NMDAR-mediated Cellular Senescence Eliminates The Anti-fibrosis Effect Of BM-MSCs In Bleomycin-induced Pulmonary Fibrosis

Idiopathic pulmonary fibrosis is a special type of interstitial pneumonia in which patients gradually develop progressive,irreversible scarring of the lungs and eventually die from respiratory failure,and lung transplantation remains the only effective treatment for idiopathic pulmonary fibrosis.In-depth exploration of the mechanism of pulmonary fibrosis development,discovering new strategies strategy for anti-pulmonary fibrosis treatment are still the focus of pulmonary fibrosis prevention and treatment at this stage.The N-methyl-D-aspartate receptor（NMDAR）is an important receptor for glutamate.Excessive glutamate can activate NMDAR excessively and lead to brain injury,resulting in glutamate excitotoxicity.NMDAR is not only expressed in central nervous system,but also in peripheral tissues such as lung,pancreas,heart,osteoblasts and stem cells.In recent years,the physiological significance of its expression in peripheral tissues has attracted great attention of researchers.It has been reported that NMDAR activation is involved in mediating acute lung injury,asthma,chronic obstructive pulmonary disease,pulmonary fibrosis,intrauterine pulmonary dysplasia and other lung diseases,but the specific mechanism has not been clarified.Bone marrow-derived mesenchymal stem cells（BM-MSCs）,which are the most important non-hematopoietic progenitor cells in bone marrow,are a kind of stem cells with self-renewal and multidirectional differentiation ability.Transplantation of exogenous BM-MSCs can effectively reduce the degree of pulmonary fibrosis,which is one of the hot spots in the study of pulmonary fibrosis therapy.However,little attention has been paid to the status of endogenous BM-MSCs during pulmonary fibrosis.It has been reported that in animal models,the use of busulfane in advance to suppress bone marrow has a more severe degree of pulmonary fibrosis in mice than in the normal model group.Early mobilization of endogenous BM-MSCs to the lung using colony-stimulating factors ameliorates pulmonary fibrosis in mice,suggesting that the functional status of endogenous bone marrow and BM-MSCs has a significant effect on pulmonary fibrosis.It has recently been reported that BM-MSCs in patients with idiopathic pulmonary fibrosis have found aging-related cell phenotypes.However,The mechanism of senescence of BM-MSCs in pulmonary fibrosis model has not been clarified,and the role of bone marrow endogenous BM-MSCs senescent in pulmonary fibrosis models has not been reported.Objective:（1）To observe the senescent changes of endogenous BM-MSCs during BLM pulmonary fibrosis in vivo at the overall level;（2）To observe the effect of senescent BM-MSCs on BLM-induced pulmonary fibrosis in vivo at the overall level.（3）The effect of NMDAR activation on the senescence and pulmonary fibrosis of BM-MSCs was observed at the overall and cellular levels,and the possible mechanism of NMDAR-mediated BM-MSCs senescent was preliminarily explored at the cellular level.（4）Observe the effect of senescent BM-MSCs on alveolar epithelial cells at the cellular level.In this study,the effects of NMDARs on the senescence of endogenous BM-MSCs were observed at the overall and cellular levels.In order to explore the role of senescence BM-MSCs in the pathogenesis of pulmonary fibrosis,and provide new enlightenment for the research on the prevention and treatment of pulmonary fibrosis.Methods and Results:1.Senescence of lung tissue and endogenous BM-MSCs in bone marrow of BLM-induced pulmonary fibrosis miceMethods:The mice model of pulmonary fibrosis was established by intratracheal injection of BLM in C57BL/6 mice.HE staining and Masson staining were used to detect the degree of pulmonary fibrosis.SA-β-gal staining and Western-blot were used to detect the expression of p53and p16,which were related to the senescence of lung tissue.The expression of endogenous BM-MSCs p16 protein in mouse bone marrow was detected by Western-blot,and together with SA-β-gal staining,the senescence of endogenous BM-MSCs in the bone marrow of the model group was observed.The senescent changes of lung tissue and BM-MSCs in model group was observed.Flow cytometry was used to determine changes in the number of BM-MSCs in peripheral blood and whole lung tissue.Results:HE and Masson staining of lung tissue of mice in BLM model group showed structural destruction of lung tissue,alveolar interstitial hyperplasia and collagen deposition.The positive rate of SA-β-gal staining of BM-MSCs in model group was significantly higher than that in Con group,and the expression of p53 and p16 proteins were significantly higher than those in Con group.Compared with the Con group,the number of whole lung BM-MSCs had no significant change on day 14,but decreased significantly on day 21,and the number of peripheral blood BM-MSCs had no significant difference on day 14 and