| Objective:The pathogenesis of osteoarthritis(OA) is unclear and no effective treatment is available.The aim of this study was to investigate the effects of zinc finger protein 521(Zfp521)on chondrocyte proliferation,apoptosis,and extracellular matrix catabolism and anabolism during OA,and to verify whether Zfp521 could retard articular cartilage degeneration.Meanwhile,the intrinsic molecular mechanism and the effective ways to enhance the efficacy of Zfp521 in OA were further investigated.Methods:1.We overexpressed or knocked down the expression of Zfp521 in chondrocytes by Zfp521 adenovirus and small interfering RNA,combined with Ed U proliferation assay,TUNEL staining and immunohistochemical staining of in vivo rats to observe the effect of Zfp521 content changes on chondrocyte proliferation and apoptosis.Besides,proteomics analysis was performed by adenovirus intra-articular injection in rats to investigate the possible signaling pathways activated by Zfp521 overexpression in chondrocytes.2.After 9 weeks of adenovirus intra-articular injection,we analyzed the effects of Zfp521 on the catabolism and anabolism of extracellular matrix in articular cartilage by gait analysis,X-ray imaging,fluorescence molecular tomography,biomechanical testing,Indian ink staining,Safranin O/fast green staining and immunohistochemical staining,and evaluated its efficacy on osteoarthritis.3.The molecular mechanism of Zfp521 in the treatment of OA was further investigated by analyzing the structural domains of the differential proteins and the KEGG signaling pathway in combination with adenovirus co-infection of chondrocytes and Western blotting experiments.4.Zfp521 was co-infected with HDAC4 full-length,HDAC4-N-terminal fragment and HDAC4-C-terminal fragment,and injected into the knees of rats OA model to investigate the therapeutic effect of Zfp521 combined with HDAC4 on osteoarthritis.Also,it was further investigated whether Zfp521 could achieve the desired therapeutic effect with only a partial HDAC4 fragment.Results:1.Zfp521 adenovirus can effectively infect chondrocytes and articular cartilage.Combined with the proteomics results,Zfp521 can regulate chondrocytes proliferation and apoptosis by regulating the cell cycle phase transition.Knockdown of Zfp521 resulted in proliferation decrease and apoptosis increase.The proteomics results also suggested that Zfp521 may be involved in the regulation of the extracellular matrix.2.Zfp521 can improve the biomechanical properties of articular cartilage,reduce joint pain and bone redundancy,and ultimately attenuate the progression of osteoarthritis.3.Zfp521 upregulation had no effect on the total amount of HDAC4 protein,but it caused the transfer of HDAC4 from the cell plasma to the nucleus by inhibiting the phosphorylation level of HDAC4.Inhibition of HDAC4 into the nucleus greatly diminished the function of Zfp521 in proliferation,apoptosis,and matrix metabolism in chondrocytes.The role of Zfp521 was dependent on HDAC4 in the nucleus,not in the cytoplasm.4.In osteoarthritis,Zfp521 co-expressed with HDAC4 had better efficacy than Zfp521 alone,and the combination of Zfp521 and HDAC4 can more effectively improve cartilage biomechanical properties,inhibit extracellular matrix loss,reduce articular cartilage osteoid formation and periarticular ossification,and ultimately attenuate the progression of osteoarthritis.The HDAC4-N terminus may weaken the protective effect of Zfp521 on articular cartilage,while the HDAC4-C terminus has no significant effect on articular cartilage based on current evidence.Conclusion:Zfp521 has an important role in attenuating the progression of osteoarthritis by promoting chondrocyte proliferation and inhibiting apoptosis,which improves OA by avoiding the reduction of chondrocytes.The therapeutic effect of Zfp521 in OA is dependent on HDAC4 in the nucleus,and co-expression of Zfp521 with HDAC4 could better attenuate the progression of OA.Compared with HDAC4 in some fragments,full-length HDAC4 can produce better therapeutic effects. |
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