The Role And Mechanism Of BLK Cross-linking Stress Network Inhibiting BMSCs Apoptosis On The Tissue-engineered Bone Repairing Steroid-induced Osteonecrosis Of The Femoral Head

Objective:Steroid-induced osteonecrosis of the femoral head（SONFH）is a common and refractory articular dysfunction in orthopedics.Currently,there is a lack of accurate and effective early cure methods.Bone marrow mesenchymal stem cells（BMSCs）combined with tissue-engineered bone provide a new idea and method for the treatment of early SONFH.However,oxidative stress in the osteonecrosis area limits the efficacy of BMSCs transplantation,and intervention strategies need to be found.In previous studies,we found that a small number of transplanted BMSCs have oxidative stress resistance and could survive in the oxidative stress microenvironment in the osteonecrosis area.However,the mechanism by which these BMSCs acquire oxidative stress resistance remains unclear.This study aims to find the key regulatory molecules that mediate BMSCs to acquire oxidative stress resistance,clarify its regulatory mechanism,and use the regulatory mechanism to make BMSCs generally acquire oxidative stress resistance,so as to improve the survival rate of transplanted BMSCs.It provides a new strategy to solve the problem of stress-induced apoptosis of BMSCs and improve the efficacy of tissue-engineered bone transplantation.Methods:1.The SONFH model of Sprague-Dawley（SD）rats was established by glucocorticoids（GCs）combined with endotoxin method.At 2 and 4 weeks（w）after drug injection,the expression levels of nicotinamide adenine dinucleotide phosphate oxidase 1（NOX1）,catalase（CAT）and reactive oxygen species（ROS）in the femur head were detected by immunofluorescence staining,and the necrosis of the femoral head was detected by magnetic resonance imaging（MRI）,hematoxylin-eosin staining（HE staining）and micro computed tomography（micro-CT）,so as to evaluate the level of oxidative stress in the femoral head region during the occurrence and development of SONFH.The tissue-engineered bone was constructed with BMSCs to repair SONFH model of SD rats.At 48 hours（h）after the tissue-engineered bone transplantation,the ROS levels in transplanted BMSCs were detected by dihydroethidium（DHE）,and the survival of transplanted BMSCs was detected by In Vivo Imaging System of animals and Td T-mediated d UTP nick-end labeling（TUNEL）.At 12w after the tissue-engineered bone transplantation,HE staining and micro-CT were used to detect the repair of osteonecrosis,and to observe the level of oxidative stress in the transplanted BMSCs and the effect of oxidative stress on the survival and repair efficacy of transplanted BMSCs.To study the level of oxidative stress and its influence during the occurrence,development and repair of SONFH.2.The combination of proteomics and phosphorylated proteomics was used to analyze oxidative stress-resistant BMSCs and oxidative stress-sensitive BMSCs treated with hydrogen peroxide（H₂O₂）,and to screen the differential proteins and phosphorylated proteins.Then,the kyoto encyclopedia of genes and genomes（KEGG）and gene set enrichment analysis（GSEA）were performed for differential proteins and phosphorylated proteins to select relevant signaling pathways enriched in redox and apoptotic regulatory networks.Finally,the core molecule regulating redox and apoptosis signaling networks,B lymphoid tyrosine kinase（BLK）,was identified through the Kinase-Substrate Interaction Networks analysis of the core proteins of these key signaling pathways.The real-time quantitative polymerase chain reaction（q PCR）and Western blotting were used to detect the expression and phosphorylation levels of BLK in oxidative stress-resistant BMSCs and oxidative stress-sensitive BMSCs,respectively,and to verify the combined omics data.To screen and validate the key regulatory molecule BLK that mediates BMSCs to acquire oxidative stress resistance.3.In vitro,BLK was overexpressed in oxidative stress-sensitive BMSCs by gene transfection overexpression technique,or BLK was knocked down in oxidative stress-resistant BMSCs by short hairpin RNA（sh RNA）interference technique.Then,under oxidative stress,ROS levels in BMSCs were detected by DHE fluorescent probe,and apoptosis of BMSCs was detected by flow cytometry and TUNEL staining.The expression levels of apoptosis-related proteins[B-cell lymphoma 2（Bcl-2）,Bcl-2 associated death promoter（Bad）,and Survivin protein]and antioxidant enzymes[CAT,heme oxygenase-1（HO-1）,and manganese superoxide dismutase（Mn