The Regulation And Its Mechanism Of Mitophagy Protecting Against The Oxidative Stress-induced Injury In Bone Marrow Mesenchymal Stem Cells

Part 1:Extraction,culture and identification of bone marrow mesenchymal stem cells from SD ratsObjective:To establish the extraction,purification and culture methods of bone marrow mesenchymal stem cells（BMSCs）from SD rats,and to observe the cell morphology,detect the multi-direction differentiation ability and determine the immune phenotype of stem cells.methods:SPF male Sprague-Dawley rats（4-8 weeks of age）were used as the experimental subjects.Under aseptic conditions separation of rat femur and tibia,rinse marrow cavity with precooling ahead of the culture medium,through improved the whole bone marrow adherent culture method uniform will blow out of the bone marrow after inoculation to the culture bottle,after training early frequent change of fluid and extend the control quantities of trypsin and exposure time,to achieve purification of BMSCs.The morphological changes of BMSCs were observed,and the expression of molecular markers on the cell surface was detected by flow cytometry.Third generation（P3）bone marrow mesenchymal stem cells（BMSCs）with good growth status were taken and exposed to different induction media to detect the differentiation ability of stem cells in three lines（osteoblast,adipoblast,and chondroblast）.Results:After isolation and purification,BMSCs were mainly spindle and polygonal cells,which showed typical spiral cell colony and active growth.After induction in osteogenic medium for 3 weeks,alizarin red staining was positive.After 3 weeks of induction on adipogenic medium,the oil red O staining was positive.After 28 days of induction in chondroblast medium,Alcian blue staining was positive.Flow cytometry showed positive expression of CD29 and CD90（99.88%and 92.66%,respectively）,but negative expression of CD45（0.53%）.Conclusions:The modified whole bone marrow culture method can efficiently and succinctly obtain rat BMSCs with good homogeneity.The cultured cells isolated and purified by this method have the biological characteristics of mesenchymal stem cells in terms of morphology,expression of cell surface markers and multidirectional differentiation ability,and were identified as rat bone marrow mesenchymal stem cells.Part 2:The effects of mitophagy on oxidative stress induced apoptosis in BMSCsObjective:The oxidative stress microenvironment after BMSCs transplantation was simulated in vitro to clarify the effects of oxidative stress on the apoptosis of BMSCs and the level of mitochondrial autophagy,and to explore the activation of mitochondrial autophagy in the oxidative stress microenvironment and its self-protective role in the apoptosis process of BMSCs.methods:BMSCs were treated with H₂O₂ at a gradient concentration in vitro for 24 h to simulate the microenvironment of oxidative stress.Cell activity was detected by CCK-8 to determine LD50,and the apoptosis model of BMSCs induced by oxidative stress was established at this concentration.Cell apoptosis rate was detected by flow cytometry and TUNEL staining.Western blot was used to detect the expression of apoptosis-related proteins.Mitochondrial autophagy vesicles were observed by immunofluorescence.The changes of autophagy-related proteins were detected by Western blot blot.The changes of mitochondrial membrane potential were detected by flow cytometry and JC-1 staining.BMSCs were treated with mitochondrial autophagy promoter AMA and mitochondrial autophagy blocker Cs A,respectively,and the change of autophagy level was detected by Western blot.Cell apoptosis rate was detected by flow cytometry and TUNEL staining.The changes of mitochondrial membrane potential were detected by flow cytometry.The expression of apoptosis-related proteins was detected by Western blot.Results:In the early stage of oxidative stress injury,the mitochondrial autophagy level of BMSCs was significantly increased,the mitochondrial membrane potential（mt△ψ）was maintained at a high level,and the cell apoptosis rate was low.However,after long-term stress injury,the level of mitochondrial autophagy decreased gradually,the mt△ψdecreased significantly,the apoptosis-related proteins increased significantly,and the apoptosis rate increased significantly.When the mitochondrial autophagy inhibitors were given at the same time as oxidative stress injury,the mt△ψdecreased significantly at the early stage of stress,and the apoptotic protein increased,and the apoptosis rate increased significantly.After treatment with mitochondrial autophagy promoter,the level of mitochondrial autophagy in cells under oxidative stress condition can be further enhanced,and the decrease of mt△ψin the late stress injury can be alleviated,the expression of apoptotic related proteins can be reduced,and the apoptosis rate can be further reduced.Conclusions:At the early stage of oxidative stress injury,BMSCs can remove damaged mitochondria by activating mitochondrial autophagy,maintain the stability of the quality and quantity of mitochondria,and avoid cell apoptosis.In the late stage of oxidative stress injury,mitochondrial autophagy pathway was inhibited,mitochondrial damage was aggravated,and cell apoptosis was accelerated.Increasing the level of mitochondrial autophagy can improve the tolerance of stem cells to oxidative stress,avoid cell apoptosis,and play a protective role.Part 3:Mitophagy plays an anti-aging role during oxidative stress induced injury in BMSCsObjective:The changes of mitochondrial reactive oxygen species（mt ROS）and the activation of mitochondrial autophagy were determined by constructing an in vitro oxidative stress mt△ψaging model,and the relationship between mt ROS and the aging of BMSCs during oxidative stress injury was explored,as well as the role of mitochondrial autophagy in the self-protection of BMSCs during oxidative stress aging.methods:BMSCs were treated with sublethal dose of H₂O₂ for 1 hour,then the solution was changed and cultured for 24 hours to establish the oxidative stress aging model.Galactosidase staining