day 21.2.Effect of senescent BM-MSCs on BLM-induced pulmonary fibrosisMethods:BM-MSCs were treated with H₂O₂and single X-ray irradiation respectively,to induce cell senescence.The expression of p53and p16 protein were determined by Western-blot and the BM-MSCs were detected by SA-β-gal staining.The effects of intravenous injection of normal BM-MSCs and senescent BM-MSCs on the survival rate,weight change,lung tissue HE,Masson staining,determination of hydroxyproline content and PCR detection of CollagenⅠand CollagenⅢm RNA levels of pulmonary fibrosis mice were observed.Results:The positive rate of SA-β-gal staining and the expression of p53 and p16 protein in BM-MSCs treated with H₂O₂and X-ray were significantly higher than those in Con group.Intravenous injection of normal BM-MSCs could significantly improve the survival rate and body weight of pulmonary fibrosis mice,and the degree of pulmonary fibrosis was reduced compared with the model group.The survival rate,body weight and pulmonary fibrosis degree of senescent BM-MSCs treatment group had no difference compared with model group.3.Effect of NMDAR activation on BM-MSCs3.1 The NMDAR antagonist memantine attenuates the degree of BLM-induced pulmonary fibrosis in mice.Methods:The experiment was divided into four groups:Con group（normal saline control group）,ME group（normal saline+memantine control group）,BLM group（BLM model group）,BLM+ME（BLM+memantine treatment group）.Survival rate,body weight change,lung tissue HE,Masson staining,hydroxyproline content changes in mice were observed,and SA-β-gal were used Staining and Western-blot determined the expression of p53 and p16 proteins,and observed the senescent of endogenous BM-MSCs in bone marrow.Results:Survival rate,body weight,HE staining,Masson staining and hydroxyproline content in lung tissue of mice in Con group and ME group were not significantly abnormal.Compared with BLM group,memantine significantly reduced BLM-induced pulmonary fibrosis,decreased the content of hydroxyproline.The positive rate of SA-β-gal staining of primary BM-MSCs in BLM model group was significantly increased,and the expressions of p53 and p16 proteins related to senescent were increased.Memantine can partially improve the elevation of the aging indicators mentioned above.3.2 Expression of NMDA receptor-associated subunits in senescent BM-MSCs induced in vitro and expression of NMDA receptor-associated subunits in endogenous BM-MSCs in the bone marrow of mice with pulmonary fibrosisMethods:The expression changes of NMDAR subunit in BM-MSCs induced by aging in vitro and BM-MSCs of BLM-induced pulmonary fibrosis mice were detected by Western-blot.Results:The expression of NMDAR1,NMDAR2A and NMDAR2B proteins in BM-MSCs senescent model group induced by H₂O₂was significantly higher than that in Con group.The expression of NMDAR1protein was significantly increased in the senescent model of BM-MSCs irradiated by X-ray.The expression of NMDAR1 in endogenous BM-MSCs in BLM pulmonary fibrosis model group was significantly increased compared with that in the Con group.3.3 Continuous treatment of higher concentrations of NMDA induces senescence of BM-MSCs and its possible mechanisms.Methods:Cells were divided into three groups:PBS group,NMDA（3m M）group and NMDA+MK-801（a NMDAR antagonist）group.Cell cycle was measured by flow cytometry.PCR was used to detect m RNA expression changes of important factors（IL-1β,IL-6 and TGF-β）in senescence related secretory phenotypes.The expression changes of senescence related proteins p16,p21,proliferation-related proteins Ki67,h TERT and stem cell characteristic proteins Oct4 and Nanog were detected by Western-blot.Alkaline phosphatase（ALP）and oil red O staining were used to investigate the changes of polytropic differentiation of osteogenesis and lipids.The expression changes ofβ-catenin,p-β-catenin,Cyclin D1 and C-myc in the WNT/β-catenin pathway were detected by Western-blot.Results:Compared with the Con group,the positive rate of SA-β-gal staining and the expression of p16 and p21 of BM-MSCs were significantly increased after two weeks of continuous treatment with higher NMDA concentration.In NMDA group,cell cycle stagnated in G1phase,the m RNA expression levels of IL-1β,IL-6 and TGF-βincreased,the expression levels of proliferation-related Ki67 and h TERT proteins decreased,and the expression levels of stem cell characteristic proteins Oct4 and Nanog decreased.ALP staining and oil red O staining were less positive.The NMDAR antagonist MK-801 partially offset the above changes.NMDAR activation significantly promotedβ-catenin phosphorylation,inhibitedβ-catenin activity,and inhibitedβ-catenin nuclear translocation,while NMDAR antagonist MK-801 partially attenuated the effect of NMDA on the phosphorylation,activity,and nuclear translocation.3.4 The anti-BLM-induced pulmonary fibrosis effect of cultured BM-MSCs induced by long-term low-dose NMDA disappeared.Methods:BM-MSCs were long-term（about 5-6 