HSP60 Regulates Monosodium Urate Crystal-Induced Inflammation by Activating the TLR4-NF-κB-MyD88 Signaling Pathway and Disrupting Mitochondrial Function

Figure 2

HSP60 affects the TLR4/MyD88/NF-κB signaling pathway in MSU crystal-stimulated RAW264.7 cells. After RAW264.7 cells were transfected with HSP60 siRNA or control siRNA for 48 h, primed with LPS (100 ng/ml) for 1 h, and then treated with MSU crystals (100 μg/ml) for 4 h. (a) HSP60 protein level in RAW264.7 cells transfected with ctrl siRNA or HSP60 siRNA. (b, c) Protein levels of TLR4, MyD88, IκBα, P50, P65, phosphorylated P50 (p-P50), and phosphorylated P65 (p-P65). (d) HSP60 knockdown inhibited MSU crystal-induced P65 nuclear localization in RAW264.7 cells and analyzed by immunostaining. Blue shows nuclei staining with DAPI. Scale bar: 10 μm. Each experiment had six fields of view. The intensity of the fluorescence signal was analyzed by Image J software. (e) HSP60 protein level in RAW264.7 cell transfection with control vector or HSP60 vector for 36 h. (f) RAW264.7 cells were transfected with a control vector or HSP60 vector for 36 h, primed with LPS (100 ng/ml) for 1 h, primed with LPS (100 ng/m), and then treated with MSU crystals (100 μg/ml) for 4 h. HSP60 overexpression promoted MSU crystal-induced P65 nuclear localization in RAW264.7 cells and analyzed by immunostaining. Blue shows nuclei staining with DAPI. Scale bar: 10 μm. Each experiment had six fields of view. The intensity of the fluorescence signal was analyzed by Image J software. Values are the of 3 independent experiments. .

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