SOD）]were detected by Western blotting.To investigate the role of BLK in mediating BMSCs to acquire oxidative stress resistance and inhibiting stress-induced apoptosis of BMSCs.4.In vivo,the 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodid（Di R）and red fluorescent protein（RFP）were used to label BMSCs.Then,the tissue-engineered bone was constructed by oxidative stress-sensitive BMSCs with overexpression of BLK or oxidative stress-resistant BMSCs with BLK knocked down,which was used to transplant and repair the SONFH model of SD rats.At 48h after transplantation,the expression levels of CAT and Bcl-2 in the transplanted BMSCs were detected by immunofluorescence staining,ROS levels in the transplanted BMSCs were detected by DHE fluorescence probe,apoptosis of the transplanted BMSCs was detected by TUNEL staining,and survival of the transplanted BMSCs was detected by the In Vivo Imaging System of animals.At 12w after transplantation,the levels of osteogenic markers runt-related transcription factor 2（Runx2）and Osterix（Osx,also known as Sp7）were detected by immunofluorescence staining,new bone regeneration at the graft site was assessed by HE and Masson staining,and bone defect repair was assessed by micro-CT.To investigate the effect of BLK on the survival rate of BMSCs transplantation and the efficacy of SONFH repair.5.In terms of mechanism of BLK,phosphorylated proteomic analysis was performed on oxidative stress-resistant BMSCs with BLK knockdown and oxidative stress-sensitive BMSCs with overexpression of BLK.By KEGG and GSEA analysis,the intersection of the key signal pathways enriched after BLK knock down and the key signal pathways enriched after BLK overexpression was conducted,and four overlapping signaling pathways were obtained,extracellular signal-regulated kinase1/2（ERK1/2）,protein kinase B（AKT）,signal transducer and activator of transcription 3（STAT3）,and nuclear factor-kappa B（NF-κB）.Then,the expression and phosphorylation levels of core proteins in ERK1/2,AKT,STAT3 and NF-κB signaling pathways were detected by Western blotting in oxidative stress-resistant BMSCs and oxidative stress-sensitive BMSCs,respectively,to verify the phosphorylomics data.Subsequently,the nuclear translocations of key proteins in ERK1/2,AKT,STAT3 and NF-κB pathways were detected by immunofluorescence in oxidative stress-resistant BMSCs with knockdown BLK and oxidative stress-sensitive BMSCs with overexpression of BLK,respectively.The phosphorylation levels of ERK1/2,AKT,STAT3 and NF-κB were detected by Western blotting,as well as the expression levels of antioxidant and anti-apoptosis related proteins in the downstream of each pathway.To study the effect of BLK on activation of ERK1/2,AKT,STAT3 and NF-κB signaling pathway.6.In BMSCs that overexpressed BLK,the ERK1/2,AKT,STAT3,and NF-κB signaling pathways were blocked by signaling pathway inhibitors,respectively.Then,the expression of antioxidant and anti-apoptosin related proteins in each pathway were detected by Western blotting under oxidative stress.The flow cytometry and TUNEL staining were used to detect apoptosis of BMSCs.To study the role of ERK1/2,AKT,STAT3 and NF-κB signaling pathways in BMSCs anti-oxidative stress mediated by BLK.Results:1.At 2w after GCs injection,the expression of NOX1 was up-regulated,the CAT expression was down-regulated,and the level of ROS was increased in the femoral head of the model group compared with the control group.With the extension of time,the NOX1 expression and levels of ROS in the femoral head were further increased,and the expression of CAT was significantly decreased at 4w after injection.At this time,abnormal signals in the femoral head region were visible by MRI,and typical osteonecrosis signs such as empty bone lacunae and trabecular interruption were visible by HE and micro-CT.The tissue-engineered bone was constructed with BMSCs and used for transplantation and repair of SONFH model.At 48h after BMSCs transplantation,DHE fluorescence probe showed a large number of ROS in the transplanted BMSCs,the in vivo imaging system of animals and TUNEL staining showed a large number of stress-induced apoptosis of transplanted BMSCs,only a very small number of BMSCs could survive in the osteonecrosis area（<10%）.At 12w after BMSCs transplantation,HE staining and micro-CT showed that the bone defect area in the graft group was filled with fibrous connective tissue,and the reconstruction effect of bone trabeculae was poor.The bone mineral density（BMD）,the bone volume fraction（BVF）,the