The Role And Mechanism Of Hydrogen Peroxide Pretreated Npmscs In The Treatment Of Intervertebral Disc Degeneration

1.1 Background and Purpose:Intervertebral disc degeneration（IVDD）is a crucial cause of chronic low back pain（CLBP）and lower limb dysfunction,causing heavy economic burden to society.Although surgery is a common way to relieve these symptoms,surgical treatment does not restore the biological and mechanical support function of the disc and may even alter the biomechanical function of the entire spine,leading to further degeneration of the adjacent disc.Therefore,it is necessary to explore new feasible treatment strategies for IVDD.Nucleus pulposus mesenchymal stem cells（NPMSCs）are a kind of mesenchymal stem cells found in nucleus pulposus,which have better potential in the treatment of IVDD by cell transplantation.Compared with other types of stem cells,NPMSCs can better resist the extracellular environment of low PH,and after differentiation into nucleus pulposus cells,NPMSCs can better secrete extracellular matrix,thus maintaining the morphology and biological function of nucleus pulposus.Therefore,NPMSCs transplantation is a promising treatment for IVDD.However,due to the harsh microenvironment in the degenerated intervertebral disc,the transplanted NPMSCs showed weak proliferation ability,high apoptosis rate,and low transplantation efficiency.Therefore,it is urgent to explore feasible methods to improve the therapeutic effect of NPMSCs transplantation.Pretreatment of transplanted cells is an effective way to improve cell transplantation therapy.Previous studies have shown that optimal concentration of hydrogen peroxide（H₂O₂）can improve the ability of cell transplantation to repair skin wounds.In view of this,this study explores the effects of H₂O₂pretreatment of NPMSCs on their proliferation,anti-apoptosis and anti-oxidative stress ability and its mechanism,and further explores whether pretreatment of NPMSCs can improve the effect of IVDD treatment.1.2 Methods:1.2.1 Isolation,culture,and identification of NPMSCs（1）The 8-week-old male SD rats were selected,and the NP tissues of the rats were collected.The NPMSCs were obtained and amplified after digestion with type II collagenase.（2）Osteogenic differentiation medium,chondrogenic differentiation medium and lipogenic differentiation medium were cultured for 21 days,and then stained with alizin red dye,Alcian blue dye and oil red O dye,respectively,and the differentiation abilities of NPMSCs were observed under the microscope.（3）FITC-labelled anti-CD34,CD45,CD73,CD90 and CD105 antibodies were co-incubated with NPMSCs,and flow cytometry was used to detect the intensity of fluorescence carried by cells after incubation with different antibodies.1.2.2 Explore the influence and mechanism of H₂O₂on NPMSCs proliferation（1）CCK-8 method and Ed U555 cell proliferation assay kit were used to detect the effect of different concentrations of H₂O₂on cell proliferation for 12 h.（2）Western blotting experiments were conducted to detect the effects of different concentrations of H₂O₂on the phosphorylation degree of key proteins in the Hippo signaling pathway in NPMSCs.1.2.3 Explore the influence and mechanism of H₂O₂pretreatment on the antioxidant stress ability of NPMSCs（1）ROS specific fluorescent probe DCFH-DA was used to detect ROS content in NPMSCs treated with different concentrations of H₂O₂,and ROS content in NPMSCs treated with 75μM H₂O₂for 12 h and then subjected to oxidative stress again.（2）Mitochondrial membrane potential（MMP）specific fluorescent probe Mito Tracker Red CMXRos was used to detect the level of mitochondrial membrane potential in NPMSCs treated with different concentrations of H₂O₂.（3）Western blotting experiments were conducted to detect the effects of different concentrations of H₂O₂on key proteins in the Keap1-Nrf2 signaling pathway in NPMSCs.1.2.4 Explore the influence and mechanism of H₂O₂pretreatment on the anti-apoptosis ability of NPMSCs（1）Annexin VPE and 7-AAD were used to detect the apoptosis rate of NPMSCs treated with different concentrations of H₂O₂for 12 h,and to detect the apoptosis rate of NPMSCs treated with 75μM H₂O₂for 12 h after receiving apoptosis-induced stimulation again.（2）Western blotting experiments were conducted to detect the effects of different concentrations of H₂O₂on the contents of key proteins in mitochondria-related apoptotic pathways in NPMSCs.1.2.5 Explore whether H₂O₂preconditioning can improve the ability of NPMSCs transplantation to treat IVDDThe rat tail IVDD model was constructed by acupuncture method,and NPMSCs pretreated with different concentrations of H₂O₂were injected into the degenerated intervertebral disc.Then,the survival of NPMSCs after transplantation was evaluated by small animal fluorescence imaging apparatus.The percentage of disc height index（DHI）（DHI%）was calculated by X-ray of the rat tail vertebra.The intervertebral discs of rats in each group were made into paraffin sections and stained with hematoxylin eosin（HE）and saffranine O-solid green to evaluate the effects of different treatments on the morphology of deformed intervertebral discs in rats.1.3 Results:1.3.1 NPMSCs exhibit typical mesenchymal stem cell characteristicsThe NPMSCs showed helical growth after culture and passage.Alizarin red staining showed the formation of mineralized nodules after osteogenic induction of