摘要
目的建立非酒精性脂肪性肝病模型,观察非酒精性脂肪性肝病大鼠血清转氨酶、血糖,肝内脂质,SOD、MDA、GSH-PX以及肝脏形态学、肝内PKB、P-PKB表达的变化以及N-乙酰半胱氨酸(NAC)、褪黑素(MT)对上述指标的影响。
方法1.(NAC部分)80只雄性Wistar大鼠随机分成5组每组16只:正常对照组予普通饲料饮食、模型组和NAC低、中、高3个剂量组予高脂饲料饮食10周。各NAC组分别给予NAC10 mg/(kg·d)、20 mg/(kg·d)、40 mg/(kg·d),正常组和模型组分别给予等量的生理盐水,每日一次定时灌胃。10周后处死,处死前各组随机选取半数大鼠门静脉注射胰岛素,剂量为2.5U。称肝湿重并计算肝指数,观察肝脏病理形态学改变,生化法检测大鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆固醇(TC)、甘油三酯(TG)、血糖(GLU),肝匀浆TC、TG,超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽—过氧化物酶(GSH—PX)、免疫组化法测定肝脏蛋白激酶B、磷酸化蛋白激酶B(PKB、P-PKB)的表达以及P-PKB/PKB比值。2.(MT部分)分组、给药及标本收集来源前期研究,具体方法:将50只雄性Wistar大鼠随机分成5组每组10只:正常对照组给予普通饮食、模型组和低、中、高3个剂量褪黑素组给予高脂饮食。褪黑素组分别给予褪黑素2.5 mg/(kg·d)、5.0mg/(kg·d)和10.0 mg/(kg·d)剂量,正常组和模型组每日一次给予等量的生理盐水(内含1%无水乙醇)腹腔注射。12周后处死,检测指标同NAC部分。
结果1.(NAC部分)10周高脂饮食成功复制大鼠NAFL/NASH模型。与正常对照组比较,模型组大鼠肝湿重及肝指数增加(P＜0.05),肝细胞呈明显脂肪变性,肝小叶内见炎性细胞浸润和点状坏死,炎症积分明显升高(P＜0.01);血清ALT、AST、TC、GLU以及肝匀浆TC、TG、MDA值升高(P＜0.05),肝匀浆SOD、GSH-PX值降低(P＜0.05)。未注射胰岛素和注射胰岛素的模型组肝内PKB、P-PKB表达及P-PKB/PKB比值均分别低于未注射胰岛素和注射胰岛素的正常组(P＜0.05)。胰岛素注射正常组PKB、P-PKB表达及P-PKB/PKB比值分别较非胰岛素注射正常组明显升高(P＜0.05)。胰岛素注射模型组PKB、P-PKB表达及P-PKB/PKB比值分别为较非胰岛素注射模型组升高,但差异无显著性(P＞0.05)。与模型组比较,NAC中高剂量组肝湿重、肝匀浆MDA值降低,肝匀浆SOD,GSH-PX水平升高(P＜0.05);NAC高剂量组肝指数、血清AST、血GLU、肝匀浆TC,TG降低(P＜0.05)。未注射胰岛素和注射胰岛素的NAC中、高剂量组大鼠组肝内PKB、P-PKB表达及P-PKB/PKB比值均分别高于未注射胰岛素和注射胰岛素的模型组(P＜0.05)。胰岛素注射的NAC中、高剂量组PKB、P-PKB表达及P-PKB/PKB比值分别较非胰岛素注射的NAC中、高剂量组明显升高(P＜0.05)。
2.(MT部分)与正常对照组比较,模型组大鼠肝湿重及肝指数明显增高(P＜0.01);肝细胞呈脂肪变性,肝小叶内见灶性炎细胞浸润和点状坏死,炎症积分明显升高(P＜0.01),血清ALT、血GLU(P＜0.05),AST,TC值(P＜0.01)以及肝匀浆TC、TG、MDA值明显升高(P＜0.05);肝匀浆SOD、GSH-PX水平明显降低(P＜0.05);肝脏PKB、P-PKB表达,P-PKB/PKB比值明显下降(P＜0.05)。与模型组相比,MT中、高剂量组肝湿重及肝指数(P＜0.05,P＜0.01),MT高剂量组血清AST降低(P＜0.05)。MT中、高剂量组肝脏TC和高剂量组TG低于模型组(P＜0.05,P＜0.01。P＜0.05);肝细胞脂肪变性、肝小叶内炎症细胞浸润明显减轻(P＜0.01)。肝匀浆SOD、GSH-PX水平升高、MDA值降低(P＜0.01)。MT中、高剂量组PKB、P-PKB表达及高剂量组P-PKB/PKB值升高(P＜0.05)。
结论NAFL/NASH大鼠肝组织PKB、P-PKB的表达以及P-PKB/PKB比值下降,对胰岛素的敏感性下降,提示NAFL/NASH大鼠肝内存在胰岛素抵抗和PKB表达异常。NAC、MT可降低NAFL/NASH大鼠血清AST水平、血GLU、TC,减少肝内脂质沉积以及改善肝脏病理学变化,对NAFL/NASH具有保护作用,这种作用可能与抗氧化应激和上调肝内PKB表达或/和改善肝内胰岛素抵抗有关。
Objective
To establish the model of nonalcoholic fatty liver disease and investigate the expression of level of serum transaminase,glucose,lipid SOD,MDA,GSH-PX, hepatic morphology and hepatic PKB,P-PKB,P-PKB/PKB,effects of N-acetylcysteine and melatonin.on above-mentioned indexs.
