Protective effect of bone marrow mesenchymal stem cells derived exosomes on alcohol-induced hepatic injury

Objective 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. Key words: Liver diseases, alcoholic; Inflammation; Immunity; Bone mesenchymal stem cells; Exosome; Oxidative stress