Experimental Study Of DJ-1 Enhancing BMSCs’ Anti-oxidative Stress Through Nrf2 Signaling Pathway

Objective:To study DJ-1 through the Nrf2 signaling pathway to enhance the anti-oxidative stress effect of BMSCs and its mechanism.Methods:（1）Extraction,purification,purification,purification,cultivation,and cultivation of BMSCs to the third generation,using flow cytometry to detect the purity of the cell surface antigen CD44,identified its purity;Induced differentiation,alkaline phosphatase identification of its bone differentiation pote ntial,oil red O dyeing identification of its lipid potential,Alishen blue staining identified its soft bone potential;（2）Transfection with DJ-1 gene in transfection with BMSCs using a lentiviral vector.Stabilocarbromercinal screen was used to detect DJ-1 m RNA amount and DJ-1 m RNA amount and DJ-1 protein expression in the cells using q PC R and Western Blot;H₂O₂ treatment of 500μmol/L was treated with H₂O₂,and the experimental points 4 Group:Group A（BMSCs）,Group B（BMSCs+H₂O₂）,C group（BMSCs/Lv-EGFP+H₂O₂）,D group（BMSCs/Lv-DJ-1-EGFP+H₂O₂）,by detecting various groups of cells Internal oxidative damage（ROS content,Annexin V/PI detection apoptosis）and antioxidant enzyme（Mn SOD,CAT,GP x）protein expression quantitative DJ-1 on BMSCs antioxidative stress loss injury;（3）adopting The slow viral vector DJ-1 m RNA,Nrf2si RN A gene transfected BMSCs,was screened with a stable strain with a stericin,and the experiment was divided into 4 groups:E group（BMSCs+NCsi RNA）,F group（BMSCs+H₂O₂）,G group（BMSCs/Lv-DJ-1+H₂O₂）,H group（BMSCs/Lv-DJ-1/Nrf2si RN A+H₂0₂）,Western Blot detects changes in DJ-1,Nrf2 and antioxidant enzymes（Mn SOD,CAT,GP x）protein expression under different conditions When the silencing Nrf2 and overexpressed DJ-1,the effect of Mn SOD,CAT,GPx protein expression in BMSCs;immunoprecipitation（CO-IP）detection of DJ-1 over-expression under oxidative stress conditions to BMSCs cytoplasm-Keap1 dissociation;Western Blot detects DJ-1 overexpression of DJ-1 on Nrf2 nuclear transfer changes in BMSCs nuclei under oxidative stress conditions;（4）SPSS22.0 statistical software package for single-factor variance analysis,P<0.05 has statistics Learning significance.Results:（1）Successfully extracted,separated,cultured,recovered and ide ntified by BMSCs,BMSCs purity>99%;（2）DJ-1 gene expression and protein expression results showed that DJ-1 overexpression group compared to air virus group,m RNA The quantity is adjusted（3.36±0.16）times,and the amo unt of DJ-1 protein is adjusted（1.71±0.14）times,and the difference has statistically significant（P<0.05）;（3）Measurement of the Group BMSCs in the group:Group A（8.22±2.35）times,group B（99.32±5.32）times,C group（97.44±4.55）times,D group（13.40±2.21）times,D group compared to C group,the ROS content is significantly reduced,the difference has statistics Learning significance（P<0.05）;B group compared to A,the ROS content increased significantly,and the difference was statistically significant（P<0.05）;the D group was compared with Group B and C groups without statistical differences.（2）Annexin V/PI detection BMSCs apoptosis rate:group A（5.20±1.08）%,Group C group（56.40±3.87）%,D group（9.39±2.08）%;D group and C group Compared to the significant reduction in apoptosis,the difference has statistically significant（P<0.05）;Compared to A,the apoptosis rate increases,and the difference has statistically significant（P<0.05）;D group and group A There was no significant difference in comparison between group B and C.