Puerarin Improves The Effect Of Hydrogen Peroxide On Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells Through NRF2 Signaling Pathway

Objective:To explore the effect of puerarin（Pue）on bone marrow mesenchymal stem cells（BMSC）and its mechanism of osteogenic differentiation under oxidative damage conditions,and to explore the role of NRF2 signaling pathway in the above process.Methods:BMSC from SD rats were isolated and purified by total bone marrow attachment and gradient centrifugation and cultured up to 4 passages to observe cell homogeneity and attachment.The survival rate of cells under hydrogen peroxide（H₂O₂）and Pue was detected by CCK8,and change process of reactive oxygen species（ROS）was detected by flow cytometer,testing over time and screening for appropriate concentrations,timing of Pue and H₂O₂interventions.The well-grown P4 generation BMSCs were divided into normal control group,H group（300μmol/L H₂O₂）,P group（0.1μmol/L Pue）,P+H group（0.1μmol/LPue+300μmol/L H₂O₂）,M+P+H group（0.1μmol/L NRF2 signaling pathway inhibitor ML385+0.1μmol/L Pue+300μmol/L H₂O₂）.The contents of malondialdehyde（MDA）and reduced glutathione（GSH）in cells were detected,and Bcl2-related x protein（Bax）,cysteinyl aspartate protease-3（Caspase3）,B lymphocytes Cytoma-2（Bcl2）,nuclear factor E2-related factor-2（NRF2）,heme oxygenase-1（HO1）,quinone oxidoreductase-1（NQO1）factor were detected by PCR and WB.WB detection of osteogenesis-related proteins runt-related transcription factor-2（RUNX2）,bone morphogenetic protein-2（BMP2）,alizarin red staining of calcium nodules with the naked eye and CPC solution for dissolving mineralized nodules.Results:1.The isolated and purified BMSC cells showed uniform arrangement and good adherence.The rate of positivity of the CD90 surface antigen molecule detected by the flow-through technique was 98.32%.And CD29 molecule expression rate was99.76%,CD45 molecule expression rate was 1.08%and CD34 molecule expression rate was 0.89%.Alkaline phosphatase（ALP）staining showed irregular black granular precipitates between cells,Alizarin Red S showed opaque nodular substances within cells.and Alizarin Red S showed good osteogenic differentiation.Oil red O staining showed good adipogenic differentiation.2.（1）The survival rate of the cells in the 400,500μmol/L H₂O₂decreased after12h of intervention（P<0.01）,and when the intervention was 24 h,the cells in the 300,400,and 500μmol/L H₂O₂groups.The survival rate decreased（P<0.01）,the survival rate of cells in the 400 and 500μmol/L H₂O₂groups decreased（P<0.01）at 48 hours of intervention.The cell survival rates in the 300μmol/L H₂O₂at 12,24,48h is（77.7±12.7）%,（74.7±2.1）%,and（62.7±21.7）%.（2）Compared with the control group,at 6 and 12 hours of intervention,the levels of ROS in each concentration of H₂O₂group increased（P<0.05）,among which the 300 and 400μmol/L groups were the most significant,reaching a peak at 12 hours and decreasing to a lower level at 24 hours.（3）The effect of Pue on the survival rate of BMSC cells under oxidative damage:The Pue pretreatment groups with different concentrations had an inverted"U"shape,and the 0.1μmol/L Pue pretreatment group had the most obvious improvement on the cell survival rate.With the H₂O₂group,the survival rate of the 0.1μmol/L Pue pretreatment group was increased at 24,48,and 72 h（P<0.05）.3.Detection of each index in 5 experimental groups:（1）Changes of MDA and GSH content:MDA in H group increased and GSH decreased（both P<0.01）.Pue pretreatment reduced the degree of cell damage.Compared with the H group,MDA decreased and GSH increased in the P+H group（both P<0.01）.After administration of ML385,the protective effect of Pue was partially inhibited.Compared with the P+H group,MDA increased and GSH decreased（both P<0.01）.（2）Bax,Caspase3,Bcl2 gene and protein expression:Compared with NC,pro-apoptotic gene and protein Bax,Caspase3 in H was increased,and the expression of anti-apoptotic gene and protein Bcl2 was decreased（all P<0.01）.Compared with the H group,the gene and protein expressions of Bax and Caspase3 in the P+H group were decreased,and the gene and protein expressions of Bcl2 were increased（all P<0.01）.Compared with the P+H group,the gene and protein expressions of Bax and Caspase3 in the M+P+H group were increased,and the expression of the Bcl2 gene and protein was decreased（all P<0.01）.（3）NRF2,HO1,NQO1 gene and protein expression:Compared with NC group,HO1,NQO1 gene and protein expression in H group increased（P<0.05）.Compared with group H,the expression of NRF2 gene,nuclear NRF2 protein,HO1,NQO1 gene and protein increased in P+H group（P<0.01）,plasma NRF2 protein decreased（P<0.01）.Compared with the P+H group,the gene and protein expressions of NRF2,nuclear NRF2,plasma NRF2,HO1,NQO1 in the M+P+H group were all decreased（P<0.01）.（4）RUNX2 and BMP2 protein expression:Compared with NC,RUNX2 and BMP2 in H group decreased（P<0.01）,the expression of RUNX2increased in the P group（P<0.01）.Compared with the H group,the expressions of RUNX2 and BMP2 in the P+H group were decreased（P<0.01）;compared with the P+H group,the expression of osteogenic protein was significantly decreased after ML385 blockade（P<0.01）.（5）Determination of mineralization:cell mineralization decreased after H₂O₂（P<0.01）.After the intervention of Pue alone,the degree of mineralization of cells increased（P<0.01）.After Pue pretreatment of cells in oxidative stress state,its degree of mineralization of cells was significantly improved compared with H₂O₂group（P<0.01）.After the addition of inhibitor,the degree of mineralization in M+P+H was not only lower than P+H（P<0.01）,but also lower than that in group H（P<0.01）.Conclusions:1.When the concentration of puerarin was 0.1μmol/L,it could promote the proliferation of BMSC cells and improve their anti-oxidative,anti-apoptotic and osteogenic differentiation abilities.2.Pue has a protective effect on cells damaged by oxidative stress,and NRF2signaling path way inhibitor ML385 can block part of Pue.The mechanism may be:Pue can exert anti-oxidative and anti-apoptotic effects through the NRF2 pathway,and promote the survival rate and osteogenic differentiation ability of BMSC cells.