Protective Effects Of Total Flavonoids From Litsea Coreana On Alcoholic Fatty Liver In Rats And The Potential Mechanism

Alcohol abuse is one of the main causes of morbidity and mortality throughout theworld. Chronic consumption of alcohol can cause a spectrum of liver abnormalitiesranging from simple fatty liver or steatosis to steatohepatitis, cirrhosis, andhepatocellular carcinoma. Chronic alcohol exposure commonly stimulateshyperlipidemia and hypercholesterolemia in several animal studies, and accumulating oftriglycerides in hepatocytes results in the development of fatty liver (steatosis).Alcoholic fatty liver (AFL) is reversible condition, but it can potentiate the developmentof alcoholic hepatitis and even cirrhosis via increasing oxidant generation, which is oneof the key pathogenic factors and could result in alcoholic liver disease (ALD).Prevention of hepatic steatosis may protect a large number of individuals at risk ofadvanced liver disease.Total flavone of Litsea coteana (TFLC) is an active constituent isolated from thefruit of Litsea coteana (L.), a Chinese herbal medicine, which has been used in civilianas a hypolipidemic drug in southern China for hundreds of years. Evidencesdemonstrated TFLC exhibits biological and pharmacological properties such as anti-inflammation, antioxidation and lipid-lowering activities. However, the detailedmechanisms of TFLC on hepatic steatosis have not been fully understood. In the presentstudy, through a model of alcoholic fatty liver in rats fed alcohol by a physiologicalfeeding way, we focused on the effect of TFLC on alcoholic fatty liver (AFL) rats toevaluate the effects of TFLC on AFL rats in vivo and in vitro, and study the potentialmechanism.The main contents are generalized as follows:1. Protective effects of TFLC on alcoholic fatty liver in ratsA model of alcoholic fatty liver in rats was established by intaking different dosesof alcohol (concentration from5%to40%) over12weeks.The alcohol containingdrinks composition of18%sucrose. Sucrose was also set as control and GSH was usedas positive control. From7weeks, TFLC(100,200,400mg/kg) was administrated byintragastric injection (ig) daily for6weeks, at the end of12weeks, rats were fastedovernight and sacrificed. Body weight was monitored throughout the12weeks protocol.Liver index was obtained by dividing liver weight by rat weight×100. Liver specimenswere taken immediately after the rats were sacrificed. Hepatic specimens were usedH&E staining, Sudan Ⅲstaining, and Electron microscopy for morphological analysis.The concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT)in the serum were measured by commercial analysis kits.Compared with the model group, rats in TFLC groups kept a steady body weightgain during the whole schedule and liver index was improved markedly. Serum levelsof AST, ALT and liver MDA contents were significantly decreased in the TFLC groups,while liver homogenate contents of SOD and GSHpx were significantly increased.Morphological evaluation revealed that rats fed with alcohol for12weeks developed ahigher degree of steatosis in model group, with a mean grade of2, meanwhile, theTFLC groups had much less as well as lower size vacuoles compared with the model group. TFLC has the protective effects on AFL rats; the mechanism may be involved inanti-lipid peroxidative.2. Effects of TFLC on serum levels of glucose, lipids, adipocytokines and insulin inAFL ratsFrom7weeks TFLC was administrated by intragastric injection (ig) daily for6weeks, the concentrations of totalcholesterol (TC), triglyceride (TG), free fatty acid(FFA), glucose, low density lipoprotein (LDL-C), and high density lipoprotein (HDL-C)in the serum were measured according to those commercial analysis kits. Serum levelsof adipocytokines such as leptin and insulin were measured by radioimmunoassay kit,and serum levers of tumor necrosis factor (TNF-α) and apolipoprotein B (APOB) wereexamined by enzyme linked immunosorbent assay method.The results showed that treated with TFLC for6weeks, the levels of