Protective Effect Of Exosomes Derived From Human Liver Stem Cells On ConA-induced Acute Liver Injury In Mice

Objective: To investigate the protective effect of human liver stem cells-exosomes（HLSC-exo）intravenous injection at different times on acute liver injury induced by Concanavalin A（Con A）in mice.Methods: HLSC-exo was extracted by differential centrifugation.The expression of CD9 and CD63 marker proteins was detected by Westernblot,and particle size distribution was analyzed by NTA.A total of 56 male C57BL/6 mice were randomly divided into normal control group（group C）,Con A model group（group M）and HLSC-exo treatment group（group E）.Group M and group E were divided into 3-hours groups（M1 and E1 groups）,6-hours groups（M2 and E2 groups）and12-hours groups（M3 and E3 groups）according to the interval time of phosphate buffer or HLSC-exo injection and Con A injection.The levels of ALT,AST,TNF-α and IL-10 in serum were detected.The changes of liver gross and pathological tissue in each group were compared.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups,and P<0.05 was considered statistically significant.Results HLSC-exo is a vesicle body with a diameter of90-110 nm.HLSC-exo showed a clear saucer-like structure under electron microscope,and its specific marker proteins CD9 and CD63 were significantly expressed.The levels of ALT,AST,IL-10 and TNF-α in group C were 31.81±6.74 U/L,69.75±8.30 U/L,313.51±10.97 pg/ml and476.05±7.31 pg/ml,respectively.The levels of ALT,AST,IL-10 and TNF-α were 1989.32±347.67 /L,2534.27±644.72U/L,266.20±8.15pg/ml,551.31±17.70pg/ml,respectively,in group M1,2208.33±349.96U/L,2437.13±288.13U/L,264.41±9.12pg/ml,556.21±11.15pg/ml,respectively,in group M2,2223.12±318.48U/L,2289.88±274.59U/L,260.23±4.56pg/ml,564.42±4.08 pg/ml,respectively,in group M3.The levels of ALT,AST,IL-10 and TNF-α were 225.58± 115.59 U/L,303.53±126.68U/L,331.61±10.46 pg/ml,478.26±12.99 pg/ml,respectively,in group E1,1174.71±203.30 U/L,1340.70±262.56U/L,288.13±10.74 pg/ml,515.58±7.18 pg/ml,respectively,in group E2,1990.00±302.59 U/L,2293.36±341.96 U/L,260.10±7.25 pg/ml,559.85±12.02 pg/ml,respectively,in group E3.The levels of ALT,AST and TNF-α in group M were higher than group C.The levels of ALT,AST and TNF-α in E1 and E2 groups were significantly lower than M1 and M2 groups,and the levels in E1 group were lower than E2 group（all P<0.001）.The level of IL-10 in E1 and E2 groups was higher thanM1 and M2 groups（P<0.001）.There were no significant differences in ALT,AST,TNF-α and IL-10 levels between M3 and E3 groups（all P>0.05）.The gross and pathological results of liver showed that the necrosis degree of liver cells in E1 and E2 groups was significantly less than M1 and M2 groups,and the liver lobules in E1 group was basically intact,with only scattered spotty necrosis.Compared with M3 group,liver cell necrosis was not improved significantly in E3 group.Conclusion Intravenous injection of HLSC-exo can alleviate acute liver injury induced by Con A in mice,and the protective effect is most significant by injection 3h in advance.Regulation of cytokines is one of the important mechanisms of HLSC-exo to reduce liver injury.