trabecular number（Tb.N）,and the trabecular thickness（Tb.Th）were lower than those of the normal group（P<0.05）.The results indicated that during the occurrence and development of SONFH,the imbalance of redox homeostasis in the femoral head region was gradually aggravated,and a large number of transplanted BMSCs suffered oxidative stress in the osteonecrosis area,and further apoptosis occurred,which limited the efficacy of BMSCs transplantation.2.A total of 128 proteins with significantly different expression levels and 1163significantly different phosphorylated peptides（corresponding to 528 proteins）were screened between oxidative stress-resistant BMSCs and oxidative stress-sensitive BMSCs using proteomics and phosphorylated proteomics analysis（Fold change>2,P<0.05）.According to KEGG and GSEA analysis,the redox and apoptosis signaling pathways in which differentially phosphorylated proteins are significantly enriched are as follows:STAT,NF-κB,forkhead box O（FOXO）and AMP-activated protein kinase（AMPK）,AKT and ERK,etc.A core molecule:BLK that may regulate redox and apoptotic signaling networks has been identified by performing the protein-protein interaction network analysis of core proteins in these signaling pathways.According to Predict Protein（https://predictprotein.org）platform,amino acids at positions 313,367,368,450,454 and 466 in the kinase domain of BLK are cysteine,which has the structural basis to respond to ROS.Similarly,these modification sites also exist in the human BLK gene sequence,which is conserved.q PCR and Western blotting results confirmed that the level of BLK expression and phosphorylation in oxidative stress-resistant BMSCs was significantly higher than that in oxidative stress-sensitive BMSCs,and the level of BLK expression and phosphorylation in oxidative stress-resistant BMSCs was further significantly increased after treatment BMSCs with H₂O₂.This is consistent with the results of omics,suggesting that BLK may be a key regulatory molecule mediating the acquisition of oxidative stress resistance in BMSCs.3.Up-regulation of BLK expression in oxidative stress-sensitive BMSCs significantly increased the expression levels of Bcl-2,Survivin,p-Bad,HO-1,CAT and Mn SOD,and decreased the intracellular ROS levels under oxidative stress.Meanwhile,TUNEL staining and flow cytometry showed that overexpression of BLK reduced the apoptosis rate of the oxidative stress-sensitive BMSCs under oxidative stress.On the contrary,knockdown of BLK gene in oxidative stress-resistant BMSCs resulted in down-regulated expressions of Bcl-2,Survivin,p-Bad,HO-1,CAT and Mn SOD,significantly increased intracellular ROS levels,and increased damage of deoxyribonucleic acid（DNA）（TUNEL⁺cell ratio increased）.BLK knockdown significantly increased the apoptosis rate of the oxidative stress-resistant BMSCs under oxidative stress.These results suggest that overexpression of BLK can promote BMSCs to acquire oxidative stress resistance and inhibit stress-induced apoptosis,while BLK knockdown can lead to loss of oxidative stress resistance of BMSCs.4.At 48h after BMSCs transplantation in rat SONFH model,immunofluorescence staining showed that compared with the control oxidative stress sensitive BMSCs group,the expression levels of CAT and Bcl-2 in the sensitive BMSCs that overexpressed BLK were significantly up-regulated,the intracellular ROS levels in the transplanted area were decreased,the percentage of TUNEL⁺cells was decreased,and the Di R fluorescence intensity in the transplantation area showed no obvious attenuation compared with 0h after transplantation.In contrast,after BLK gene knockdown in oxidative stress-resistant BMSCs,compared with control resistant BMSCs,BLK gene knockdown resulted in down-regulated CAT and Bcl-2 expression levels and increased intracellular ROS levels in BMSCs.TUNEL staining and in vivo imaging system of animals showed that the survival rate of transplanted BMSCs in the transplantation aera was significantly decreased.At 12w after transplantation,immunofluorescence staining further showed that the expression levels of Runx2 and Osx,the osteogenic markers of oxidative stress-sensitive BMSCs in the transplantation area were lower,HE and Masson staining showed less bone area and new bone area in the defect area,micro-CT showed large bone defect.BMD,BVF,Tb.N and Tb.Th were significantly lower than those in normal group（P<0.05）;However,after BLK was overexpressed in the sensitive BMSCs,the level of osteogenetic markers in the graft area was