weeks）cultured with a low dose of NMDA（0.2 mmol/L）in vitro.The expression of senescence related protein p16 was detected by Western-blot and the positive rate of SA-β-gal staining was used to identify senescence.The senescent BM-MSCs were injected into mice with pulmonary fibrosis through the tail vein.The survival rate,body weight,lung tissue HE,Masson staining and hydroxyproline content were observed.The m RNA level of CollagenⅠandⅢwere detected by PCR.Results:The positive rate of SA-β-gal staining and the expression of p16 protein in BM-MSCs were significantly higher than those in Con group after low-dose and long-term NMDA.In BLM model group,alveolar structure damage,interstitial hyperplasia,Collagen deposition and CollagenⅠandⅢm RNA levels were increased.The survival rate,body weight and pulmonary fibrosis degree of aging BM-MSCs treatment group had no significant difference compared with model group.4.Effects of memantine on EMT in pulmonary fibrosis model with BLM and Effects of senescent BM-MSCs on alveolar epithelial cellsMethods:The experiment was divided into four groups（same as 3.1）:Con group,ME group,BLM group,BLM+ME group.Western-blot analysis were performed to detect the changes of mesenchymal associated proteinα-SMA,vimentin and epithelial phenotype E-cadherin and Ep CAM in lung tissues of each group.To observe the effect of memantine on EMT in pulmonary fibrosis model with BLM.BM-MSCs cells were divided into three groups:PBS group,NMDA group and NMDA+MK-801 group.The above three groups of cells were co-cultured with alveolar epithelial cells MLE-12 respectively.The viability of MLE-12 cells was detected by CCK-8,the Changes of mesenchymal phenotypic markers（vimentin andα-SMA proteins）and epithelial phenotypic markers（E-cadherin and Ep CAM proteins）were detected by immunofluorescence and Western-blot.Results:Memantine decreased the expression ofα-SMA and vimentin and increased the levels of E-cadherin and Ep CAM proteins in lung tissue.of pulmonary fibrosis model with BLM.Co-culture with NMDA-induced senescent BM-MSCs resulted in increased activity of MLE-12 cells and decreased E-cadherin and Ep CAM proteins,and increased vimentin andα-SMA expression.Epithelial cells co-cultured with BM-MSCs in the NMDA+MK-801 treatment group showed partial inhibition of cell viability,partial promotion of vimentin andα-SMA expression,and partial inhibition of E-cadherin and Ep CAM protein reduction.Conclusion:1.The senescence of bone marrow endogenous BM-MSCs in BLM mice with pulmonary fibrosis was increased and the expression of NMDAR was up-regulated.The NMDAR antagonist memantine could reduce the senescence of BM-MSCs and reduce the degree of pulmonary fibrosis,suggesting that NMDAR is involved in mediating the senescence of bone marrow BM-MSCs in BLM mice with pulmonary fibrosis.One of the protective effects of memantine against pulmonary fibrosis is that memantine reduces the senescence of endogenous BM-MSCs.2.NMDAR activation,H₂O₂and X-ray can induce BM-MSCs senescence in vitro.senescent BM-MSCs lost protective effect on BLM-induced pulmonary fibrosis in mice,which further suggested that the senescence of BM-MSCs is an important mechanism for the loss of the endogenous protective mechanism against pulmonary fibrosis.3.NMDAR activation promote phosphorylation ofβ-catenin protein in the WNT classical pathway of BM-MSCs,and inhibit the activity ofβ-catenin protein and nuclear translocation.It may be one of the signal transduction pathways that mediate the activation of NMDAR to promote the aging of BM-MSCs,but this theory needs further study.4.Senescent BM-MSCs promote pulmonary epithelial mesenchymal transformation in vitro,which may be the mechanism of the loss of the protective effect of senescent BM-MSCs against pulmonary fibrosis.Conclusion:1.The senescence of bone marrow endogenous BM-MSCs in BLM mice with pulmonary fibrosis was increased and the expression of NMDAR was up-regulated.The NMDAR antagonist memantine could reduce the senescence of BM-MSCs and reduce the degree of pulmonary fibrosis,suggesting that NMDAR is involved in mediating the senescence of bone marrow BM-MSCs in BLM mice with pulmonary fibrosis.One of the protective effects of memantine against pulmonary fibrosis is that memantine reduces the senescence of endogenous BM-MSCs.2.NMDAR activation,H₂O₂and X-ray can induce BM-MSCs senescence in vitro.senescent BM-MSCs lost protective effect on BLM-induced pulmonary fibrosis in mice.It is further suggested that the senescence of BM-MSCs is an important mechanism to weaken the endogenous protective mechanism against pulmonary fibrosis.3.NMDAR activation inhibitsβ-catenin activity and nuclear translocation by promotingβ-catenin phosphorylation in the classic WNT pathway.It may be one of the mechanisms that mediate NMDAR activation and promote BM-MSCs senescent.4.Senescent BM-MSCs promote EMT in lung epithelial cells.Senescent BM-MSCs may promote the development of pulmonary fibrosis.