NPMSCs.After induction of cartilage differentiation,alcian blue staining showed obvious proteoglycan sulfide staining.Oil red O staining showed that lipid droplets of different sizes were formed in the cells after induction of lipid differentiation.Flow cytometry showed that CD73,CD90 and CD105 were positive,while CD34 and CD45 were negative.1.3.2 Appropriate concentration of H₂O₂promotes the proliferation of NPMSCs by inhibiting the Hippo signaling pathwayCCK-8 experiment showed that 75μM and 100μM H₂O₂treatment significantly promoted the proliferation of NPMSCs,while 300μM H₂O₂significantly inhibited the proliferation of NPMSCs.Ed U staining showed that Ed U-positive cells were significantly increased after 75μM H₂O₂treatment compared with the 0μM H₂O₂group.The Ed U-positive cells decreased significantly with 300μM H₂O₂.Western blotting results showed that75μM H₂O₂could promote the expression of cyclin D1 and inhibit the expression of P16.In contrast,300μM H₂O₂treatment significantly inhibited the expression of cyclin D1 and promoted the expression of P16.Western blotting results showed that 75μM H₂O₂reduced the phosphorylation levels of key proteins in the Hippo pathway,including phosphorylated large tumor suppressor kinase 1（p-Mst1）,phosphorylated mercapto pyruvate thiotransferase 1（p-Lats1）and phosphorylated YES1-related transcriptional regulator（p-Yap）.The NPMSCs treated with 300μM H₂O₂showed opposite results.1.3.3 Pretreatment of 75μM H₂O₂enhanced the antioxidant stress ability of NPMSCs by activating the Keap1-Nrf2 signaling pathwayWith the increase of H₂O₂treatment concentration,the intracellular ROS concentration also increased.However,ROS content of NPMSCs pretreated with 75μM H₂O₂decreased significantly when they were exposed to 300μM H₂O₂again,compared with 300μM H₂O₂pretreatment group.The mean fluorescence intensity of CMXRos in NPMSCs was significantly decreased after 300μM H₂O₂treatment.However,the fluorescence intensity of NPMSCs treated with75μM H₂O₂and then treated with 300μM H₂O₂did not decrease significantly.When NPMSCs pretreated with 75μM H₂O₂were exposed to 300μM H₂O₂,the content of Keap1 and Nrf2 were significantly decreased and increased.We found that Brd4 content decreased significantly when NPMSCs pretreated with 75μM H₂O₂were exposed to 300μM H₂O₂compared with 300μM H₂O₂alone.The content of Brd4 in JQ-1 treatment group and75μM H₂O₂pretreatment group was decreased.At the same time,the content of Keap1 and Nrf 2 decreased significantly.1.3.4 Pretreatment with 75μM H₂O₂enhanced the anti-apoptotic ability of NPMSCs by inhibiting the apoptotic mitochondrial pathwayWhen H₂O₂concentration was lower than 150μM,the apoptosis rate of NPMSCs did not change significantly.The apoptosis rate of NPMSCs was significantly increased after treatment with 200 and 300μM H₂O₂.However,when NPMSCs were pretreated with 75μM H₂O₂for 12 h and re-exposed to 300μM H₂O₂,the apoptosis rate of the cells was significantly reduced.When treated with 75μM H₂O₂,the ratio of Bcl-2 to Bax in NPMSCs was the highest.When the concentration of H₂O₂exceeded 100μM,the cytochrome C level increased significantly.Cleaved-caspase 3 levels increased when H₂O₂concentration exceeded 150μM.However,exposuring of 75μM H₂O₂pretreated NPMSCs to 300μM H₂O₂increased Bcl-2/Bax ratio,decreased cleaved-caspase 3 and cytochrome C content when compared with 300μM H₂O₂treated NPMSCs.1.3.5 Pretreatment with 75μM H₂O₂can enhance the ability of NPMSCs transplantation for IVDDOn day 60 after NPMSCs transplantation,DHI%in all cell transplantation groups was higher than that in the untreated group and PBS injection group.The treatment effect of the 75μM H₂O₂pretreatment NPMSCs group was better than other cell therapy groups.The fluorescence imaging results showed that the tail intervertebral disc of rats transplanted with NPMSCs was obviously fluorescent.The fluorescence intensity of 75μM H₂O₂pretreated NPMSCs rats was higher than that of 50μM H₂O₂pretreated NPMSCs group,100μM H₂O₂pretreated NPMSCs group and NPMSCs group.The results of histological staining and analysis showed that the reduction of disc contents and fibrous disorganization were observed in both untreated and PBS groups.Histological scores were lower in the 4-cell graft group than in the untreated group,and the75μM H₂O₂pretreated NPMSCs group showed the best improvement in disc height compared with the NPMSCs group.1.4 Conclusion:The results of in vitro cell experiments showed that the appropriate concentration of H₂O₂may enhance the proliferation ability of NPMSCs by inhibiting Hippo signaling pathway.Pretreatment with 75μM H₂O₂may enhance the antioxidant stress capacity of NPMSCs by activating Keap1-Nrf2 pathway.Pretreatment with 75μM H₂O₂may enhance the anti-apoptotic ability of NPMSCs by protecting mitochondrial morphology and function.The results of animal experiments showed that the cells pretreated with 75μM H₂O₂showed better survival ability in the degenerated intervertebral disc of the rat,and could significantly improve the morphology and structure of the degenerated intervertebral disc.In conclusion,75μM H₂O₂pretreatment is the best concentration to improve the proliferation ability,antioxidant stress and anti-apoptosis ability of transplanted NPMSCs.This study is expected to provide a new feasible method for improving the therapeutic effect of IVDD cell transplantation.