Knockdown of NEK7 alleviates anterior cruciate ligament transection osteoarthritis (ACLT)-induced knee osteoarthritis in mice via inhibiting NLRP3 activation.

Osteoarthritis is thought to be a NLRP3-related disease. NEK7 is an essential mediator for NLRP3 inflammasome activation. This study aimed to demonstrate whether NEK7 has regulatory roles in the pathogenesis of osteoarthritis. C57BL/6 mice were subjected to anterior cruciate ligament transection osteoarthritis (ACLT) for constructing animal models of osteoarthritis. Injection of adeno-associated virus (AAV) expressing NEK7-specific shRNA into the knee joints of mice, following of which immunohistochemistry, qRT-PCR, western blotting, Safranin-O Fast Green staining, ELISA, and co-immunoprecipitation were performed to determine the effects of NEK7. NEK7 was highly expressed in the joint tissues of ACLT mice. As compared with shScr, AAV delivery of NEK7 shRNA significantly inhibited cartilage degeneration, OARSI score, and serum CTX-II and COMP levels. AAV delivery of NEK7 shRNA downregulated the expression of matrix-degrading enzymes (ADAMTS-4, MMP3, and MMP13) and upregulated the expression of ECM-related molecules (SOX9, collagen II, and aggrecan). In addition, AAV delivery of NEK7 shRNA alleviated ACLT-induced synovial inflammation, as was evidenced by the decreased levels of TNF-α, IL-6, IL-1ß, and IL-18 and increased levels of IL-10. In the joint tissues of ACLT mice, NEK7 interacted with NLRP3 proteins. AAV delivery of NEK7 shRNA inhibited the protein interaction, and thereby inhibited the activation of the NLRP3 inflammasome. AAV delivery of NEK7 shRNA has no significant effects on cartilage degeneration and synovial inflammation in Nlrp3-/- mice. In conclusion, knockdown of NEK7 exerted anti-osteoarthritic effects, possibly via inhibiting the activation of the NLRP3 inflammasome. This study provided a novel mechanism of NEK7-NLRP3 interaction affecting osteoarthritis.

NEK7 is highly expressed in ACLT-induced mouse models of osteoarthritis.Knockdown of NEK7 alleviates ACLT-induced cartilage degeneration and inflammation.NEK7 mediates the activation of NLRP3 inflammasome in ACLT mouse models.Knockdown of NEK7 alleviates ACLT-induced osteoarthritis by inhibition of NLRP3.

Constructing adeno-associated virus-TGFbeta3 and comparing its biological effect on proteoglycan synthesis in dedifferentiated nucleus pulpous cells with adenovirus-TGFbeta1.

OBJECTIVE: To construct adeno-associated virus (AAV) expression system for transforming growth factor beta3 (TGFbeta3 ) and detect its biological effect on proteoglycan synthesis of the earlier and later dedifferentiated rabbit lumbar disc nucleus pulpous (NP) cells, which was compared with that of adenovirus (AV) expression system for TGFbeta1. METHODS: TGFbeta3 gene was obtained using PCR. Its upstream contained restriction enzyme site Kpn I, and its downstream contained restriction enzyme site Sal I. Using the restriction enzyme sites of PCR product of TGFbeta3 and the corresponding multiple cloning site (MCS) in plasmid AAV, TGFbeta3 was subcloned into AAV. The recombinant plasmid AAV-TGFbeta3 was transfected into H293 cells with Lipofectamine 2000, and the expression of TGFbeta3 gene was detected using immunofluorescent analysis. After AAV-TGFbeta3 virus particle with infectious activity was packaged, TGFbeta3 expression in NP cells was detected by immunoblotting, and its biological effect on proteoglycan synthesis was detected by antonopulos method and compared with that of AV-TGFbeta1 in the earlier and later dedifferentiated NP cells. RESULTS: For the earlier dedifferentiated NP cells, AAV-TGFbeta3 slowly and stably enhanced proteoglycan synthesis, but AV-TGFbeta1 rapidly and transiently enhanced its synthesis. For the later dedifferentiated NP cells, AAV-TGFbeta3 stably enhanced proteoglycan synthesis, but AV-TGFbeta1 inhibited its synthesis. CONCLUSION: AAV expression system can mediate TGFbeta3 gene to be expressed stably, and AAV-TGFbeta3 can enhance proteoglycan synthesis of the earlier and later dedifferentiated NP cells.

Effects of adeno-associated virus (AAV) of transforming growth factors beta1 and beta3 (TGFbeta1,3) on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated nucleus pulposus (NP) cells.

The effects of AAV-TGFbeta(1) and AAV-TGFbeta(3) on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFbeta(1) or AAV-TGFbeta(3), their biological effects on promoting synthesis of glycosaminoglycan or collagen type II were detected and compared by the methods of (35)S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFbeta(1) and AAV-TGFbeta(3) could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type II, and the effect of AAV-TGFbeta(1) was better than that of AAV-TGFbeta(3). For the later dedifferentiated NP cells, the AAV-TGFbeta(3) could promote their synthesis, but AAV-TGFbeta(1) could slightly inhibit their synthesis. Therefore, AAV-TGFbeta(1) and AAV-TGFbeta(3) could be used for the earlier dedifferentiated NP cells, and the TGFbeta(3) could be used as the objective gene for the later dedifferentiated NP cells.