貂皮樟总黄酮对酒精性脂肪肝大鼠防治作用及部分机制研究
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摘要
酒精性肝病（alcoholic liver disease，ALD）是因长期、大量饮用乙醇所致肝脏的损害性病变，在组织病理学上主要表现为三种形式：酒精性脂肪肝、酒精性肝炎和肝纤维化，这三种形式可单独或混和存在。酒精性脂肪肝（Alcoholic fattyliver，AFL）是ALD早期阶段，进一步发展为肝纤维化和肝硬化的危险性比单纯性脂肪肝高。AFL的发病机制可能与乙醇及其代谢产物对肝脏的损伤以及脂质过氧化损伤有关，酒精本身具有热量，长期大量摄入，容易造成能量堆积，营养失衡，酒精不仅损害脂肪氧化，也可刺激脂肪形成，引起脂质代谢紊乱。
豹皮樟，又名老鹰茶，系樟科木姜子属毛豺皮樟(Litsea Coreana level)的嫩梢和叶片，是我国南方各民族民间长期饮用的一种植物代用茶，具有清凉止渴、解毒消肿、明目健胃等功效。老鹰茶中含有多种活性成分，经我院天然药物化学教研室提取分离鉴定，确定了豹皮樟的有效部位为总黄酮(Total flavonoids)，总黄酮含量超过51.5%。我们前期研究发现豹皮樟总黄酮(Total Flavonoids of Litsea Coreana Level，TFLC)具有显著的抗炎免疫、降糖、调血脂作用和抗肝纤维化等作用。
本研究以生理途径摄入酒精建立AFL大鼠模型，肝脏灌流获得原代肝细胞，观察TFLC对AFL大鼠和AFL大鼠肝细胞的防治作用并探讨其作用机制。具体内容概括如下：
1、TFLC对AFL大鼠防治作用生理途径摄入酒精制备AFL大鼠模型，设置正常组，AFL模型组，TFLC (100，200，400mg/kg)组，阳性药谷胱甘肽（Glutathion，GSH）组，另设蔗糖对照组。第7周开始给药，连续6周。测定血清中ALT、AST水平，肝匀浆中MDA、SOD和GSHpx水平，HE染色、苏丹III染色以及透射电镜方法观察大鼠肝脏病理形态学的改变。结果显示：与模型组比较，TFLC用药后可改善大鼠一般状况，减轻明显增加的肝脏指数，降低AFL大鼠血清过高的ALT、AST水平，降低肝脏升高的MDA水平，同时使其降低的SOD和GSHpx水平有所恢复。病理检查结果显示，与AFL模型组比较，TFLC组大鼠肝脏脂肪变性和炎症明显减轻。TFLC对大鼠AFL的形成具有防治作用，可能与其抗脂质过氧化的作用有关。
2、TFLC对AFL大鼠血糖、血脂以及脂肪细胞因子和胰岛素抵抗的作用造模第7周开始， TFLC ig连续6周。与AFL模型组比较，TFLC能明显降低大鼠血清TG、TC、FFA和LDL-C水平，对低下的HDL-C水平有一定的提升作用，对升高的血糖水平有一定的降低作用。ELISA法检测血清APOB、TNF-α水平，放射免疫法测定血清中Leptin、Insulin的含量。与模型组相比，TFLC能降低大鼠血清过高瘦素和胰岛素水平，也能降低AFL大鼠血清中增高的TNF-α和APOB水平RT-PCR法测定AFL大鼠肝脏脂肪细胞因子TNF-α、Leptin mRNA的表达。结果显示，与AFL模型组比较，TFLC可明显降低肝脏TNF-α、Leptin mRNA表达水平。TFLC可明显改善AFL脂质代谢紊乱状况，与其调节脂肪细胞因子和改善高胰岛素血症的作用有关。
3、TFLC调节AFL大鼠脂质代谢相关指标ADRP、PPAR γ和Sir2的作用脂质代谢相关指标ADRP、PPAR γ和Sir2在AFL发生发展中其重要作用。RT-PCR和Western blotting检测AFL大鼠肝脏ADRP、PPAR γ和Sir2的表达。以β-actin作为内标，TFLC用药后ADRP、PPAR γ蛋白和mRNA表达有所下降，Sir2蛋白和PGC-1α、Sir2mRNA相对含量明显增高。