Methods
1.(the part of N-acetylcysteine) eighty male male Wistar rats were randomly divided into 5 groups with 16 rats in each group.The rats in the normal control group were fed with normal diet while that in the model group,the N-acetylcysteine in low dose group,moderate dose,high dose group with high-fat diet.The rats in various groups were treated with N-acetylcysteine 10 mg /(kg·d)、20mg /(kg·d)、40mg / (kg·d)and equal doses of saline to control and model group once a day by intragastric administration respectively.After 10 weeks feeding,before to put to death, half mice from each group were either injected insulin(2.5 U) or similar volume of NS in the portal vein,5 min before the resection of the liver,liver humid weight and liver index and Histopathological changes in liver were observed.The level of alanine aminotransferase(ALT)and aspartate aminotransferase(AST) in serum,the level of total cholesterol(TC)and triglyceride(TG) in liver tissue homogenates were measured by biochemical method.The expression of PKB,P-PKB and the ratio of P-PKB/PKB in hepatocytes were tested by immunohistochemistry.
2.(The part of melatonin)The dividing of group,the administration and the materials of drawing from animals were from the research of the early days.The concret method is as follows:50 male Wistar rats were randomly divided into 5 groups with 10 rats in each group.The rats in the normal control group were fed with normal diet while that in the model group,the melantonin in low dose group,moderate dose,high dose group with high-fat diet.The rats in various melatonin groups were treated with melatonin 2.5 mg /(kg·d)、5.0mg /(kg·d)、10.0 mg /(kg·d)by intraperitoneal injection respectively.After 12 weeks feeding,the other indexs were detected as the part of acetylcysteine.
Results
1.(the part of acetylcysteine) the NAFL/NASH rat model was successfully established with high fat diet for 10 weeks.compared with control group,liver humid weigh,liver index increased in model group and developed steatosis and focus infiltration of inflammatory cells and punctiform necrosis in hepatic lobules.The level of serum ALT,AST,TC,GLUand TC,TG;MDA in liver tissue homogenates increased significantly(P＜0.05).simultaneously with decrease on liver SOD,GSH-PX(P＜0.05). The expression of hepatic PKB、P-PKB and the ratio of the two in the model group with or without injecting insulin were lower than the control group (P＜0.05).The expression of hepatic PKB、P-PKB and the ratio of the two in the control group with injecting insulin were higher than the same group without injecting insulin(P＜0.05).The expression of hepatic PKB、P-PKB and the ratio of the two in the model group with injecting insulin were higher than the same group without injecting insulin,But the diffence between two has no significant meaning(P＞0.05).Compared with model group,N-acetylcysteine in dose of moderate and high group improved the pathological changes in liver with steaosis and the decrease of histological activity index(P＜0.05),the level of liver humid weigh,MDA SOD,GSH- PX in liver tissue homogenates(P＜0.05).N-acetylcyeteine in dose of high group improved the level of liver index,serum AST,GLU;TC,TG in liver tissue homogenates.The expressions of hepatic PKB、P-PKB and the ratio of the two in the moderate and high dose of NAC were higher than the model group with or without injecting insulin(P＜0.05).The expressions of hepatic PKB、P-PKB and the ratio of the two in the moderate and high dose of NAC with injecting insulin are higher than the same group without injecting insulin(P＜0.05).
2.(The part of melatonin) Compared with control group,the model group developed steatosis and foucus infiltraion of inflammatory cells and punctiform necrosis in hepatic lobules,with significant increase on liver humid weight,liver index,serum ALT,GLU(0.05),AST,TC(P＜0.01),andTC,TG,MDA in liver tissue homogenate(P＜0.05),simultaneously with decrease on liver SOD,GSH-PX(P＜0.05).The expression of PKB,P-PKB,the ratio of P-PKB and PKB in liver was increased.Compared with model group,melatonin improved the pathological changes in liver with steaosis and the decrease of histological activity index(P＜0.05),the level of MDA in liver tissue homogenate,liver index,liver humid weight(P＜0.05, P＜0.01),simultaneously with the increased of SOD,GSH-Px in liver tissue homogenate(P＜0.05)in moderate and high dose of MT group.The level of serum AST, liver tissue homogenate TC,TG in high dose of MT group and the level of liver tissue homogenate TC in moderate dose of MT group was lower than model group (P＜0.05).The expressions of PKB,P-PKB in moderate and high dose of MT group and the ratio of the two were improved in high dose of MT group.
Conclusions The expression of hepatic PKB,P-PKB、P-PKB/PKB decreased in rats with NAFL/NASH.N-acetylcysteine and Melatonin can improve lipid both in serum and liver tissue homogenates,hepatic morphology.Its protective mechanism may be correlated with anti-oxidative stress and up-regulating the expression of hepatic PKB or(and) improving hepatic insulin sensitivity.
引文
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