ω-3鱼油脂肪乳对肝细胞氧化损伤的修复作用及其机制

摘要

目的：观察ω-3鱼油脂肪乳对肝细胞氧化损伤的修复作用，并探讨其机制。方法体外培养人肝细胞系HL7702细胞。取对数生长期的HL7702细胞分成对照组、模型组和观察组，每组9孔。对照组仅加培养基常规培养；模型组、观察组在培养基中加入500μmol ／L过氧化氢（H2 O2），培养1 h后观察组加入0．5％ω-3鱼油脂肪乳；另设空白对照组，只加培养基，无细胞。各组继续培养4h后收集细胞，油红O染色观察各组细胞内脂滴变化， CCK-8法测算各组细胞存活率，DCFH-DA荧光探针法检测各组细胞内活性氧簇（ROS），比色法检测各组细胞培养上清液丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST），COD-PAP单试剂比色法检测各组细胞内甘油三酯（TG）、总胆固醇（TC），硫代巴比妥酸法检测各组细胞内丙二醛（MDA），黄嘌呤氧化酶法检测各组细胞内超氧化物歧化酶（SOD），实时荧光定量PCR法检测各组细胞中过氧化物酶体增生物激活受体（PPAR）αmRNA，Western blotting法检测各组细胞中肝型脂肪酸结合蛋白（L-FABP）相对表达量。结果光镜下可见脂滴染为橘红色。对照组组HL7702肝细胞排列紧密，仅见少许橘红色脂滴。模型组肝细胞可见大量橘红色脂滴并伴有脂滴融合现象。观察组肝细胞可见较多橘红色脂滴，但与模型组比较显著减少。对照组、模型组、观察组细胞存活率分别为67．25％±4．68％、71．72％±2．47％、100％。观察组细胞存活率高于模型组（P＜0．05），但低于对照组（P＜0．05）。与对照组比较，模型组细胞内ROS增高（P＜0．05），细胞培养上清液ALT、AST增高（P均＜0．05），细胞内TG、TC 、MDA增高（P均＜0．05），细胞内 SOD 降低（P＜0．05），PPARαmRNA 降低（P＜0．05），L-FABP 相对表达量降低（P＜0．05）。与模型组比较，观察组细胞内ROS降低（P＜0．05），细胞培养上清液ALT、AST均降低（P均＜0．05），细胞内TG、TC 、MDA均降低（P均＜0．05），细胞内SOD活性增高（P＜0．05），PPARαmRNA增高（P＜0．05），L-FABP相对表达量增高（P＜0．05）。结论ω-3鱼油脂肪乳可修复人肝HL7702细胞氧化损伤，其机制可能与其上调L-FABP ／PPARα信号通路相关因子的表达、维持肝细胞脂质稳态有关。%Objective To observe the repair function ofω-3 fish oil fat emulsion on oxidative damage in liver cells and to explore its mechanism.Methods Human hepatic cell line HL7702 was cultured in vitro.HL7702 cells were di-vided into the control group,model group and observation group,and each group was divided into 9 holes.The control group was cultured with conventional culture medium,in the model and the observation groups,we added 500 μmol/L hy-drogen peroxide (H2O2)to the culture medium,after 1 hour,the observation group was added with 0.5%ω-3 fish oil fat emulsion,and then we set a blank control group which was only added with the culture medium without cells.After 4 h of culture,the cells in each group were collected.We observed the intracellular lipid droplets by oil red O staining and calcu-lated the cell survival rate by CCK-8,measured reactive oxygen species (ROS)in the cells of each group by DCFH-DA fluorescence probe,detected the alanine aminotransferase (ALT)and aspartate aminotransferase (AST)in the supernatant of each group by colorimetric assay,the triglyceride (TG)and total cholesterol (TC)by COD-PAP single reagent colori-metric method,malondialdehyde (MDA)by thiobarbituric acid and superoxide dismutase (SOD)by xanthine oxidase,the relative expression of peroxisome proliferator-activated receptor-α(PPAR-α)mRNA by real-time fluorescence quantitative PCR,and the relative expression of liver fatty acid-binding protein (L-FABP)protein by Western blotting.Results Un-der microscope,the intracellular lipid droplets were orange.The HL7702 liver cells in the control group arrayed closely, and only a few orange red lipid droplets appeared.In the model group,a large number of orange red lipid droplets were ob-served and accompanied by lipid droplets.In the observation group,the liver cells showed more orange lipid droplets,but they were significantly decreased as compared with those of the model group.The cell survival rates of the control group, the model group and the observation group were 100%,67.25%±4.68%,and 71.72%±2.47%.The survival rate of the observation group was higher than that of the model group (P<0.05),but was lower than that of the control group (P<0.05).Compared with the control group,the intracellular ROS of the model group was increased (P<0.05),the ALT and AST in cell culture supernatants were increased (all P<0.05 ),and the intracellular TG,TC,MDA were increased (all P<0.05),the SOD was decreased (P<0.05),the PPARαmRNA was decreased (P<0.05)and the relative ex-pression of L-FABP protein was decreased (P<0.05).Compared with the model group,the intracellular ROS of the ob-servation group was decreased (P<0.05),the ALT and AST in cell culture supernatants were decreased (all P<0.05), the intracellular TG,TC,MDA were decreased (all P<0.05),the SOD was increased (P<0.05),the PPARαmRNA was increased (P<0.05)and the relative expression of L-FABP protein was increased (P<0.05).Conclusions ω-3 fish oil fat emulsion can repair oxidative damage in human hepatic cell line HL7702,and its mechanism may be related to the up-regulation of the expression of L-FABP/PPARαsignaling pathway and the maintenance of lipid homeostasis in liver cells.