Protective Effect Of Vitamin A On Oxidative Damaged Canine Bone Marrow Mesenchymal Stem Cells Induced By Hydrogen Peroxide

Bone marrow mesenchymal stem cells（BMSCs）are a kind of adult stem cells derived from mesoderm.Due to their multi-directional differentiation potential,ability of immunomodulation,they have great potential in cell and tissue engineering and regenerative medicine.Vitamin A is an indispensable fat-soluble vitamin in the body,which plays an important role in animal reproduction,embryonic development,bone development,immunity,and antioxidant.At present,many in vivo and in vitro tests have confirmed that vitamin A is an effective antioxidant.And,vitamin A has the effect of promoting the proliferation of BMSCs and maintaining the characteristics of stem cells.This article aims to explore the biological effects of vitamin A on canine BMSCs cultured in vitro and its effects on oxidative damage of canine BMSCs.Experiment 1 Effects of vitamin A on proliferation,senescence,apoptosis and immunity of canine BMSCs In this experiment,canine BMSCs were isolated from canine bone marrow,and the effects of different concentrations（0.5,1,2,4,8,16,32μmol/L）of VA pre-culture on the proliferation,senescence,apoptosis,and immunity of canine BMSCs were studied to provide a theoretical basis for subsequent anti-oxidation research.In this test,the cell viability and the survival rate was measured by CCK-8 method after 24 and 48 hours of VA pretreatment of canine BMSCs;β-galactosidase stain was used to calculate the proportion of senescence;The related expression of proliferation,senescence,apoptosis and immune genes were detected by RT-q PCR.The results are as follows:VA up-regulated the proliferation genes PCNA,CCND1,Nanog,and down-regulated P27 to promote the proliferation of canine BMSCs.With the increase of VA concentration,the cell survival rate and the relative expression of proliferation gene mRNA both increased,of which 16μmol/L and 32μmol/L are the most obvious;when the VA concentration is below 16μmol/L,it does not have the toxic effect of inducing aging and apoptosis of BMSCs in dogs,but 32μmol/LVA will promote the expression of its aging genes P16,P53,and the apoptotic gene Bax.VA promotes the increase of relative expression of IL-6,IL-8,TNF-α,TGF-β1,VEGF,and CXCL12 mRNA in canine BMSCs,and promotes BMSCs to play an immunomodulatory role.Experiment 2 Effect of vitamin A on H₂O₂-induced oxidative damage model of canine BMSCs This experiment explored the protective effect of VA pretreatment on canine BMSCs oxidative damage model.First,100-1800μmol/L H₂O₂ was used to treat canine BMSC cells for 2h,6h,and 18h respectively.The CCK-8 method andβ-galactosidase senescence staining were used to select the 1300μmol/L H₂O₂ treated for 2h group to establish an oxidative damage model;then 0.5,1,2,4,8,16,32μmol/LVA pretreatment 24h,48h,add1300μmol/L H₂O₂ treatment for 2h,CCK-8 method to detect cell viability,β-galactosidase senescence staining,screening out 16μmol/L VA concentration for follow-up experiments;follow-up experiments are divided into four groups（16μmol/L VA pretreatment alone for 24h group;16μmol/L VA pretreatment for 24h+1300μmol/L H₂O₂treatment for 2h group;1300μmol/L H₂O₂ treatment for 2h group;none treatment control group）,CCK-8 method to detect cell viability,β-galactosidase senescence stain,apoptosis and necrosis staining to calculate the apoptosis rate and necrosis rate,antioxidant index SOD,MDA,GSH-Px,CAT content detection;RT-q PCR to detect the expression of aging,apoptosis genes and antioxidant-related genes.The results showed that:4,8,16,32μmol/L VA pretreatment for24h can significantly increase the survival rate of H₂O₂-induced canine BMSC cell oxidative damage model and reduce its aging rate;among them,16μmol/L VA can reduce the canine BMSCs oxidative damage model aging ratio and relative expression of P16 gene,reduce cell necrosis rate and relative expression of apoptosis genes Bax,Caspase3,Caspase8,increase the expression of anti-apoptotic gene Bcl-2,and at the same time can increase antioxidant enzymes SOD,CAT,GSH-Px content,reduce MDA content;VA can activate the antioxidant transcription factor Nrf2,promote the downstream phase II detoxification enzymes GCLM,NQO1,GPx1,TrxR1 gene transcription,together play an antioxidant effect.Conclusion:8-32μmol/L VA can promote the proliferation of canine BMSCs and promote the expression of its immune-related genes;16μmol/L VA can protect H₂O₂-induced oxidative damage of canine BMSCs by activating the Nrf2 signaling pathway.