| Background and PurposeOsteoarthritis(OA)is an irreversible degenerative joint disease that causes patients to suffer from severe consequences such as joint pain,stiffness,and even disability worldwide.According to statistics,10% of elderly men suffer from osteoarthritis,and the prevalence rate of elderly women has reached an astonishing 18%.With such a high incidence rate and serious complications,we have to attach great importance to this disease.At present,the commonly used conservative treatment methods for osteoarthritis include the use of nonsteroidal anti-inflammatory drugs,hyaluronic acid,etc.Joint replacement surgery can largely solve patients’ pain,but the lifespan of artificial joints is a noteworthy issue.Research has shown that articular cartilage and ear cartilage are usually not renewable,but in recent years,scholars have found that cartilage seed cells in the human body,known as soft bone marrow/progenitor cells(CSPCs),have the differentiation ability of stem cells such as osteogenic and chondrogenic cells.Aging of cartilage stem/progenitor cells can be an important pathogenesis of osteoarthritis.Therefore,we try to slow down aging by regulating the N-methyl-D-aspartic acid receptor and its subunits,so as to promote the selfrenewal of chondrocytes and provide new ideas and research basis for the early treatment of osteoarthritis.Method:First,high-throughput sequencing was performed on the specimens collected from patients with osteoarthritis who underwent knee varus arthroplasty to detect the differentially expressed genes in cartilage tissue of medial tibial platform(MTP)and lateral tibial platform(LTP),and then verified at the tissue and cell levels.Subsequently,cartilage stem/progenitor cells of LTP and MTP were extracted separately.Detect surface related biomarkers such as CD90,CD29,etc.of CSPCs by flow cytometry.Specific staining and Western Blot were used to detect the osteogenic and chondrogenic differentiation abilities of CSPCs.Detect age-related biomarkers,cell cycle of CSPCs,and cloning ability to demonstrate differences in the degree of aging between inner and outer CSPCs.Western blot and immunofluorescence were used to detect the differences in the expression levels of GLu N2 B and age-related proteins.In addition,we detected whether the circadian rhythm of senescent CSPCs on the degenerative side was destroyed by RT q PCR.Finally,we treated the lateral and medial CSPCs with N-methyl-D-aspartic acid(NMDA)and memantine hydrochloride(MEM)respectively,and then observed whether the aging,apoptosis and chondrogenic differentiation of CSPCs were regulated by NMDAR.Results:1.The differentially expressed genes related to osteoarthritis were analyzed by high-throughput sequencing,including the NMDAR subunit Glu N2 B and its direct downstream DLG4.Subsequently,tissue immunofluorescence and Western blot analysis revealed that the expression level of GLu N2 B was higher on the medial side than on the lateral side,and safranine O staining also showed that MTP was stained shallower due to cartilage damage.2.Flow cytometry revealed positive expression of CD29,CD90,and CD146,while negative expression of CD45 in the extracted CSPCs.In addition,after osteogenic induction and differentiation of CSPCs,alizarin red S staining showed that the MTP group had more calcified nodules than the lateral ones,and the protein expression levels of ALP and RUNX-2 were higher in the MTP group.At the same time,toluidine blue staining showed that the cartilage matrix components in the cartilage spheres formed by LTP group CSPCs were more than those in MTP,and the expression levels of COLL-2 and SOX9,which were closely related to cartilage components,were higher in LTP CSPCs at the m RNA level.3 β-Galactosidase activity staining showed that the aging degree of MTP CSPCs was heavier than that of LTP,and the expression levels of aging related proteins P21 and P53 were higher in MTP CSPCs.The cell cycle of CSPCs with MTP is stagnant and their cloning ability decreases.Immunofluorescence showed that the expression level of type II collagen in CSPCs of LTP was higher than that in CSPCs of MTP.4.Through high-throughput sequencing,it was found that the expression of NAMPT was down regulated in MTP group,and then the relationship between the expression level of NAMPT and circadian rhythm was explored through RT q PCR.It was found that the circadian rhythm fluctuation of CSPCs in LTP group was more obvious than that in MTP group,and the expression level of proteins related to circadian rhythm was also lowered in CSPCs in MTP group.5.The NMDAR inhibitor MEM and the agonist NMDA were used to act on the CSPCs of the MTP group and the LTP group,respectively,to detect changes in aging degree,aging related protein expression,NAD+content,and chondrogenic ability.MEM has a alleviating effect on the aging of MTP CSPCs and contributes to cartilage renewal.At the same time,NMDA promotes the aging of CSPCs by activating NMDAR.Conclusion:1.NMDAR expression is upregulated in degenerative cartilage,accompanied by aging of cartilage stem/progenitor cells,cycle arrest,and weakened renewal ability.2.The expression of NAMPT decreased,so the circadian rhythm was destroyed,which led to cartilage degeneration.3.Inhibiting NMDAR can to some extent slow down the aging of cartilage stem/progenitor cells,and NMDAR can serve as an effective molecular target for regulating the pathogenesis of osteoarthritis. |
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