was used to observe cell aging and quantify cell aging.Western blot was used to detect the changes in the expression of age-related proteins,and flow cytometry was used to detect the apoptosis of stem cells to confirm the successful establishment of BMSCs aging model.The formation of mitochondrial autophagy vesicles was observed by immunofluorescence,and the expression of autophagy-related proteins was detected by Western blot.The changes of mt ROS were detected by Mito-Sox Red probe,and the changes of mt△ψwere observed by JC-1 staining.After BMSCs were treated with mitochondrial autophagy inhibitor Cs A,the expression of autophagy related proteins was detected by Western blot,and the formation of autophagy vesicles was observed by immunofluorescence.The cell senescence was observed by galactosidase staining and the cell senescence rate was quantified.The expression of senescence related proteins was detected by Western blot.The changes of mt ROS were detected by Mito-Sox Red probe,and the changes of mt△ψwere observed by JC-1 staining.Results:After H₂O₂ treatment,the number of BMSCs aging staining increased significantly,and the expressions of age-related proteins p21 and p16 increased,while the apoptosis rate of cells detected by flow cytometry did not significantly change,indicating that the oxidative stress aging model was successfully constructed.The number of mitochondrial autophagosomes increased and the expression of autophagy-related proteins was up-regulated by immunofluorescence.Mito-Sox Red probe showed an increase in mt ROS and a significant decrease in mt△ψof JC-1 staining.After treatment with the mitochondrial inhibitor Cs A,mitochondrial autophagy level decreased significantly,mitochondrial activity increased further,and mt△ψdecreased significantly.The number of stem cells stained with galactosidase was significantly increased,and the expression of p21 and p16 protein was also significantly up-regulated.Conclusions:BMSCs treated with sublethal dose of H₂O₂ can construct oxidative stress aging model.In the aging process of BMSCs,mitochondrial mt ROS gradually increase,mitochondria are damaged,and the autophagy pathway of mitochondria is initiated to remove the damaged mitochondria.When the mitochondrial autophagy pathway was blocked,the extent of mitochondrial damage was aggravated,mt ROS were significantly increased,and the degree of cell senescence was further enhanced.These results indicate that BMSCs can remove damaged mitochondria through initiation of mitochondrial autophagy,reduce mt ROS,improve cellular oxidative stress tolerance and avoid cellular senescence.Part 4:Pink1/Parkin-mediated mitophagy protects against the oxidative stress induced senescence in BMSCsObjective:In the process of oxidative stress aging of BMSCs,the activation of PINK1 and Parkin and the mechanism by which PINK1 and Parkin mediate the initiation of mitochondrial autophagy were determined.To clarify the role of PINK1/Parkin-mediated mitochondrial autophagy in oxidative stress-induced aging.methods:The expression of PINK1 and Parkin in mitochondria was detected by Western blot after the cells were treated with sublethal doses of H₂O₂.Parkin lentivirus was constructed to reduce the expression of Parkin,and the changes of mitochondrial autophagy,senility and mt ROS and mt△ψwere detected.Mitochondrial autophagy level,aging changes,mt ROS and mt△ψwere detected by giving Parkin promoter Sal while interfering with Parkin,so as to confirm that oxidative stress induced mitochondrial autophagy was dependent on the activation of Parkin.Lentivirus of PINK1 was constructed to reduce the expression of PINK1.Immunofluorescence and Western blot were used to detect the changes in mitochondrial autophagy and the expression level of Parkin in mitochondria.The aging phenotype was detected by galactosidase staining and Western blot.JC-1 staining was used to detect mt△ψ,and Mito-Sox Red was used to detect Mtros.After PINK1 was knocked out and Parkin promoter Sal was given,mitochondrial autophagy level and senescence changes,mt ROS level and mt△ψwere detected,so as to clarify the effects of PINK1 on mitochondrial autophagy,recruitment of Parkin on mitochondria and cell senescence.To comprehensively evaluate whether the initiation of mitochondrial autophagy in the process of oxidative stress injury mainly depends on the activation of PINK1/Parkin signaling pathway and its influence on premature aging of BMSCsResults:（1）Expression of PINK1 and Parkin on mitochondria were up-regulated during oxidative stress aging of BMSCs.（2）After decreasing Parkin expression,mitochondrial autophagy level and mt△ψwere significantly decreased,while mt ROS level was increased,and the senescence of BMSCs was increased.（3）The use of Parkin promoter Sal can up-regulate the level of mitochondrial autophagy,reduce the degree of mitochondrial damage and control the content of mt ROS,suggesting that the initiation of mitochondrial autophagy in BMSCs induced by oxidative stress is dependent on the activation of Parkin and plays a protective role in cells.（4）After decreasing PINK1 expression,mitochondrial autophagy level and mt△ψwere significantly decreased,while mt ROS level was increased,and the senescence of BMSCs was increased.（5）The use of Parkin promoter Sal could not reverse this trend,suggesting that the activation of PINK1 is the primary condition for the initiation of mitochondrial autophagy,and Parkin needs to be recruited by PINK1 to initiate autophagy above the mitochondria.These results indicate that the initiation of mitochondrial autophagy in the oxidative stress aging process of BMSCs is mainly dependent on the activation of PINK1/Parkin signaling pathway.Conclusions:Oxidative stress activates PINK1 and recruits Parkin to the mitochondria,which in turn initiates the mitochondrial autophagy pathway.BMSCs can remove damaged mitochondria by activating PINK1/Parkin-mediated mitochondrial autophagy,maintain the metabolic stability of mitochondria,improve the tolerance to oxidative stress,avoid premature cell aging and play an anti-aging role.