（3）Measurement of the relative expression of the antioxidant enzyme（Mn SOD,CAT,GPx）protein in each group:A group of Mn SOD is relatively expressed（1.18±0.12）,CAT relative expression quantity（1.20±0.13）,GPx content（0.97±0.11）times（0.21±0.11）times（0.21±0.11）times（0.27±0.13）times,GPx relative expression quantity（0.28±0.13）times（0.28±0.13）times（0.28±0.13）times（0.28±0.13）times（0.22±0.08）Double,CAT relative expression（0.22±0.09）times,GPx is relatively expressed（0.28±0.09）times,the amount of Mn SOD in group D（1.11±0.16）times,CAT relative expression（1.05±0.14）,GPx The relative expression quantity（0.91±0.13）times;D group compared to the C group,the relative expression of antioxidant e nzymes increased significantly,and the d ifference has statistically significant（P<0.05）;B group compared to A,antioxidant relative expression The amount is significantly reduced,and the difference has statistically significant（P<0.05）;there is no statistically significant difference between the D group and the Group B and Group C.（4）The relative expression of antioxidant enzymes in BMSCs under different relative expression levels of DJ-1 and Nrf2 in each group was measured:the relative expression of Nrf2 in group E（0.87±0.10）times,the relative e xpression of DJ-1（0.47±0.09）Times,relative expression of Mn SOD（0.88±0.10）times,relative expression of CAT（0.86±0.11）times,relative expression of GPx（0.55±0.12）times,relative expression of Nrf2 in group F（0.84±0.07）times,DJ-1 relative expression level（0.23±0.11）times,relative expression level of Mn SOD（0.21±0.10）times,relative expression level of CAT（0.25±0.12）times,relative expression level of GPx（0.09±0.07）times,relative expression of Nrf2 in group G（0.87±0.11）times the relative expression amount of DJ-1,（1.01±0.11）times the relative expression amount of DJ-1,（1.11±0.12）times the relative expression amount of Mn SOD,（0.98±0.09）times the relative expression amount of CAT,and（0.90±0.09）times the relative expression amount of GPx（0.90±0.11）times,the relative expression of Nrf2 in group H（0.08±0.03）times,the relative expression of DJ-1（0.96±0.09）times,the relative expression of Mn SOD（0.22±0.13）times,the relative expression of CAT（0.27±0.05）The relative expression of GPx was（0.25±0.07）times;the relative expression of antioxidant enzymes was significantly higher in group G and H,and the difference was statistically significant（P<0.05）;compared with group E,the antioxidant enzyme The enzyme content was significantly reduced,and the difference was statistically significant（P<0.05）.（5）The relative expression of free Keap1 in BMSCs measured by immunoprecipitation:the relative expression of group A（1.15±0.11）times,the relative expression of group B（0.84±0.12）times,the relative expression of group C（0.72±0.10）times,The relative expression of group D was（0.13±0.07）times.Compared with group C,the relative expression of Keap1 in group D was significantly reduced,and the difference was statistically signi ficant（P<0.05）.（6）In the C-Nrf2 protein group,the relative expression of group A was（0.65±0.05）times,the relative expression of group B was（0.45±0.06）times,the relative expression of group C was（0.41±0.07）times,and the relative expression of group D was measured.（0.02±0.02）times;in the N-Nrf2 protein group,the relative expression of group A（0.33±0.04）times,the relative expression of group B（0.52±0.05）times,the relative expression of group C（0.56±0.06）times,D The relative expression of the group was（0.90±0.09）times;under the condition of DJ-1 overexpression,the relative expression of N-Nrf2 protein was significantly greater than the content of C-Nrf2protein,and the difference was statistically significant（P<0.05）.Conclusions:DJ-1 promotes the expression of antioxidant enzymes（Mn SOD,CAT and GPx）through the Keap-Nrf2-ARE antioxidant molecular signaling pathway,and enhances the ability of BMSCs to resist oxidative stress.