The Protective Effect Of Protocatechuic Aldehyde And Its Mechanism On The Cartilage And Subchondral Bone In Mice With Osteoarthritis

Objective:The differentiation of mouse bone marrow-derived monocytes(BMMs)into osteoclasts was induced by nuclear factor-KB receptor activator ligand(RANKL),To investigate the role of Protocatechuic aldehyde(PCA)in inhibiting osteoclast differentiation and its molecular mechanism;Interleukin-1β(IL-1β)was used to induce chondrocytes to mimic osteoarthritis in mice,To investigate the role of PCA in protecting chondrocytes and its molecular mechanism;Also to establish a mouse knee osteoarthritis(OA)model by anterior cruciate ligament transection(ACLT),To investigate whether PCA plays a role in protecting cartilage and subchondral bone in vivo.To provide an effective experimental basis for the treatment of OA by PCA.Method:1.The safe concentration range of PCA for BMMs was determined by the Cell Counting Kit-8(CCK-8)method;Interventions with different concentrations of PCA,the changes in the transcript levels of nuclear factor of activated T cells 1(NFATC1),Cathepsin K(CTSK),cellular oncogene FOS(C-FOS),and dendritic cell-specific transmembrane protein(DC-STAMP)genes in osteoblasts formed by RANKL induction were detected by real-time quantitative PCR(qPCR)technique;Changes in the synthesis levels of osteoclast differentiation marker proteins by Western blot(WB);and changes in the nuclear factor κB(NF-κB)signaling pathway.2.The safe concentration range of PCA on chondrocytes was determined by the CCK-8 method;after intervention with different concentrations of PCA,qPCR was used to detect changes in IL-1β-stimulated chondrocytes of pro-inflammatory factors:interleukin-6(IL-6)and tumour necrosis factor-α(TNF-α),inflammatory factors:cyclooxygenase-2(COX-2)and inducible nitric oxide synthase(iNOS)gene transcript levels;WB technique was used to detect changes in the levels of inflammatory factors:COX-2,iNOS,catabolic factors:a disintegrin and metalloproteinase with thrombospondin motifs-5(ADAMTS-5)and matrix metalloproteinase-13(MMP-13),cartilage matrix proteins:aggregable proteoglycan(Aggrecan),type Ⅱ collagen(COL-Ⅱ),and changes in NF-κB signaling pathways.3.The animal experiments were divided into control group(Sham group),surgical group(ACLT group),low intervention group(ACLT+1Omg/kg PCA,Low Group),high intervention group(ACLT+20mg/kg PCA,High group);The mice were operated according to the group and different concentrations of PCA were injected into the mice intraperitoneally for intervention,and knee joint tissue sections were performed after 12 weeks of intervention.To observe the structure of subchondral bone and osteoclast number and size by tartrate-resistant acid phosphatase(TRAP)staining,By hematoxyeosin(H&E)and Safranin O(SO),Observe the cartilage tissue structure and the morphological changes of chondrocytes,The degree of cartilage damage was also assessed with the International Society for Osteoarthritis research society international(OARSI)score.Results:1.PCA concentration ≤15 μM was not toxic to mouse BMMs;compared with the RANKL-induced group,5 μM PCA inhibited the expression of key genes for osteoclast differentiation(C-FOS,NFATC1,CTSK,DC-STAMP)and the synthesis of key proteins for osteoclast differentiation(C-FOS,NFATC1,CTSK),and also inhibit the phosphorylation level of p65 in NF-κB signaling pathway.2.PCA concentration<20 μM was not toxic to mouse chondrocytes;compared with the IL-1β-stimulated group,5 μM PCA could inhibit the expression of pro-inflammatory factors IL-6 and TNF-α,inflammatory factors COX-2 and iNOS genes in chondrocytes,and suppress the synthesis of inflammatory factors COX-2,iNOS and catabolic factors ADAMTS-5 and MMP-13 protein synthesis in chondrocytes.while it can promote the synthesis of extracellular matrix Aggrecan,COL-Ⅱ protein in chondrocytes;and also inhibit the phosphorylation level of IκBa,p65 in NF-κB-type transduction pathway.3.In animal experiments,TRAP staining showed that:an increased number of osteoclasts and sparse bone trabeculae could be observed in the subchondral bone of the knee joint in the ACLT group compared with the Sham group;mice in the Low and High groups showed reduced subchondral bone destruction and osteoclasts in the knee joint after PCA intervention,and the protective effect of High was more pronounced.H&E and SO staining showed that:In the ACLT group compared with the Sham group,the transparent cartilage layer was reduced,the calcified cartilage layer was significantly thickened and some cartilage was exfoliated in H&E staining,and some chondrocyte staining was significantly reduced and the tide line was blurred or even disappeared in SO staining;the cartilage damage in the knee joint was better in the Low and High groups compared with the ACLT group,and the protective effect of High was more obvious.The arthritic cartilage damage OARSI score showed the same results.Conclusion:1.In cellular experiments,PCA reduced the synthesis of proteins associated with osteoclast differentiation,reduced inflammatory factors secreted by IL-1β-stimulated chondrocytes,and increased matrix protein synthesis by inhibiting the phosphorylation of IκBa and p65 in the NF-κB signaling pathway.2.In animal experiments,PCA exhibited effects of inhibiting osteoclast activation in subchondral bone,reducing subchondral bone destruction and mitigating cartilage damage,indicating that cartilage damage and subchondral bone destruction in OA mice can be protected by PCA.