摘要目的 探讨骨髓间充质干细胞源性外泌体(mesenchymal stem cells derived exosomes,MSC-exosomes)对酒精性肝损伤的保护作用.方法 将18只6～8周龄雄性C57BL/6小鼠分成对照组、模型组和外泌体组,每组6只.模型组和外泌体组小鼠Lieber-DeCarli词料饲养4周并在第26、27和28天分别进行乙醇灌胃构建酒精性肝损伤模型,而对照组小鼠给予等热量的葡聚糖麦芽糖.外泌体组小鼠分别在实验第14和26天尾静脉注射MSC-exosomes进行治疗.实验后,检测各组小鼠ALT和AST水平,观察肝组织病理改变,蛋白质印迹法检测核因子相关因子2(nuclear factor erythroid 2-related factor 2,Nrf-2)、血红素加氧酶-1(heme oxygenase-1,HO-1)、CD63、CD81、TSG101和细胞色素C蛋白质表达情况,实时定量PCR分别检测Nrf-2、HO-1、IL-10和IL-17的mRNA变化,试剂盒分别检测血清IL-10、IL-17水平和肝组织丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)、超氧化物歧化酶(superoxide dismutase,SOD)氧化应激指标,流式细胞仪分析肝脏Treg和Th17数量.各组之间比较采用单因素方差分析.结果 MSC-exosomes表达阳性标志物CD63、CD81和TSG101,但不表达阴性标志物细胞色素C.模型组小鼠血清ALT和AST分别为(87.3±25.1)U/L和(223.2±43.5)U/L,外泌体组小鼠血清ALT和AST分别为(47.7±12.0)U/L和(128.2±33.6)U/L,两组比较差异均有统计学意义(F值分别为12.818和12.226,均P<0.05).与对照组相比,模型组小鼠肝脏SOD活性和GSH水平明显降低,MDA水平明显升高;而外泌体组的SOD活性和GSH水平明显高于模型组,MDA水平明显低于模型组,比较差异均有统计学意义(F值分别为4.245、24.074和36.675,均P<0.05).与对照组相比,模型组小鼠肝内Nrf-2和HO-1蛋白表达显著降低,而经MSC-exosomes治疗后表达明显升高,比较差异均有统计学意义(F值分别为33.623和14.960,均P<0.05);各组间Nrf-2及HO-1 mRNA表达趋势与蛋白质表达趋势相同(F值分别为20.784和276.336,均P<0.05).正常组Treg/Th17比值为4.3±0.9、模型组为0.4±0.2、外泌体组为3.4±0.5,各组间比较差异有统计学意义(F=64.227,P<0.05).与对照组相比,模型组小鼠血清IL-17蛋白水平和肝内IL-17基因表达明显升高,而MSC-exosomes治疗后血清IL-17蛋白水平和肝内IL-17基因表达显著降低,比较差异均有统计学意义(F值分别为15.581和40.095,均P<0.05).模型组小鼠血清IL-10蛋白水平和肝内IL-10基因表达明显降低,而MSC-exosomes治疗后血清IL-10蛋白水平和肝内IL-10基因表达显著升高,比较差异均有统计学意义(F值分别为98.268和153.743,均P<0.05).结论 MSC-exosomes可能通过激活Nrf-2/HO-1抗氧化通路及调节肝脏Treg/Th17比例,从而改善酒精诱导的肝脏损伤.

abstractsObjective To evaluate therapeutic effects of bone marrow mesenchymal stem cells(MSC)derived exosomes on alcohol-induced liver injury.Methods Eighteen male C57BL/6 mice aged 6 to 8 week were randomly divided into control group,model group and exosomes group,with 6 mice in each group.The mice in the model group and the exosomes group were fed with Lieber-DeCarli ad libitum diet(Dyets Inc.)for 4 weeks,followed by gavage a bolus of ethanol at day 26,27 and 28.The mice in the control group matched the alcohol-derived calories with dextran-maltose.Meanwhile,the mice in exosomes group were injected with MSC-exosomes via the tail vein at day 14 and 26.After the experiment,serum levels of alanine aminotransferase(ALT)and aspartate aminotransaminase(AST)were detected,and the pathological changes of liver tissues were observed.The expressions of nuclear factor erythroid 2-related factor 2(Nrf-2),heme oxygenase-1(HO-1),CD63,CD81,TSG101 and Cytochrome C were analyzed by Western blot,and mRNA levels of Nrf-2,HO-1,interleukin(IL)-10 and IL-17 were analyzed by real-time polymerase chain reaction(RT-PCR).The commercial kits were used to detect serum IL-10,IL-17 levels and liver tissue malondialdehyde(MDA),glutathione(GSH),superoxide dismutase(SOD)oxidative stress indicators.The numbers of regulatory T cell(Treg)and help T(Th)17 cells in the liver were analyzed by flow cytometry.One-way analysis of variance was used for comparison between groups.Results MSC-exosomes expressed positive markers CD63,CD81 and TSG101,but did not express the negative markers Cytochrome C.The serum ALT and AST levels in model group were(87.3±25.1)U/L and(223.2±43.5)U/L,respectively,while those in exosomes group were(47.7±12.0)U/L and(128.2±33.6)U/L,respectively.The differences between the two groups were both statistically significant(F=12.818 and 12.226,respectively,both P<0.05).Compared with control group,the SOD activity and GSH level in the model group significantly decreased with statistically significant differences(F=4.245 and 24.074,respectively,both P <0.05).Lieber-DeCarli ethanol feeding significantly increased intrahepatic MDA level in the model mice,which was reversed by MSC-exosomes supplementation,and the difference was statistically significant(F=36.675,P <0.05).Compared with control group,the intrahepatic protein expressions of Nrf-2 and HO-1 in model group were significantly decreased,while the expressions in exosomes group were obviously increased.The differences were statistically significant(F=33.623 and 14.960,respectively,both P <0.05).The expression trends of Nrf-2 and HO-1 mRNA were the same as those of protein expressions(F=20.784 and 276.336,respectively,both P <0.05).The proportions of liver Treg/Th17 in the control group,model group and exosomes group were 4.3±0.9,0.4±0.2,and 3.4±0.5,respectively.The differences among groups were statistically significant(F=64.227,P <0.05).Compared with control group,the serum protein and intrahepatic gene expression of IL-17 in the model group were significantly increased,which were reversed by MSC-exosomes treatment.The differences were statistically significant(F=15.581 and 40.095,respectively,both P<0.05).Serum IL-10 protein levels and intrahepatic IL-10 gene expression were significantly decreased after Lieber-DeCarli ethanol feeding,which were lower than the exosomes group.The differences were statistically significant(F=98.268 and 153.743,respectively,both P <0.05).Conclusions MSC-exosomes transplantation may relieve alcohol-induced liver injury.The mechanism could involve reduction of oxidative stress in the liver via regulating Nrf-2/HO-1 and normalizing the balance of Treg and Th17 cells.