岩藻黄素对小鼠非酒精性脂肪性肝病的修复作用

Repairing Effect of Fucoxanthin on Non-alcoholic Fatty Liver Disease in Mice

目的:探究岩藻黄素对高脂饮食诱导的C57BL/6小鼠非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)的修复作用及其机制。方法:将52只C57BL/6小鼠随机分成4组,正常组14只,其余为实验组,实验组给予8周的高脂饮食喂养之后,再分为模型组、岩藻黄素低剂量和高剂量组,给予灌胃6周,每日1次。每周记录小鼠的体质量,第14周结束时小鼠禁食12 h后全部处死。后续分别测定各组小鼠血清中谷草转氨酶(aspartate aminotransferase,AST)、谷丙转氨酶(alanine aminotransferase,ALT)活力,总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、低密度脂蛋白胆固醇(lowdensitylipoproteincholesterol,LDL-C)、高密度脂蛋白胆固醇(highdensitylipoproteincholesterol,HDL-C)、游离脂肪酸、脂联素和瘦素含量;检测肝脏组织匀浆液中超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽(glutathione,GSH-Px)、丙二醛(malondialdehyde,MDA)、过氧化氢酶(catalase,CAT)、白细胞介素-1β、白细胞介素-6和肿瘤坏死因子α含量;透射电镜下观察肝脏的组织病理学变化并采用蛋白免疫印迹法检测肝脏中5’-单磷酸腺苷活化蛋白激酶(adenosine 5’-monophosphate-activated protein kinase,AMPK)、核因子红系2相关因子2(nuclear factor erythroid2-related factor 2,Nrf2)和Toll样受体4(toll-like receptors 4,TLR4)信号通路蛋白表达情况。结果:与模型组相比,岩藻黄素组中的TC、TG、LDL-C、ALT、AST显著降低(P<0.05),HDL-C显著升高(P<0.05),且降低了瘦素水平,增加了脂联素分泌,GSH-Px、SOD、CAT升高(P<0.05),MDA降低(P<0.05),且炎症因子的水平降低。苏木精-伊红染色、油红O染色、糖原染色和透射电镜结果显示岩藻黄素组肝脏组织学结构好转,趋近于正常组。蛋白免疫印迹法结果显示岩藻黄素治疗可上调AMPK信号通路中磷酸化5’-单磷酸腺苷活化蛋白激酶和过氧化物酶体增殖物激活受体α、磷酸化乙酰辅酶A羧化酶、肉碱脂酰转移酶1,下调固醇调节元件结合蛋白-1c、脂肪酸合酶表达;抑制Kelch样环氧氯丙烷相关蛋白1/Nrf2信号通路中Keap-1的水平,提高Nrf2及其下游抗氧化蛋白血红素氧合酶1、NAD(P)H-醌氧化还原酶1和谷氨酸-半胱氨酸连接酶的表达水平;下调TLR4信号通路中TLR4蛋白的表达,抑制髓样分化因子88、磷酸化人核因子κB抑制蛋白α和磷酸化核因子κB蛋白(p65)的表达。结论:岩藻黄素可通过调节脂质代谢、减少氧化应激和调节炎症等途径修复高脂饮食诱导的小鼠非酒精性脂肪性肝病。

Objective:To explore the repairing effect and underlying mechanism of fucoxanthin on non-alcoholic fatty liver disease(NAFLD)induced by a high-fat diet(HFD)in C57BL/6 mice.Methods:Fifty-two C57BL/6 mice were randomly divided into four groups,including one normal group(n=14)and three experimental groups(n=38).The normal group was fed a regular diet,and the experimental groups were fed a HFD.After feeding for eight weeks,two animals were selected from the experimental groups for serum biochemical assays and liver histological observation,and the other 36 were divided into three groups(n=12 each):model,low-dose and high-dose fucoxanthin,which were then administrated with physiological saline or fucoxanthin by gavage once a day for six weeks.Body mass was recorded weekly,and all mice th were killed after fasting for 12 h at the end of the 14 week.The serum levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C),high density lipoprotein cholesterol(HDL-C),free fatty acid(FFA),adiponectin,and leptin were measured.In addition,the levels of superoxide dismutase(SOD)activity,glutathione(GSH-Px),malondialdehyde(MDA),catalase(CAT),interleukin-1β(IL-1β),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)in liver homogenate were also determined.Furthermore,hepatic histopathological changes were observed under microscope,and the protein expressions of the adenosine 5’-monophosphate-activated protein kinase(AMPK),nuclear factor erythroid 2-related factor 2(Nrf2)and toll-like receptors 4(TLR4)signaling pathways in liver tissues were detected by Western blot.Results:Compared with the model group,the levels of TC,TG,LDL-C,ALT and AST in the fucoxanthin-treated groups were significantly decreased(P<0.05),Leptin was decreased,while the levels of HDL-C and adiponectin were significantly increased(P<0.05).Moreover,the levels of GSH-Px,SOD and CAT in the fucoxanthin-treated groups were significantly increased(P<0.05),leptin was decreased,while the levels of MDA and inflammatory cytokines were significantly decreased(P<0.05)compared with the model group.The results of hematoxylin-eosin(H&E)staining,oil red O staining,periodic acid-schiff staining(PAS),and transmission electron microscopy(TEM)showed that the histological structure of the liver in the fucoxanthin-treated groups recovered to almost normal.The results of Western blot showed that fucoxanthin treatment upregulated the protein expression of phosphorylated adenosine 5’-monophosphate-activated protein kinase(p-AMPK),peroxisome proliferators-activated receptorα(PPARα),phosphorylated acetyl-CoA carboxylase(p-ACC),and carnitine acyl transferase 1(CPT-1)in the AMPK signaling pathway,downregulated the expression of sterol regulatory element binding protein-1c(SREBP-1c)and fatty acid synthase(FAS),inhibited the level of Kelch-like epichlorohydrin-associated protein-1(Keap-1)in the Keap-1/nuclear factor-erythroid 2-related factor 2(Nrf2)signaling pathway,increased the expression of Nrf2 and its downstream antioxidant proteins heme oxygenase-1(HO-1),NAD(P)H quinone oxidoreductase 1(NQO1),and glutamate cysteine ligase modifier(GCLM),and downregulated the expression of TLR4,myeloid differentiation factor 88(MyD88),phosphorylated nuclear factorκB inhibitory proteinα(p-IκBα),and phosphorylated nuclear factorκB(p65)(p-NF-κB(p65))in the TLR4 signaling pathway.Conclusion:Fucoxanthin can repair HFD-induced NAFLD in mice through regulating lipid metabolism,reducing oxidative stress and suppressing inflammation.