| Background and ObjectiveOsteoarthritis(OA)is a degenerative joint disease,in which cartilage degradation is the core player in progression of OA.Deletion of autophagy plays key roles in the pathogenesis of OA.Vitamin D,a fat-soluble hormone,is essential for bone health.Patients with OA are often accompanied by vitamin D deficiency,and vitamin D supplementation helps patients achieve improvement of pain and alleviate the progression of joint structures.However,the role of vitamin D in OA and its related mechanism are still unclear.The aim of this study was to explore the role of vitamin D in OA and its effect on the chondrocyte autophagy.MethodsWe isolated chondrocytes from tibial surface cartilage of patients undergoing arthroplasty for OA,and identified the cells using toluidine blue staining and immunohistochemistry.A rat model of OA,the destabilization of the medial meniscectomy(DMM),was constructed by medial meniscectomy.The proliferation of chondrocytes treated with vitamin D was detected by CCK8 and HE staining in vitro and vivo,and the apoptosis of chondrocytes was detected by TUNEL staining,the expression of apoptosis-related proteins including Caspase3,Bcl-2 and Bax were detected by Western blot(WB).Safranin-O/fast green staining was used to detect the expression of cartilage proteoglycans under the addition of vitamin D,and the expression of OA-related proteins and mRNA including Collagen type Ⅱ alpha 1(COL2A1),matrix metalloproteinase-13(MMP-13)and Vascular endothelial growth factor(VEGF)were detected by WB and qRT-PCR.After determining the effect of vitamin D on OA,we examined autophagy-related proteins including p62,Beclin-1 and the ratio of LC3Ⅱ/LC3Ⅰ by WB.In order to further explore the effect of vitamin D on the autophagy,the expression of LC3 in chondrocytes was detected by m RFP-GFP-LC3 adenovirus transfection,the lysosome activity was detected by Lyso-Tracker Red staining,and the ultrastructure of autophagic lysosome was observed by transmission electron microscopy.We observed the expression of LC3 in rat articular cartilage by immunohistochemistry.After the determining the role of autophagy through AMPK/ mTOR signaling pathway by WB,we used small interfering RNA(siRNA)to knock down AMPK and mTOR in vitro and vivo,respectively,the protein and mRNA expression related to autophagy and OA was detected by WB and qRT-PCR.The expression of apoptosis and proteoglycan were detected by HE and Safranin-O/fast green staining,respectively.To verify the effect of NF-κB on autophagy in chondrocytes,we knocked down NF-κB in chondrocytes by intra-articular injection of siRNA in rat knee.The protein and mRNA expression related to autophagy and OA was detected by WB and qRT-PCR.The expression of apoptosis and proteoglycan were detected by HE and Safranin-O/fast green staining,respectively.To determining the specific mechanism of NF-κB,we detected the expression of p65,p-p65 and vitamin D receptor(VDR)in total and nucleus by WB and qRT-PCR.The expression of p-p65 was detected by immunohistochemistry and NF-κB nucleation experiment.We used the immunoprecipitation to detect the interaction of NF-κB with VDR.Meanwhile,the expression of IκBα and p-IκBα were detected by WB and qRT-PCR.We used the siRNA to knocked down the VDR and IκBα,the protein and mRNA expression related to autophagy and OA was detected by WB and qRT-PCR.The expression of apoptosis and proteoglycan were detected by HE and Safranin-O/fast green staining,respectively.Results1.Toluidine blue staining shows that a small number of heterochromatic granules appear in the periphery of chondrocytes,the cytoplasm is blue-purple,the nucleus is dark blue,and the nucleoli is obvious.Immunohistochemical staining of type Ⅱ collagen shows regular morphology of chondrocytes,brown heterochromatic granules,dark brown nuclei and light brown cytoplasm.2.Vitamin D promotes the proliferation of chondrocytes in OA,and reduces the apoptosis of chondrocytes in vivo and in vitro.3.Vitamin D reduces IL-1β and DMM-induced catabolism in the ECM,including promotes the expression of COL2A1 and proteoglycan and inhibits the expression of VEGF and MMP-13.4.Vitamin D significantly promotes the expression of autophagy related protein LC3 Ⅱ in chondrocytes,activates lysosome activity,promotes the formation of autophagy lysosomes,which play a role in the degradation of intracellular organelles and unnecessary substances,and maintain homeostasis.5.Vitamin D participates in the activation of chondrocyte autophagy through AMPK/mTOR signaling pathway.The knockdown of mTOR activates autophagy of chondrocytes,reduces the loss of articular cartilage and proteoglycans in rats,and delays progression of OA.Knockdown of AMPK,which activates the phosphorylation of mTOR,or directly inhibits autophagy,it decreases the number of chondrocyte and increases proteoglycan loss in OA.6.Inhibition of NF-κB is able to delay OA progression by reducing the apoptosis of chondrocytes and degradation of ECM though regulation of AMPK/mTOR signaling pathways.7.Vitamin D inhibits the phosphorylation of NF-κB by the interaction of NF-κB and VDR,then decreases the phosphorylation of NF-κB entering into the nucleus.8.Vitamin D could directly inhibit the phosphorylation of IκBα or regulate the mRNA expression of IκBα through VDR transcription factors,promote the interaction of IκBα with NF-κB,and reduce the p-NF-κB entering into nucleus.9.Knockdown of VDR and IκBα inhibit autophagy in chondrocytes and induce chondrocyte apoptosis and degradation of ECM.Conclusions1.Vitamin D may play a key role in OA by inhibiting NF-κB-induced autophagy pathway.2.Vitamin D mainly inhibits NF-κB activity in two ways: one is to stabilize NF-κB through the interaction of VDR and NF-κB;the other is to regulate IκBα expression through VDR,increase the binding of IκBα and NF-κB,to inhibit activation of NF-κB.3.Vitamin D protects OA through regulating AMPK/mTOR signaling pathway to activate chondrocyte autophagy by inhibition of NF-κB.Vitamin D may be effective in the treatment of OA. |
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