Protective Effect Of Astaxanthin On H₂O₂-induced Oxidative Stress In Bone Marrow Mesenchymal Stem Cells

Objective:Bone defect repair is a common problem in clinic.Traditional application of autologous and allogeneic bone grafts has achieved clinical efficacy,but its application is limited.The rapid development of tissue engineered bone has become a new method for bone defect repair,solving a large number of preparation and large area repair problems.This method can overcome the shortcomings of autologous and allogeneic bone transplantation,including the problem of donor damage,immune rejection after transplantation and pathogenicity,etc.,and has become a research hotspot in recent years.Bone mesenchymal stem cells（BMSCs）are self-renewing and multifunctional biological cells that can be isolated,expanded and cultured in vitro and in vivo.BMSCs can not only efficiently differentiate osteoblasts,chondrocytes and various adipocytes,but also produce a variety of regulatory factors on bone metabolism.In recent years,the number of reports on the efficacy of BMSCs in bone tissue engineering and regenerative medicine has been increasing.Due to its therapeutic pluripotency,anti-inflammatory and immune regulation advantages,BMSCs have been widely used in the treatment of a series of diseases such as heart,lung,nerve,hematopoiesis,tendon,ligament and musculoskeletal tissue repair.BMSCs are widely considered as an ideal seed cell for bone tissue engineering.Oxidative stress is the main cause of cell apoptosis after transplantation,so it is very important to find a way to reduce the level of oxidative stress after transplantation.Oxidative stress of the body is mainly a pathological state in which oxygen free radicals and their related metabolites gather in large quantities due to the excessive generation of free radicals in the body and the damage of the internal antioxidant prevention system of the cell,resulting in a series of effects in the cell.Astaxanthin（AST）is a fat-soluble orange ketone carotenoid widely existed in medium Marine organisms of shrimp,crabs and algae.It is mainly used as a food colorant.It is non-toxic and harmless,and plays anti-inflammatory,anti-cancer and anti-oxidation effects in multiple organ systems of human body,and has been applied in clinical research in many fields.Astaxanthin,due to its unique molecular structure,plays an important role in antioxidant activity.This experiment by in vitro culture between human bone marrow mesenchymal stem cells,with hydrogen peroxide（H₂O₂）cell oxidative stress model is set up,astaxanthin pretreatment cell,by detecting level of cell survival and apoptosis,oxidative stress level and the expression of related pathway Nrf2/ARE,explore the protective effect of astaxanthin on H₂O₂-induced bone marrow mesenchymal stem cells（BMSCs）and possible mechanism in oxidative stress.Methods:（1）Human bone marrow mesenchymal stem cells were selected as the research object,cultured and amplified in vitro.CCK-8 assay was used to detect the survival rate of cells induced with different concentrations of H₂O₂（0,100,200,300,400,500,600umol/L）,and BMSC oxidative stress model was established.（2）Cells were treated with different concentrations of astaxanthin culture medium（0,12.5,25,50,100,200,400,800,1600,3200umol/L）,and the survival rate of cells was detected by CCK-8 method to explore the optimal astaxanthin treatment concentration.（3）The cells were preprotected by astaxanthin medium（0,25,50,100,200umol/L）for2h,and then induced by H₂O₂for 24h.The survival rate of cells was determined by CCK-8 method.（4）Cell grouping:the four groups were（1）control group,（2）astaxanthin group,（3）H₂O₂model group,and（4）astaxanthin pre-protection+H₂O₂group.The contents of glutathione（GSH）,superoxide dismutase（SOD）and malondialdehyde（MDA）in astaxanthin pretreated BMSCs were determined by biochemistry method.（5）Annexin V-FITC/PI flow cytometry was used to detect the effect of astaxanthin pretreatment on BMSC cell apoptosis.（6）The expression of protein Nrf2,HO-1 and NQO1 in cells were detected by Western blotting.Results:（1）The results of H₂O₂treatment with different concentrations showed that the optimal concentration and time of H₂O₂solution treated with 500umol/L for 24h was to establish the oxidative stress model.（2）The results of different concentrations of astaxanthin culture solution showed that the astaxanthin solution of 25-200umol/L was in the safe concentration range.（3）The results of CCK-8 method showed that,the viability of cells in H₂O₂model group was significantly decreased vs control（P<0.05）;Compared with H₂O₂model group,the viability of BMSC cells in astaxanthin preprotection group was significantly higher than that in oxidation group,with statistical significance（P<0.05）.The viability of cells in the astaxanthan-containing medium preprotected group at 200umol/concentration was the highest.（4）The SOD activity and GSH content in H₂O₂model group were significantly decreased vs control（P<0.05）,and the MDA content in H₂O₂model group was significantly increased（P<0.05）.Compared with H₂O₂model group,the SOD activity and GSH content in cells in astaxanthin preprotection group were significantly increased（P<0.05）,while the MDA content in cells was significantly decreased（P<0.05）.（5）Annexin V-FITC/PI flow cytometry results showed that the apoptosis rate of cells in H₂O₂model group was significantly increased vs the control（P<0.05）.Compared with H₂O₂model group,the apoptosis rate in astaxanthin preprotection group was significantly decreased（P<0.05）.（6）The protein expressions of Nrf2,HO-1 and NQO-1 in H₂O₂model group were significantly decreased vs control（P<0.05）by Western blotting assay.The protein expression of astaxanthin preprotection group was increased compared with that of H₂O₂model group（P<0.05）.Conclusions:Astaxanthinin preprotection may reduce H₂O₂-induced BMSCs oxidative stress by activating the Nrf2/ARE pathway,improving the survival rate of H₂O₂-induced cells,increasing the activity of SOD and GSH in cells,reducing the content of MDA in cells,and reducing the rate of cell apoptosis.