卫生研究

目的 评价番茄红素是否可以改善D-半乳糖诱导的肝脏衰老及其相关分子机制。方法 将45只2月龄雌性CD-1小鼠适应性喂养1周后，按随机数字表法分为3组，每组15只，对照组：标准饲料+腹腔注射生理盐水；D-半乳糖组：标准饲料+腹腔注射150 mg/kg D-半乳糖；番茄红素干预组：番茄红素饲料(0.03%番茄红素，均匀混在标准饲料中)+腹腔注射150 mg/kg D-半乳糖，共8周。小鼠衰老相关β-半乳糖苷酶(senescence associated-beta-galactosidase, SA-β-gal)染色及酶联免疫分析(ELISA)试剂盒检测肝脏组织SA-β-gal含量、Western blots检测肝脏组织P21及P53蛋白表达，明显高于对照组，即判定衰老小鼠造模成功。试剂盒测定血清中谷丙转氨酶(glutamate pyruvate transaminase, GPT)、谷草转氨酶(glutamic oxaloacetic transaminase, GOT)、碱性磷酸酶(alkaline phosphatase, ALP)活性，肝脏组织中过氧化氢(H_2O₂)、总抗氧化能力(total antioxidant capacity, T-AOC)和丙二醛(malondialdehyde, MDA)活性；ELISA试剂盒检测小鼠肝脏组织成纤维细胞生长因子21(fibroblast growth factor 21,FGF21)、白细胞介素(interleukin, IL)6、8和10含量；免疫荧光检测肝脏组织活性氧(reactive oxygen species, ROS)水平；免疫组化检测肝脏组织线粒体融合蛋白2(mito-fusion 2,MFN2)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平。透射电子显微镜观察肝脏线粒体形态；Western blots检测线粒体融合蛋白1(mito-fusion 1,MFN1)和线粒体分裂蛋白1(fission 1,FIS1)的表达情况。结果 与对照组相比，D-半乳糖组小鼠肝脏SA-β-gal含量升高(P<0.01),P21及P53蛋白表达水平上调(P<0.01),即衰老小鼠造模成功。同时血清GPT(P<0.05)、GOT(P<0.01)及ALP(P<0.05)活性显著上调；T-AOC活性下调(P<0.05),MDA(P<0.05)和H_2O₂(P<0.01)含量升高以及ROS水平上调；透射电子显微镜观察到D-半乳糖组小鼠肝脏线粒体双层膜结构被破坏且肿胀；FGF21水平下调(P<0.05),线粒体分裂基因FIS1蛋白表达下调(P<0.01),线粒体融合基因MFN1及MFN2蛋白表达上调(P<0.05);肝脏组织中促炎因子TNF-α、IL-6(P<0.01)、IL-8(P<0.05)表达上调，以及抗炎因子IL-10表达下调(P<0.01)。与D-半乳糖组相比，番茄红素干预组小鼠肝脏SA-β-gal含量降低(P<0.01)、P21及P53蛋白表达水平下调(P<0.01);血清中GPT(P<0.05)、GOT(P<0.05)及ALP(P<0.01)活性显著下调；T-AOC活性上调(P<0.01),MDA(P<0.01)和H_2O₂(P<0.01)含量降低以及ROS水平下调；透射电子显微镜观察到番茄红素干预组小鼠肝脏线粒体双层膜结构有所改善；FGF21水平上调(P<0.05),线粒体分裂基因FIS1蛋白表达上调(P<0.01),线粒体融合基因MFN1及MFN2蛋白表达下调(P<0.05);肝脏组织中促炎因子TNF-α、IL-6(P<0.05)、IL-8(P<0.05)表达下调，且抗炎因子IL-10表达上调(P<0.05)。结论 番茄红素可以改善D-半乳糖诱导的肝脏衰老、氧化应激、炎症及肝功能损伤，这可能与其对肝脏FGF21信号的激活以及对线粒体功能的增强相关。

OBJECTIVE Evaluation of whether lycopene(LYC)ameliorates D-galactose-induced liver senescence and its associated molecular mechanisms.METHODSForty-five 2-month-old female CD-1 mice were acclimatized and fed for 1 week and randomly divided into 3 groups(15 mice/group): Control group receiving 0.9% physiological saline(intraperitoneally injected, i.p.) plus basal chow diet; D-galactose group injected with 150 mg/kg D-galactose(i.p) plus basal chow diet; D-galactose+LYC group receiving 150 mg/kg D-galactose(i.p) plus 0.03% LYC(W/W, mixed with standard diet) for consecutive 8 weeks. Mouse senescence associated-beta-galactosidase(SA-β-gal) staining and enzyme-linked immunoassay(ELISA) kit were used to detect the SA-β-gal content in liver tissues, and western blots detected the protein expression of P21 and P53 in liver tissues, which were significantly higher than those in the control group, it was determined that the modeling of senescent mice was successful. The kit was used to determine glutamate pyruvate transaminase(GPT), glutamic oxaloacetic transaminase(GOT), alkaline phosphatase(ALP) activities in serum, hydrogen peroxide(H_2O₂), total antioxidant