Abstract:

Objective

To evaluate the therapeutic effects of human umbilical cord mesenchymal stem cell-derived exosomes （hucMSC-Exo） in inhibiting the activation of hepatic stellate cells （HSCs） and improving liver fibrosis in vivo and in vitro.

Methods

The exosomes were isolated and purified by ultracentrifugation and characterized by transmission electron microscopy（TEM）， nanoparticle size analysis， and Western blot. For in vivo experiments， 40 male C57BL/6J mice were divided into 4 groups： normal control group， model group， exosome treatment group，and colchicine treatment group （n = 10）. The hepatic fibrosis model was built by intragastric administration of CCl₄-olive oil （1 ： 3）， 2 times/week for 8 weeks. After 4 weeks of modeling，intervention treatments were carried out： exosomes were proceeded by intravenous injection administration （300μg/100g body weight）， and colchicine was given by intragastric administration（0.01mg/100g body weight）. At the end of 8 weeks， levels of ALT， AST， IV-C， LN， PC-III， and HAase in serum were measured using biochemical assays and ELISA methods. Liver histopathological changes were assessed with H&E and Masson staining， and α-smooth muscle actin （α-SMA） and Collagen-Ⅰ expression in liver tissue were detected using immunohistochemistry. For in vitro experiments， the human HSC cell line LX-2 was treated with TGF-β₁ and hucMSC- Exo， and named blank control， TGF-β₁ induction group， TGF-β₁ induction+hucMSC-Exo group. Immunofluorescence was used to analyze the expression of α-SMA， and Western blot was performed to detect the expression levels of α-SMA and Collagen-Ⅰ proteins. One-way analysis of variance was used for comparison between multiple groups， and LSD-t test was used for pairwise comparison between groups.

Results

Compared to the normal control group， the levels of ALT， AST， Collagen-Ⅳ， LN，PC-Ⅲ， and HAase were increased （all P ＜ 0.01）. Compared to the model group， the levels of ALT[（3.17 ± 0.70） vs （ 6.34 ± 0.37） U/ L］， AST [（2.91 ± 0.60） vs （7.11 ± 0.41）U/ L］， Collagen-Ⅳ[（116.38 ± 4.96）vs （143.79 ± 6.71） μg/ L］， LN [（762.96 ± 120.45） vs （950.86 ± 29.77） μg/ L］，PC- Ⅲ[（14.88 ± 0.83）vs （18.39 ± 2.57） μg/ L］and HAase [（109.92 ± 11.32）vs （187.58 ±10.42） ng/L］ were decreased significantly in the hucMSC-Exo treatment group （all P ＜ 0.01）.Immunohistochemical results of liver tissues showed that the level of the expression of α-SMA and Collagen-Ⅰ was increased （all P ＜ 0.01）. Compared to the model group， the expression of α-SMA（ 337.05 ± 39.13 vs 2 681.83 ± 312.63）， and Collagen-Ⅰ expression （479.32 ± 86.25 vs 1 022.57 ±102.13）were decreased in liver tissues in hucMSC-Exo intervention group （all P ＜ 0.01）. The Western blot data of LX-2 cell lysate indicated that hucMSC- Exo inhibited the expression of α-SMA and Collagen-Ⅰ in LX-2 cells compared to the model group [（0.87 ± 0.09 vs 1.01 ± 0.34） and（0.46 ± 0.07 vs 0.75 ± 0.22）］（all P ＜ 0.05）.

Conclusion

Early stage transplantation of hucMSCExo ameliorates hepatic fibrosis induced by CCl₄ in rats， which may be induced by suppressing HSCs activation and the synthesis and secretion of collagen.

Key words: Human umbilical cord mesenchymal stem, Exosomes, Hepatic stellate cell, Hepatic fibrosis