Therapeutic Effects And Mechanism Of Calculus Bovis Sativus On Fructose-Induced Non-Alcoholic Fatty Liver Disease

Part Ⅰ: Establishment and evaluation of a mice model for non-alcoholic fatty liver disease induced using fructoseObjective: To establish a mice model of non-alcoholic fatty liver disease(NAFLD)by fructose and investigate its features.Methods: NAFLD model was established by continuous feeding C57BL/6 mice with 30%(w/v)fructose solution for eight weeks,while normal control mice were fed with distilled water.The body weight of mice was recorded;liver weight was measured and liver index was calculated after animals were sacrificed at the 2nd,4th,6th and 8th week.The indices of lipid metabolism in the serum were detected by corresponding kits.Homeostasis model assessment-insulin resistance index(HOMA-IR)was obtained by measuring the serum levels of glucose and insulin.The histopathology of livers was analyzed by oil red staining and HE staining.Results: Compared with normal control group,30%(w/v)fructose made the body weight,liver weight and liver index increase obviously(P<0.05).The serum level of triglyceride,total cholesterol,low density lipoprotein and free fatty acid significantly was raised,while that of high density lipoprotein markedly decreased(P<0.05).HOMA-IR was elevated gradually in model mice and the value is 5.8 fold of normal control at the 8th week(P<0.05).The histopathological results displayed small lipid droplets were observed in liver of model mice at the 2nd week,and diffused macrovesicular lipid droplets and fat infiltration were seriously developed as the disease progressed.Conclusion: 30% fructose can establish the mice model of NAFLD with the pathological feature,biochemical indices similar to the disease of human,which can be used for the study of pathogenesis and therapy of fructose-induced NAFLD.Part Ⅱ: Protective effects and mechanism of Calculus Bovis Sativus on fructose-induced NAFLD in miceObjective: To preliminary evaluate the protective effects of Calculus Bovis Sativus(CBS)on fructose-induced non-alcoholic fatty liver disease mice.And investigate the pharmacological mechanism of CBS on fructose-induced NAFLD via lipid metabolism,oxidative stress,and cell apoptosis.Methods: The NAFLD model was established in C57BL/6 mice by exclusively feeding fluids that contain 30% fructose for eight consecutive weeks.After these eight weeks,mice were given CBS(50 mg/kg/day or 100 mg/kg/day)or GW4064(30 mg/kg/day)for two consecutive weeks.The liver index was calculated by measuring the body and liver weight of mice.The insulin sensitivity of mice was evaluated by measuring fasting blood-glucose and fasting insulin in mice.The OGTT test was used to evaluate the function of islet β-cells and the mice’s ability to regulate blood glucose.HE staining and oil red O staining of mice liver were performed to observe liver microstructural changes and lipid accumulation.The liver levels of TG,TC,FFA were determine.Moreover,the content of TG,TC,LDL-C and HDL-C in serum was also measured.Oxidative stress indicators such as reactive oxygen species(ROS),superoxide dismutase(SOD)and malondialdehyde(MDA)were detected in liver tissues.Western blot was used to detect the protein expression of FAS,HSL,nuclear Nrf2,NF-κB,CASPASE-3,BAX and BCL-2.The liver of the mice was stained with Tunel to detect the apoptosis of hepatocytes.Results: Calculus Bovis Sativus significantly alleviate the weight gain caused by high fructose diet and reduce liver index.CBS can effectively reduce serum fasting blood-glucose,fasting insulin,TG and TC levels in NAFLD mice,and increase HDL-C levels.CBS treatment also significantly decreased levels of TG,TC,and FFA in the liver.It can be seen from the results of HE staining and oil red O staining that CBS alleviates liver structural damage and lipid accumulation caused by fructose.CBS effectively reduce the protein expression of FAS,thereby reducing hepatic de novo lipogenesis.In terms of antioxidants,the drug enhances SOD enzyme activity in the liver,reduces ROS and MDA levels,and decreases nuclear receptor Nrf2 protein expression.Tunel staining showed that CBS significantly reduced the proportion of hepatocyte apoptosis.The protein expressions NF-κB,CASPASE-3,and BAX were significantly decreased after CBS treatment.Conclusion: CBS treatment exerted therapeutic effects in the liver of mice