and protection of mitochondrial injury.Part II:Effect of bicyclol on high fat diet (HFD) induced fatty liver in rat and related mechanismsAfter 4 week feeding with HFD, rat had severe fatty liver as evidenced by hepatic TG and CHO accumulation, abnormal serum lipids and pathological changes (including microvesicualr steatosis, hepatocyte hydropic degeneration and inflammatory cell infiltration). Oral administration of bicyclol (150,300mg/kg/day×2 to 4 weeks) could significantly inhibit the increase of hepatic lipid contents, regulate the decreasing of serum TG level, and alleviated hepatic pathological damage mentioned above.SREBP-lc is an important transcription factor that regulating fatty acid and TG synthesis. After feeding with HFD for 4 weeks, both precursor and matured protein of SREBP-1c were increased to 3.5 and 2 fold of control respectively. There was no alteration in mRNA expression of SREBP-lc. Pretreatment and treatment with bicyclol (150,300mg/kg) significantly reversed the elevation of precursor and matured SREBP-1c protein to normal level.Acetyl-CoA carboxylase (ACC), fatty acid synthesis (FAS) and stearoyl-CoA desaturase (SCD) are key enzymes responsible for fatty acid synthesis and are regulated by SREBP-lc. The mRNA expression of hepatic ACC, FAS and SCD were elevated significantly after feeding with HFD for 4 weeks. The protein of ACC and FAS were also increased to 1.84 and 3.1 fold of control. Bicyclol showed protective effect on the indexes mentioned above to different extent.Insulin induced genes (Insigs) are important factors participating in the activation process of SREBP-1c. There expression could result in the detention of SREBP-SCAP (SREBP cleavage-activating protein, SCAP) in Endoplasmic Reticulum (ER). The mRNA expression of rat hepatic Insig-1 was reduced significantly after feeding with HFD for 4 weeks, whereas Insig-2a mRNA expression showed no alteration. Pretreatment and treatment with bicyclol showed no effect on mRNA expression of Insig-1 and Insig-2a.In summary, bicyclol had significant protective effect on HFD-induced fatty liver. Its mechanisms were related to the inhibition on SREBP-lc expression in protein level, thereby suppressing the transcription of enzymes responsible for fatty acid synthesis.In conclusion, bicyclol had a notable protective effect on tetracycline-induced acute fatty liver in mice and HFD-induced chronic fatty liver in rat. It can not only significantly inhibit the elevation of serum transaminase and the accumulation of hepatic triglyceride and cholesterol, but also improve pathological changes, including microvesicualr steatosis, hepatocyte hydropic degeneration and inflammatory cell. The possible mechanisms included:1) Inhibition of oxidative stress:attenuate lipid peroxidation, decrease the conpensable elevation of GSH content and SOD activity.2) Attenuation on mitochondrial dysfunction:maintain mitochondrial membrane integrity and normal membrane potential, restore mitochondrial ATPase activity, increase MRC I and MRC IV activity.3) Regulation on the expression of PPARa and its target genes:inhibit the decrease expression of PPARa, increase ACD, AOX, CYP2E1 and CYP4A at mRNA and enzyme activity level.4) Inhibition SREBP-lc regulatory pathway:inhibit the precursor and matured protein of SREBP-1c, reduce the mRNA and protein expression of SREBP-lc target genes responsible for fatty acid synthesis, including ACC, FAS and SCD.With the results mentioned above, the present study will provide valuable experimental evidences for further investigation on the hepatoprotective effect of bicyclol and the possibility of clinical application in the treatment of NAFLD.