TG, TC,LDL-C, and APOB in serum were significantly decreased, while HDL-C levels wereelevated, which indicated that TFLC could efficiently regulate disturbance of lipidmetabolism induced by chronic alcohol consumption. TFLC treatment couldsignificantly decrease serum levels of insulin, TNF-α, leptin, and glucose. Meanwhile,the expressions of TNF-α and Leptin mRNA detected by Reverse TranscriptasePolymerase Chain Reaction (RT-PCR) were suppressed in TFLC-treated groups. TFLCtreatment could improve hyperinsulinemia, hyperleptinemia, and lipid metabolicdisturbance.3. Regulatory effects of TFLC on serum levels of lipid metabolism relatedindicators such as ADRP, PPAR γ and Sir2in AFL ratsLipid metabolism related indicators such as ADRP, PPAR γ and Sir2have animportant relationship with hepatic steatosis, and insulin resistance. ADRP is anintrinsic lipid storage protein found in lipid droplets, and has been recognized to be a reliable and sensitive marker for lipid droplets in alcohol-induced fatty liver.Expression of ADRP, PPAR γ and Sir2in the liver were evaluated by Westernblotting, Immunohistochemistry and RT-PCR, respectively. After6weeks TFLCadministration, protein and mRNA levels of lipid metabolism related indicators in AFLrat livers corrected by β-actin was analysed. Compared with that in AFL model group,the expression of ADRP and PPAR γ were effectively down-regulated in TFLC treatedgroups, but expression of hepatic Sir2protein and hepatic PGC-1α,Sir2mRNA weresignificantly increased.Expression of hepatic ADRP and PPAR γ assessed by immunohistochemistry wereshowed that ADRP expression was significantly increased in model rats, predominantlyin the centrilobular regions of the liver. ADRP, visualized as brown granules in cellmembrane. Meanwhile PPAR γ expression was also significantly increased in modelrats, mainly in cell nucleus and cytolymph. Rats treated with TFLC showed apronounced reduction in the number and degree of ADRP-positive hepatocytes and wassimilar to PPAR γ expression.TFLC has protective effects on AFL rats; the potential mechanism may beassociated with lipid-lowering and regulation lipid metabolism related indicators suchas ADRP, PPAR γ and Sir2.4. In vitro study the effects of TFLC on AFL hepatocytesHepatocytes were isolated from AFL rats by a modified two-step collagenaseperfusion method in situ. Hepatocytes viability, as assessed by trypan blue exclusion,exceeded80%, and hepatocytes were identified using Cytokeratin18antibodies stainingwith SP. Hepatocytes were cultured in DMEM medium containing10％fetal bovineserum. And then were inoculated in6-well culture plates. Cell viability and proliferationfunction were detected by MTT assay which relies on mitochondrial metabolic capacityof viable cells. Hepatocytes were divided into normal hepatocytes (NC), AFL hepatocytes (model, M), AFL hepatocytes with TFLC(1,10,100mg/L), AFLhepatocytes with GSH or Nic, respectively. After cultured16h, TFLC or GSH or Nicwas added into cultured supernatant. Immunohistochemistry and RT-PCR were used todetermine protein and mRNA expression of ADRPand PPAR γ, Western Blotting wasadopted to detect protein expression of Sir2. After administrated TFLC, proliferationfunction of hepatocytes was improved in TFLC treated group. RT-PCR showedexpressions of ADRP and PPAR γ mRNA were significantly decreased, and expressionof hepatic ADRP and PPAR γ assessed by immunohistochemistry shown that thenumber and color degree of ADRP and PPAR γ-positive cell expression weresignificantly decreased in TFLC treated group, meanwhile expression of Sir2proteinand mRNA were markedly increased.Taken as a whole, the results above demonstrate that TFLC has protective effectson AFL rats both in vivo and in vitro, the potential mechanism may be involved in itsproperties such as anti-inflammatory, anti-lipid peroxidative, modification lipidmetabolic disturbance, regulation lipid metabolism related indicators such as ADRP,PPAR γ and Sir2. TFLC might be beneficial for the prevention of excessive lipidaccumulation such as AFL.