Silencing of Angiopoietin‐Like Protein 4 (Angptl4) Decreases Inflammation, Extracellular Matrix Degradation, and Apoptosis in Osteoarthritis via the Sirtuin 1/NF‐κB Pathway

Figures

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  Figure 1. The expression of Angptl4 was increased in osteoarthritic human and mouse articular cartilage. (a, b) Representative H&E staining, Safranin O staining, immunofluorescence staining, and immunohistochemistry staining images of human articular cartilage from healthy control and OA patients (bar: 50 μm). (c, d) Representative H&E staining, Safranin O staining, immunofluorescence staining, and immunohistochemical staining images of mouse knee articular cartilage from the sham and DMM models (bar: 50 μm). (e, f) The protein expression of Angptl4 in chondrocytes obtained from normal and degenerated human specimens. (g, h) The protein expression of STING in chondrocytes collected from normal group and DMM (degenerated) groups. All data are presented as the mean ± SD (n = 5); ∗∗P < 0.01.

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  Figure 2. TNF-α promoted the expression of angiopoietin-like protein 4 (Angptl4) in chondrocytes. (a, c, e) The chondrocytes were treated with TNF-α at 0, 25, 50, 100, and 200 (ng/mL) for 24 hours. The protein and mRNA expression of Angptl4 was determined using Western blotting assay and qRT-PCR, respectively. (b, d, f) The chondrocytes were treated with TNF-α (50 ng/mL) for 0, 6, 12, 24, and 48 h. The protein and mRNA expression of Angptl4 was determined using Western blotting and qRT-PCR, respectively. All data were presented as the mean ± SD (n = 5); ns: no significance, ∗P < 0.05, ∗∗P < 0.01.

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  Figure 3. Angptl4 promotes ECM degradation, inflammation, and apoptosis in chondrocytes. The chondrocytes were treated with TNF-α (50 ng/mL) except for the control group. (a, b, e, f) Results of Western blotting showing the protein expression level of Angptl4, MMP3, MMP13, ADAMTS5, Collagen II, and IL-6 in chondrocytes treated as above. (c, d, g, h) Results of Western blotting showing the protein expression level of Angptl4, BAX, Bcl-2, C-caspase3, and Cyto-C in chondrocytes treated as above. All data are presented as the mean ± SD (n = 5); ∗∗P < 0.01 vs. control group, #P < 0.01 vs. TNF-α group.

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  Figure 4. Angptl4 promoted ECM degradation and inflammation through NF-κB activation. The chondrocytes were treated with TNF-α (50 ng/mL) except for the control group. (a, b) The phosphorylation level of P65 and IκBα in chondrocytes as determined by Western blotting. (c, d) Immunofluorescence staining of P65 in chondrocytes as treated above (bar: 10 μm). All data are presented as the mean ± SD (n = 5); ∗∗P < 0.01 vs. control group, $P < 0.05, $$P < 0.01 vs. TNF-α group.

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  Figure 5. Angptl4 knockdown regulated ECM degradation and inflammation through the SIRTUIN 1/NF-κB pathway. (a, b) Expression level of P65, Lamin B, SIRTUIN 1, and GAPDH in chondrocytes as determined by Western blotting. (c, d) Confirmation of the successful knockdown of SIRTUIN 1 via siRNA. (e, f) The expression level of IL-6, Collagen II, ADAMTS5, MMP3, and MMP13 in chondrocytes as determined with Western blotting. (g, f) Immunofluorescence staining of P65 in chondrocytes after the indicated groups (bar: 10 μm). (i, j) Immunofluorescence staining of Collagen II, MMP13 and IL-6 in chondrocytes as treated above (bar: 20 μm). All data are presented as the mean ± SD (n = 5); ns: no significance, ∗∗P < 0.01.

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  Figure 6. Angptl4 knockdown regulated apoptosis through theIRT1/NF-κB pathway. (a, b) The protein expression level of BAX, Bcl-2, C-caspase3, and Cyto-C in chondrocytes after treatment as determined with Western blotting. (c, d) TUNEL staining assay was conducted on the chondrocytes, as treated above (bar: 50 μm). All data are presented as the mean ± SD (n = 5); ns: no significance, ∗P < 0.05 vs. control group, ∗∗P < 0.01.

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  Figure 7. Angptl4 knockdown ameliorated osteoarthritis in vivo. Establishment of a mouse model of osteoarthritis surgical destabilization of the medial meniscus (DMM). After 8 weeks, the pathology of OA was assessed by X-ray, H&E staining, and Safranin O staining. (a) Digital X-ray image of mouse knee joints from different experimental groups. (b) Representative immunohistochemistry images of Angptl4, HE, and S-O staining results of the cartilage and synovitis in the four groups at eight weeks postsurgery (bar: 50 or 25 μm). (c) Quantitative analysis of immunohistochemical results of Angptl4. (d, e) The OARIS scores of cartilage and scores of synovitis in the four groups. All data are presented as the mean ± SD (n = 10), ns: no significance, ∗∗P < 0.01.

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  Figure 8. Angptl4 knockdown ameliorated the degradation of ECM, inflammation, and apoptosis by inhibiting NF-κB in vivo. (a, b) The expression of p-P65, MMP13, and IL-6 in mouse cartilage as examined by immunohistochemistry (bar: 20 μm). (c, d) Results of the TUNEL staining assay in mouse cartilage (bar: 50 μm). All data are presented as the mean ± SD (n = 5); ns: no significance, ∗∗P < 0.01.

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  Figure 9. Schematic graph of the role of Angptl4 in TNF-α-induced chondrocyte ECM catabolism, inflammation, and apoptosis.