Separation and identification of hASCs and exosomes Under the inverted microscope, hASCs were fibrous, uniform in size and grew in an eddy-shaped manner. Flow cytometry showed that CD molecules on the surface of hASCs were positive for CD90, CD44, CD105 and CD73, but negative for CD34, CD19 and CD45. After cultured in lipid-induced medium, hASCs were stained with oil red O. Red lipid droplets were observed under optical fiber microscopy. After the differentiation of hASCs was induced by osteogenic induction medium, a large number of red nodules could be seen in intracellular calcium nodules stained with Alizarin red.   Under scanning electron microscopy, exosomes were observed as uniform round cup shapes with vesicular structures between 30 and 150 nm in size. The diameter of exosomes was 30-150 nm by Nanosight granulometer. The results of antibody microarray showed that FLOT1 (flotillin 1), ICAM1 (intercellular adhesion molecule 1), ALIX, CD81, CD63, EpCAM (epithelial cell adhesion molecule), ANXA5 (annexin A5), TSG101 (tumor susceptibility gene 101) were all positive, while GM130 (cis-Golgi matrix protein) was negative. Histogram statistical analysis showed that the gray values of FLOT1, ICAM, ALIX, CD81, CD63, EpCAM, ANXA5 and TSG101 were all higher than those of the blank group (data not shown).   Effect of exosomes on proliferation and apoptosis of activated HSCs The expression level of α-SMA in HSCs stimulated by TGF-β1 was significantly higher than that of HSCs. It indicates that TGF-β1 could activate HSCs (Figure 1A). Compared with the PBS group, the proliferation of activated HSCs was inhibited by exosome, especially at 96 hours. However, there was no significant difference between the 10 μg/L and 1 μg/L exosome treatment groups. It indicates that exosome could inhibit the proliferation of activated HSC (Figure 1B). As shown in Figure 3C, the late apoptosis percentage of activated HSCs in the high or low concentration exosomes groups and control group was 26.5%, 21.4%, and 19.9% respectively, while the early apoptosis percentage was 10.6%, 5.56%, and 6.45% respectively. The early and late apoptosis rate of high concentration exosome treatment group was higher than that of the PBS group. The late apoptosis rate in the low concentration exosome treatment group was higher than that in the PBS group, while the early apoptosis rate in the low concentration exosome treatment group was lower than that of the PBS group (Figure 1C). Compared with PBS and low-concentration exosome treatment groups, the treatment of activated HSCs with high concentration exosomes can significantly promote later apoptosis. It indicates that the apoptosis of activated HSC was significantly promoted when the exosome concentration reached a certain level (Figure 1D). "

Quantitative analysis of experimental animals All rats were involved in the result analysis.    Effect of exosomes on liver fibrosis CCl4 was injected intraperitoneally for 4 weeks to induce liver fibrosis. Sirius red and Masson trichrome staining of liver tissue showed moderate to severe liver fibrosis. Continuous tail venous injection of exosomes alleviated liver fibrosis, with the best effect at 4 weeks compared with the other groups (Figure 2A-L). After liver staining, Ishak was scored and compared statistically (Figure 2M). After intraperitoneal injection of CCl4 in rats, Ishak score reached 3-4, indicating moderate and severe fibrosis has formed. Compared with the rat models of liver fibrosis without PBS or exosome therapy, the Ishak scores of PBS treatment at 2 or 4 weeks were insignificantly different, indicating that the establishment of liver fibrosis model in rats is relatively stable. After 4 weeks of treatment with exosomes, liver fibrosis was significantly reduced. Semi-quantitative fiber statistics showed that exosomes significantly reduced the degree of liver fibrosis after 4 weeks of treatment (Figure 2N). No animals died during the experiment.  "

Effects of exosomes on serum liver function, type PIII and IV-C collagen in rats with liver fibrosis Compared with the exosome treatment group, serum ALT and AST levels were slightly higher, while serum TP and ALB levels were lower in the PBS group, indicating that exosome can improve the liver function. Serum TP level in the EXO-4W group was significantly higher than that in the PBS-4W group (P < 0.05). However, there was no significant difference in serum albumin level between exosomes and PBS groups (Figure 3A-D). It suggests that the effect of exosomes on the recovery of hepatic enzymes is not obvious in rats with liver fibrosis. Serum PIII and IV-C collagen levels in the exosome treatment group were lower than those in the PBS group, and there was statistical significance between EXO-4W and PBS-4W (P < 0.05) (Figure 3E-F). The results showed that exosomes decreased the expression of serum PIII/IV-C and alleviated liver fibrosis.    "

Effect of exosomes on the ECM protein expression in rats with liver fibrosis Immunohistochemical staining showed that the expression of α-SMA and TIMP1 was high in liver tissue of rats with liver fibrosis, while the expression of MMP9 was low. With the prolongation of exosome therapy time, the expression of α-SMA and TIMP1 in liver tissue decreased gradually, and the expression of MMP9 in ECM increased gradually. There was significant difference in protein expression in ECM between PBS-4W and EXO-4W. It indicates that exosomes could inhibit activated HSCs to produce α-SMA and TIMP1, and promote HSCs to produce MMP9. "