| Osteoarthritis(OA)is a degenerative disease caused by a variety of risk factors,including genetic and epigenetic inheritance,race,age,gender,obesity and inflammation.OA is characterized by cartilage rupture,subchondral sclerosis,bone spur,joint malformation and synovial inflammation.80% of OA patients diagnosed by X-ray examination are over 65 years old.OA usually causes joint pain,stiffness,movement disorders,and loss of function.As there are many risk factors for OA and the initiating factors are not clear,the pathogenesis of OA is still unclear.Since OA directly manifests as degeneration and wear of articular cartilage,chondrocyte metabolism has become a research hotspot,especially the molecular mechanism regulating cartilage metabolism.Cell adhesion molecule 1(CADM1,also known as RA175/Syn CAM1/TSLC1/ NECL-2/IGSF4)is a member of the immunoglobulin superfamily(Ig SF)and has been studied as a tumor suppressor gene in a variety of tumor-related studies.Currently,CADM1 has been studied in a wide range of areas,including neurological diseases and the effects on bone metabolism.In addition to inhibiting the proliferation and metastasis of most cells and promoting apoptosis,CADM1 is also associated with a variety of cell signal transduction pathways.Through bioinformation technology studies,we found that CADM1 expression increased in OA cartilage,but its function and role in the pathogenesis and development of OA have not been reported.In OA,the PI3K/Akt/mTOR signaling pathway is phosphorylated and activated,thus inhibiting the protective effect of autophagy on chondrocyte anabolic and catabolic imbalance and promoting the progress of OA.Therefore,the study investigated the role of CADM1 in the occurrence and progression of OA and related molecular mechanisms.Part 1.The expression of CADM1 in cartilage of patients with Osteoarthritis.Through the analysis of Gene Expression Omnibus database(GSE113825)by bio-information technology,it can be seen that CADM1 is obviously highly expressed in OA patients' cartilage.Meanwhile,we tested the gene expression of normal human cartilage and cartilage of OA patients.q RT-PCR results showed that the level of CADM1 was significantly increased in cartilage of OA patients.Western blotting(Wb)results showed that CADM1 protein expression level in OA cartilage was also significantly increased.Immunohistochemistry(IHC)results are consistent with the above results.These experimental results showed that CADM1 gene and protein expression levels were significantly increased in OA cartilage tissue.Part 2.Expression of CADM1 in IL-1?-induced primary human chondrocyte osteoarthritis model in vitro,and the effect of knockdown of CADM1 on anabolism,catabolism and autophagy of primary human chondrocytes Human primary chondrocytes ware extracted to construct an in vitro model of OA,so as to explore the expression and function of CADM1 in OA.Through q RT-PCR and Wb detection,we found that CADM1 gene and protein expression levels were significantly increased in chondrocytes of osteoarthritis model in vitro.This is consistent with the elevated expression of CADM1 in cartilage tissues of OA patients in part I,suggesting that CADM1 may play a role in the occurrence and development of arthritis.To further verify whether CADM1 affects the metabolic function of arthritic chondrocytes,we used small interfering RNA technology to knock down CADM1 gene in IL-1? treated human primary chondrocytes.Glycosaminoglycans(GAGs)deposition was detected by Safranin "O" staining.The results showed that knockdown of CADM1 significantly increased the deposition of GAGs in extracellular matrix.Wb results showed that knockdown of CADM1 significantly increased protein levels of Collagen II and Aggrecan in chondrocytes,while decreased protein expression levels of ADAMTS-5and MMP-13.This suggests that knockdown of CADM1 can promote anabolism of chondrocytes and inhibit catabolism of chondrocytes in vitro model of arthritis,thereby increasing extracellular matrix content.To investigate the relationship between CADM1 and autophagy in chondrocytes,we used Wb to detect the protein expression of autophagy markers and electron microscopy to observe the number of autophagosomes and autophagolysosomes.The results showed that knockdown of CADM1 significantly decreased the expression level of autophagy marker P62 protein,Beciln-1 protein expression level and LC3 II/I ratio were significantly increased,and the number of autophagosomes and autophagolysosomes were significantly increased.This suggests that knockdown of CADM1 promotes autophagy expression.In order to explore whether knockdown of CADM1 can affect chondrocyte metabolism by activating autophagy.An autophagy inhibitor Chloroquine(CQ)was used for recovery verification.The results showed that CQ could block knockdown of CADM1 to promote anabolism of chondrocytes,inhibit its catabolism and increase GAGs deposition.These results suggest that knockdown of CADM1 promotes anabolism of chondrocytes and inhibits its catabolism by activating autophagy.Part 3.Knockdown of CADM1 activates autophagy by regulating the PI3K/Akt/mTOR pathway in an in vitro osteoarthritis model of human primary chondrocytes induced by IL-1?,thereby affecting chondrocyte metabolism To further explore the regulatory mechanism of knockdown of CADM1 on autophagy,we detected the PI3K/Akt/mTOR signaling pathway closely related to OA and autophagy.Through Wb and IF,we found that knockdown of CADM1 significantly reduced the phosphorylation levels of PI3 K,Akt and mTOR in OA chondrocytes.This suggests that knockdown of CADM1 can inhibit the activity of PI3K/Akt/mTOR signaling pathway.In order to further explore the relationship between knockdown of CADM1,PI3K/Akt/mTOR signaling pathway and autophagy,we used the PI3 K pathway activator 740Y-P to perform the recovery experiment,and again used Wb to detect the protein expression of autophagy markers and electron microscope to observe the number of autophagosomes and autophagolysosomes.The results showed that activator 740Y-P reversed the autophagy activation induced by knockdown of CADM1,significantly decreased the expression of autophagy marker Beclin-1 protein and LC3II/I ratio,while increased P62 and decreased the number of autophagosomes and autophagolysosomes.This suggests that knockdown of CADM1 modulates autophagy through the PI3K/Akt/mTOR signaling pathway,thereby affecting chondrocyte metabolism.Part 4.The effects of knockdown of CADM1 on mice model of Osteoarthritis induced by destabilization of the medial meniscus(DMM).Three parts above of the paper showed that knockdown of CADM1 could activate autophagy by inhibiting PI3K/Akt/mTOR signaling pathway,and play a role in promoting anabolism and inhibiting catabolism of OA chondrocytes.To verify the role of CADM1 in animals,we established mice model of OA and knockdown of CADM1 expression in cartilage tissue by injecting Si-CADM1 into the joint.Then,the relevant indexes were detected by immunohistochemistry and the OARSI score of the mouse joints was evaluated by the Safranin "O"-Fast Green staining.The results showed that knockdown of CADM1 could significantly reduce the expression of mTOR and P62 of autophagy marker.OARSI score showed that knockdown of CADM1 could significantly inhibit the degeneration of articular cartilage in mice.Based on the above experiments,we first established CADM1 as the research target,and studied the function of CADM1 in OA and its related molecular mechanism in vivo and in vitro.The results showed that knockdown of CADM1 promoted the anabolism and catabolism of OA chondrocytes by inhibiting PI3K/Akt/mTOR signaling pathway and activating autophagy.This provides a new idea for the pathogenesis of OA and a new direction for the treatment of OA. |
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