免疫组化方法检测大鼠肝脏PPAR γ和ADRP表达，PPAR γ主要表达于细胞的胞浆和胞核，呈黄棕色颗粒；ADRP主要定位于成熟脂肪细胞胞膜上，呈棕黄色颗粒。TFLC用药6周后，与模型组比较，TFLC组大鼠肝脏PPAR γ和ADRP表达有不同程度的降低。TFLC对AFL大鼠的保护作用可能与其降血脂，调节AFL大鼠脂质代谢相关指标ADRP、PPAR γ和Sir2的作用有关。
4、TFLC对AFL大鼠肝细胞脂质代谢的影响
改良的原位二步Ⅳ胶原酶灌流法分离获得AFL大鼠原代肝细胞，SP染色的CK18鉴定肝细胞，胎盼蓝染色观察细胞活性大于80%。肝细胞分为正常组，AFL模型组，TFLC (1，10，100mg/L)，GSH组以及Nic组。肝细胞培养16h，TFLC或GSH或Nic与肝细胞共培养48h，MTT测定TFLC对肝细胞的增殖能力的影响。免疫组化和RT-PCR方法测定ADRP、PPAR γ的蛋白和mRNA表达，Western Blot检测Sir2蛋白表达。TFLC用药后可明显改善体外培养的AFL肝细胞的增殖能力，减少ADRP以及PPAR γ阳性表达的细胞数和表达量，增加AFL肝细胞Sir2蛋白和mRNA表达。
以上实验结果表明，整体动物和细胞水平研究发现，TFLC可明显大鼠减轻肝脏脂肪变性和炎症，具有降血糖，降血脂，调节脂肪细胞因子和改善高胰岛素血症的作用。能够降低ADRP和PPAR γ表达，增加PGC-1α和Sir2相对含量，作用机制可能与TFLC的抗炎、抗脂质过氧化、改善脂质代谢紊乱以及调节ADRP、PPAR γ和Sir2等脂质代谢相关指标等的作用有关。
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 rats
A 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 rats
From7weeks 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 rats
Lipid 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 hepatocytes
Hepatocytes 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.
豹皮樟，又名老鹰茶，系樟科木姜子属毛豺皮樟(Litsea Coreana level)的嫩梢和叶片，是我国南方各民族民间长期饮用的一种植物代用茶，具有清凉止渴、解毒消肿、明目健胃等功效。老鹰茶中含有多种活性成分，经我院天然药物化学教研室提取分离鉴定，确定了豹皮樟的有效部位为总黄酮(Total flavonoids)，总黄酮含量超过51.5%。我们前期研究发现豹皮樟总黄酮(Total Flavonoids of Litsea Coreana Level，TFLC)具有显著的抗炎免疫、降糖、调血脂作用和抗肝纤维化等作用。
本研究以生理途径摄入酒精建立AFL大鼠模型，肝脏灌流获得原代肝细胞，观察TFLC对AFL大鼠和AFL大鼠肝细胞的防治作用并探讨其作用机制。具体内容概括如下：
1、TFLC对AFL大鼠防治作用生理途径摄入酒精制备AFL大鼠模型，设置正常组，AFL模型组，TFLC (100，200，400mg/kg)组，阳性药谷胱甘肽（Glutathion，GSH）组，另设蔗糖对照组。第7周开始给药，连续6周。测定血清中ALT、AST水平，肝匀浆中MDA、SOD和GSHpx水平，HE染色、苏丹III染色以及透射电镜方法观察大鼠肝脏病理形态学的改变。结果显示：与模型组比较，TFLC用药后可改善大鼠一般状况，减轻明显增加的肝脏指数，降低AFL大鼠血清过高的ALT、AST水平，降低肝脏升高的MDA水平，同时使其降低的SOD和GSHpx水平有所恢复。病理检查结果显示，与AFL模型组比较，TFLC组大鼠肝脏脂肪变性和炎症明显减轻。TFLC对大鼠AFL的形成具有防治作用，可能与其抗脂质过氧化的作用有关。
