Abstract: The purpose of the experiment is to isolate and culture bovine amniotic fluid mesenchymal stem cells (AF-MSCs), and to study its therapeutic effect on alcoholic liver disease (ALD) mice and its effect on the mRNA expression of hypoxia inducible factor-1 α(HIF-1α), vascular endothelial growth factor (VEGF) and Toll-like receptor 4 (TLR4).AF-MSCs were isolated from amniotic fluid mesenchymal stem cells obtained from 4-to 5-month-old bovine embryos, and were identified by RT-PCR, immunofluorescence and induction of adipogenesis. Forty-eight ICR mice (male) were randomly divided into 4 groups:Blank control group (BC group), Model control group (MC group), AF-MSCs group (MC+AF-MSCs group) and silybin capsule (SC) group (MC+SC group), with 12 mice in each group.Except for the mice in BC group were intragastric administrated with 0.2 mL of normal saline, the rest of the groups were intragastrically administered with 56-degree Hongxing Erguotou (5 g·kg ^-1, 2 times·d ^-1) for 4 consecutive weeks; on the 29th day, the MC+AF-MSCs group was given extrahepatic injection Dil-labeled AF-MSCs (5×10⁶ cells·mL^-1), the MC+SC group was given intragastric SC twice a day for one day, total amount of SC was 2.4 mg (dissolve in 56-degree Hongxing Erguotou); after the experiment, the activities of serum glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST) and triglyceride (TG) were measured. HE staining and immunohistochemical methods were used to observe the modeling. Dil traced the colonization and distribution of AF-MSCs in the liver; qRT-PCR was used to detect the expression of HIF-1α, VEGF and TLR4 genes. RT-PCR and immunofluorescence results showed that bovine AF-MSCs were successfully isolated and had the ability to induce adipogenic differentiation. Serological results showed that the activities of ALT, AST and TG in the MC group were significantly higher than those in the BC group (P<0.01). Histopathological section showed that there were pathological changes such as steatosis, inflammatory cell infiltration and hepatic sinusoidal congestion in the liver tissue of the MC group, indicating that the 56-degree Hongxing Erguotou successfully replicated the ALD mouse model; Compared with the MC+SC group, the ALT and AST activities of the MC+AF-MSCs group were significantly reduced (P<0.01), the fatty change in the liver tissue was mitigated, and the liver sinusoids were not congested, indicating that AF-MSCs improved liver function more effective than SC; The qRT-PCR results showed that the expression of HIF-1α, VEGF and TLR4 genes in the MC+AF-MSCs group was significantly lower than that in the MC+SC group (P<0.01), indicating that AF-MSCs are involved in the regulation of HIF-1α/VEGF signaling pathway, and the effect of inhibiting TLR4 expression is better than SC; Cell tracing proved that AF-MSCs colonized the diseased site and chemotaxis more AF-MSCs migrated to the inflammation site and participated in the repair of liver tissue. In summary, this study successfully isolated bovine AF-MSCs. It can participate in the regulation of oxidative stress, angiogenesis, and inhibit the release of inflammatory factors. And the effect of improving ALD is better than SC.

Key words: AF-MSCs, ALD, HIF-1α, xenotransplantation