| Acute liver injury is a common clinical condition in pets,often caused by the intake of harmful substances due to the owner’s improper management of their diet.Acute liver injury develops rapidly and,without timely medical intervention,can easily lead to acute liver failure,but there are still no specific therapeutic drugs for this disease.Adipose-Derived Mesenchymal Stem Cells(AD-MSCs),an important part of regenerative medicine,have been shown to repair damaged liver tissue through multiple mechanisms,and this function is mainly attributed to their paracrine effect.When cultured in vitro,AD-MSCs have limited their original therapeutic potential due to changes in the culture environment while hypoxic culture can simulate the physiological environment of MSCs and improve their paracrine function.To this end,we investigated the effects of hypoxia mimetic Deferoxamine(DFO)pretreatment on the proliferation,differentiation,and paracrine properties of canine AD-MSCs,and the effects of DFO pretreated AD-MSCs conditioned medium(AD-MSC-CM)on the injured BRL cell model and the APAP-caused acute liver injury model in mice.The results were as follows:1.Effect of DFO on the biological properties of canine AD-MSCsThe effect of different DFO concentrations on the activity of AD-MSCs at 24 and48 h was examined using the CCK-8 method,and the results showed that the activity of AD-MSCs decreased with increasing DFO concentration,and 25 μM at 48 h was selected as the optimal DFO pretreatment concentration and time;comparing the lipogenic and osteogenic differentiation ability of DFO pretreated and untreated ADMSCs,it showed that DFO pretreatment inhibited the osteogenic differentiation ability;The results of q PCR showed that the gene levels of HGF,IL-6,IL-10,STC-1,TSG-6and VEGF related to hepatocyte damage and repair in AD-MSCs pretreated with DFO were significantly increased,and ELISA assay showed the protein content of TSG-6and STC-1 in its supernatant increased significantly.It is suggested that DFO pretreatment can affect the proliferation and differentiation of AD-MSCs and promote the paracrine effects of AD-MSCs associated with liver injury repair.2.Repair effect of DFO pretreated AD-MSC-CM on APAP-induced liver injuryIn this experiment,DFO-MSC-CM and MSC-CM were prepared with DFO pretreated and untreated AD-MSCs,and their effects on proliferation activity,apoptosis rate,ROS content and antioxidant-related gene expression in BRL cell injury model induced by APAP were investigated by CCK8,Annexin V flow assay,ROS flow assay and q PCR.The results showed that both DFO-MSC-CM and MSC-CM could significantly increase the activity of damaged BRL cells,reduce the rate of apoptosis and the content of ROS.The treatment of DFO-MSC-CM significantly promoted the expression of antioxidant genes Nrf2,NQO-1,and HO-1,while only the level of Nrf2 gene was significantly increased in MSC-CM.The results suggested that MSC-CM and DFO-MSC-CM could repair APAP-induced hepatocyte injury by increasing cell proliferation activity,inhibiting apoptosis,reducing ROS content and increasing antioxidant gene levels,and the latter had a stronger damage repair effect.A mouse model of acute liver injury was established by intraperitoneal injection of 300 mg/kg APAP,and the mice were treated by tail vein injection of MSC-CM and DFO-MSC-CM,the blood and liver were taken 24 h after treatment for serum liver enzyme assay,pathological histological observation,and liver tissue antioxidant enzyme assay.The results showed that injection of MSCs supernatant could reduce the necrosis and bleeding of liver lobules with the effect of DFO-MSC-CM group was more obvious;After modeling,the contents of ALT and AST increased significantly,while the contents of both in the serum of mice after injection of MSCs supernatant decreased significantly,the DFO-MSC-CM group had a downward trend compared with the MSC-CM group,but the difference was not significant;After modeling,the activities of CAT,GSH-Px and SOD in the injured liver tissue were all significantly decreased.After injection of MSCs supernatant,the activities of the three in the DFO-MSC-CM group all increased significantly while in the MSC-CM group,only the content of GSHPx increased significantly.The above findings suggest that DFO enhances the paracrine effect of AD-MSCs by simulating hypoxic environment,thus increasing the content of factors related to liver injury repair in DFO-MSC-CM and having a stronger ability to repair liver injury. |