with NAFLD,which may be associated with amelioration of metabolic disorders,enhanced antioxidant effects,and alleviation of apoptosis.Part Ⅲ: Therapeutic effects of CBS on NAFLD in vitro and related mechanismObjective: To establish a method for fructose-induced L02 cell steatosis model and evaluate the lipid synthesis of this model.Based on this model and serum pharmacology,we further investigatd the molecular mechanism of CBS alleviating lipid accumulation in vitro.Methods: L02 cells were cultured and treated with different concentrations of fructose for 24 h to induce cell steatosis.Cck-8 method was used to detect the effects of fructose on cell viability,and the changes of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the medium were determined to determine the damage of fructose on hepatocytes.Oil red O staining was used to observe the intracellular lipid droplet deposition,and the intracellular TG content was determined to determine the optimal modeling concentration.At the same time,the lipid metabolism-related proteins expression were detected to confirm the optimal modeling concentration.CBS-containing serum of mice was obtained by serum pharmacology and its effect on the proliferation of L02 hepatocytes was evaluated.The lipid reducing effects of CBS-containing serum through Nrf2 was evaluated by fructose-induced L02 hepatocyte steatosis model,Nrf2 agonist oltipraz combined intervention,cell oil red O staining and intracellular TG content.The effects of CBS-containing serum on lipid peroxidation and hepatocytes apoptosis were evaluated ROS and apoptosis assay,respectively.Real-time quantitative PCR was used to detect the relative expression of lipid synthesis-related genes and apoptosis-related genes.Results: When the fructose concentration was 4 mmol/L,a large amount of lipid droplets were formed in the L02 hepatocytes.And the intracellular TG content increased significantly,which was 1.5 times that of the normal control.4 mmol/L fructose significantly up-regulated the protein expression of carbohydrate responsive element binding protein(Ch REBP),sterol regulatory element binding protein-1c(SREBP-1c)and acetyl-Co A carboxylase 1(ACC1).CBS drug-containing serum do not have significant effect on L02 hepatocyte proliferation.Compared with the model group,CBS-containing serum can effectively reduce the formation of lipid droplets in fructose-induced L02 hepatocytes,significantly reduce intracellular TG and ROS levels,and significantly reduce hepatocyte apoptosis rate(P<0.05).Compared to the model group,Ch REBP,SREBP-1c,fatty acid synthase(FAS),ACC1,stearoyl-Co A desaturase 1(SCD1),BAX and CASPASE-3 m RNA were significantly reduced in CBS drugcontaining serum treatment group(P<0.05).All of the above effects can be reversed by oltipraz.Conclusion: The fatty degeneration of L02 cells can be successfully induced by 4 mmol/L fructose.This model is suitable for the de novo lipogenesis induced by high fructose diet in non-alcoholic fatty liver disease.CBS-containing serum can significantly inhibit the fructose-induced L02 liver fat deposition.The mechanism may related to the reducing of intracellular ROS level through the Nrf2 pathway and the improving of intracellular peroxidation state,thus reducing apoptosis.Part Ⅳ: Effect of CBS on liver bile acid profiling in NAFLD miceObjective: To investigate liver bile acid profiling in NAFLD mice model and the effect of CBS on NAFLD mice.Methods: NAFLD mice model was constructed according the method in the second chapter.After modeling,mice were orally treated with 50 mg/kg and 100 mg/kg CBS or GW4064(30 mg/kg/day).Mice livers were collected 24 h after dosing on the last day,and the profile of bile acid metabolism in mouse livers was analyzed using targeted metabolomics.Results: Liver bile acid composition and content in NAFLD mice were significantly changed compared to control mice.Concentration of CDCA,DCA,and GCDCA were increased(P<0.05),TMCA,TLCA,and TDCA were decreased(P<0.05)in NAFLD mice compared to control mice.Compared with model control,concentration of CDCA,DCA,GCDCA were decreased(P<0.05)and MCA,TMCA,TCA,GCA were increased(P<0.05)in CBS groups.Conclusion: CBS can alleviate the dysregulation of bile acid in NAFLD mice liver.This may be due to the fact that CBS can protect liver cells by increasing the combination of bile acid and converting the liver bile acid.