capacity(T-AOC) and malondialdehyde(MDA) activities in liver tissue. ELISA kits were used to detect the levels of fibroblast growth factor 21(FGF21), interleukin 6(IL-6), interleukin 8(IL-8) and interleukin 10(IL-10) in mouse liver tissues; immunofluorescence was used to detect the levels of reactive oxygen species(ROS) in liver tissues; immunohistochemistry was used to detect mito-fusion 2(MFN2) and tumor necrosis factor-α(TNF-α) in liver tissues. Transmission electron microscopy was used to observe the morphology of liver mitochondria; western blots were used to detect the expression of mito-fusion 1(MFN1) and fission 1(FIS1).RESULTS Compared with the control group, hepatic SA-β-gal content was elevated(P<0.01) and P21 and P53 protein expression levels were up-regulated(P<0.01) in the D-galactose group, successful modelling in senescent mice. Serum GPT(P<0.05), GOT(P<0.01) and ALP(P<0.05) activities were significantly up-regulated; T-AOC activity was down-regulated(P<0.05), MDA(P<0.05) and H_2O₂(P<0.01) contents were elevated, and the level of ROS was up-regulated; the mitochondria of mouse livers in the D-galactose group were disrupted and swollen in the double membrane structure, as observed by transmission electron microscopy; the level of FGF21 was down-regulated(P<0.05), the protein expression of the fission gene FIS1 was down-regulated(P<0.01), the proteins of the mito-fusion genes MFN1 and MFN2 were up-regulated(P<0.05); the expression of the pro-inflammatory factors TNF-α, IL-6(P<0.01), and IL-8(P<0.05) was up-regulated in the liver tissue and down-regulated expression of anti-inflammatory factor IL-10(P<0.01) in liver tissue. Compared with the D-galactose group, the hepatic SA-β-gal content in the D-galactose + lycopene group was reduced(P<0.01), and the protein expression levels of P21 and P53 proteins were down-regulated(P<0.01); the activities of GPT(P<0.05), GOT(P<0.05), and ALP(P<0.01) were significantly down-regulated in serum; the activity of T-AOC was up-regulated(P<0.01), reduced MDA(P<0.01) and H_2O₂(P<0.01) contents as well as down-regulated ROS levels; improved mitochondrial bilayer membrane structure in lycopene-intervened group as observed by transmission electron microscopy; up-regulated FGF21 levels(P<0.05), the protein expression of the fission gene FIS1 was up-regulated(P<0.01) and that of the mito-fusion genes MFN1 and MFN2 was down-regulated(P<0.05); the expression of the pro-inflammatory factors TNF-α, IL-6(P<0.05), IL-8(P<0.05) was down-regulated and the expression of the anti-inflammatory factor IL-10 was up-regulated(P<0.05) in liver tissue.CONCLUSION Lycopene ameliorated D-galactose-induced hepatic senescence and hepatic impairment, which may be associated with its activation of hepatic FGF21 signaling and enhancement of mitochondrial function.