2、TFLC对AFL大鼠血糖、血脂以及脂肪细胞因子和胰岛素抵抗的作用造模第7周开始， TFLC ig连续6周。与AFL模型组比较，TFLC能明显降低大鼠血清TG、TC、FFA和LDL-C水平，对低下的HDL-C水平有一定的提升作用，对升高的血糖水平有一定的降低作用。ELISA法检测血清APOB、TNF-α水平，放射免疫法测定血清中Leptin、Insulin的含量。与模型组相比，TFLC能降低大鼠血清过高瘦素和胰岛素水平，也能降低AFL大鼠血清中增高的TNF-α和APOB水平RT-PCR法测定AFL大鼠肝脏脂肪细胞因子TNF-α、Leptin mRNA的表达。结果显示，与AFL模型组比较，TFLC可明显降低肝脏TNF-α、Leptin mRNA表达水平。TFLC可明显改善AFL脂质代谢紊乱状况，与其调节脂肪细胞因子和改善高胰岛素血症的作用有关。
3、TFLC调节AFL大鼠脂质代谢相关指标ADRP、PPAR γ和Sir2的作用脂质代谢相关指标ADRP、PPAR γ和Sir2在AFL发生发展中其重要作用。RT-PCR和Western blotting检测AFL大鼠肝脏ADRP、PPAR γ和Sir2的表达。以β-actin作为内标，TFLC用药后ADRP、PPAR γ蛋白和mRNA表达有所下降，Sir2蛋白和PGC-1α、Sir2mRNA相对含量明显增高。
免疫组化方法检测大鼠肝脏PPAR γ和ADRP表达，PPAR γ主要表达于细胞的胞浆和胞核，呈黄棕色颗粒；ADRP主要定位于成熟脂肪细胞胞膜上，呈棕黄色颗粒。TFLC用药6周后，与模型组比较，TFLC组大鼠肝脏PPAR γ和ADRP表达有不同程度的降低。TFLC对AFL大鼠的保护作用可能与其降血脂，调节AFL大鼠脂质代谢相关指标ADRP、PPAR γ和Sir2的作用有关。
4、TFLC对AFL大鼠肝细胞脂质代谢的影响
改良的原位二步Ⅳ胶原酶灌流法分离获得AFL大鼠原代肝细胞，SP染色的CK18鉴定肝细胞，胎盼蓝染色观察细胞活性大于80%。肝细胞分为正常组，AFL模型组，TFLC (1，10，100mg/L)，GSH组以及Nic组。肝细胞培养16h，TFLC或GSH或Nic与肝细胞共培养48h，MTT测定TFLC对肝细胞的增殖能力的影响。免疫组化和RT-PCR方法测定ADRP、PPAR γ的蛋白和mRNA表达，Western Blot检测Sir2蛋白表达。TFLC用药后可明显改善体外培养的AFL肝细胞的增殖能力，减少ADRP以及PPAR γ阳性表达的细胞数和表达量，增加AFL肝细胞Sir2蛋白和mRNA表达。
以上实验结果表明，整体动物和细胞水平研究发现，TFLC可明显大鼠减轻肝脏脂肪变性和炎症，具有降血糖，降血脂，调节脂肪细胞因子和改善高胰岛素血症的作用。能够降低ADRP和PPAR γ表达，增加PGC-1α和Sir2相对含量，作用机制可能与TFLC的抗炎、抗脂质过氧化、改善脂质代谢紊乱以及调节ADRP、PPAR γ和Sir2等脂质代谢相关指标等的作用有关。
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 rats
A 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 rats
From7weeks 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 rats
Lipid 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 hepatocytes
Hepatocytes 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.
引文
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