significantly increased,HE and Masson staining showed significant increases in bone area and new bone area,micro-CT showed a regular arrangement of bone trabeculae,and the repair of the bone defect area was complete.BMD,BVF,Tb.N and Tb.Th also reached the level similar to that of the normal group.For the efficacy of oxidative stress-resistant BMSCs transplantation in vivo,the results were similar.The expression level of osteogenic markers in the transplantation area was significantly improved after the transplantation of resistant BMSCs,and the new bone regeneration in the transplantation area was promoted and the bone defects were repaired.However,the BLK gene knockdown of oxidative stress resistance BMSCs did not significantly improve the expression level of osteogenic markers at the graft site,and the efficacy of new bone regeneration and bone defect repair was not ideal.These confirmed that overexpression of BLK can improve the survival rate of BMSCs transplantation in vivo and promote the repair of SONFH.5.Phosphorylated proteomics was used to screen downstream targets regulated by BLK.The results showed that differential phosphorylated proteins were mainly enriched in apoptosis,ERK1/2,AKT,STAT3,NF-κB and tumor protein 53（P53）signaling pathways in oxidative stress resistance BMSCs with BLK-knockdown.In oxidative stress-sensitive BMSCs that overexpressed BLK,differentially phosphorylated proteins were mainly enriched in ERK1/2,AKT,STAT3,NF-κB,AMPK and FOXO signaling pathways.The ERK1/2,AKT,STAT3 and NF-κB pathways were simultaneously enriched,and their activation states were opposite in the two groups.Overexpression of BLK in oxidative stress-sensitive BMSCs increased the phosphorylation levels of ERK1/2,AKT,STAT3 and NF-κB,promoted the nuclear translocation of ERK1/2,nuclear factor erythroid 2-related factor 2（Nrf2）,STAT3 and NF-κB,and increased the expression of antioxidant and anti-apoptosis related proteins in the downstream of each pathway.On the contrary,when BLK gene was knocked down in oxidative stress-resistant BMSCs,the phosphorylation levels and nuclear translocation of key proteins in ERK1/2,AKT,STAT3 and NF-κB signaling pathways were reduced,pathway activation was inhibited,and the expression levels of downstream target proteins of each signaling pathway were also significantly decreased.This suggests that BLK regulates the activation of ERK1/2,AKT,STAT3,and NF-κB signaling pathways.6.In BMSCs that overexpressed BLK,the ERK1/2,AKT,STAT3,and NF-κB signaling pathways were blocked by signaling pathway inhibitors.Western blotting analysis showed that blocking ERK1/2 signaling pathway down-regulated the expression of CAT,glutathione reductase（GR）and HO-1,while blocking AKT signaling pathway down-regulated the expression of Survivin,p-Bad and phospho-caspase9（p-CASP9）,and blocking STAT3 signaling pathway down-regulated the expression of Bcl-2,Bcl-XL and cellular-myelocytomatosis oncogene（c-Myc）.The blocking of NF-κB pathway resulted in the down-regulation of Bcl-2 related protein A1（Bfl-1）,inhibitor of apoptosis 1/2（IAP1/2）and Mn SOD expression.At the same time,after blocking ERK1/2,AKT,STAT3 and NF-κB,the percentage of TUNEL⁺positive cells were increased significantly in all groups,and the flow cytometry analysis showed that the stress-induced apoptosis rate of BMSCs in each group was significantly increased,the effect of BLK on the resistance to stress apoptosis was weakened.These results confirm that BLK inhibit stress-induced apoptosis of BMSCs by targeting the activation of ERK1/2,AKT,STAT3,and NF-κB signaling pathways.Conclusions:In this study,we identified the key regulatory molecule BLK that mediates BMSCs to acquire oxidative stress resistance by combining omics in oxidative stress resistant BMSCs.As a kind of oxidative stress sensor,BLK can sense the change of redox state in cells,make BMSCs respond quickly to oxidative stress stimulation,and inhibit stress-induced apoptosis of BMSCs.In terms of mechanism,BLK is a tyrosine kinase,which can activate its tyrosine kinase activity by changing its own phosphorylation level,and can cross-link ERK1/2,AKT,STAT3 and NF-κB signaling pathways to synergistically regulate redox and apoptotic signaling networks,thereby making BMSCs acquire oxidative stress resistance and inhibit stress-induced apoptosis of BMSCs.BLK is expected to be a potential intervention target to provide new strategies for improving the survival rate of BMSCs transplantation and the repair efficacy of stem cells in early SONFH.