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. 2016 Mar 28;5(3):e003062. doi: 10.1161/JAHA.115.003062

Figure 3.

Figure 3

High‐glucose treatment suppresses macrophage activation induced by TNF‐α plus CaPO 4. A, Mmp9 mRNA expression levels in RAW264.7 cells after stimulation are shown relative to Gapdh expression. RAW264.7 cells were cultured with TNF‐α and/or CaPO 4 for 2 days, and mRNA expression in the cell lysates was determined by quantitative polymerase chain reaction. B, Representative Western blot and densitometoric analysis of Mmp9 secreted protein expression levels in culture media from RAW264.7 cells treated with TNF‐α and/or CaPO 4. The cells were treated with TNF‐α and/or CaPO 4 for 5 days, and the culture medium was then analyzed. C, Mmp9 mRNA expression levels in RAW264.7 cells after stimulation are shown relative to Gapdh expression. RAW264.7 cells were cultured for 3 days under N‐Glc (5.5 mmol/L glucose) without TNF‐α plus CaPO 4 (NT control), N‐Glc with TNF‐α plus CaPO 4, Man (5.5 mmol/L glucose and 10.5 mmol/L mannitol) and TNF‐α plus CaPO 4, and H‐Glc (15.5 mmol/L glucose) with TNF‐α plus CaPO 4. D, Representative Western blot and densitometoric analysis of secreted Mmp9 expression levels in culture media from RAW264.7 cells treated with TNF‐α and/or CaPO 4. The cells were treated with NT or TNF‐α plus CaPO 4 and N‐Glc, Man, or H‐Glc for 5 days, and then the culture medium was analyzed. E, Effect of high‐glucose treatment on cell viability. The cells were treated with NT or TNF‐α plus CaPO 4 and N‐Glc, Man, or H‐Glc for 2 days, and then the MTT assay was performed. F, MMP9 gelatinolytic activity according to gelatin zymography. The cells were treated with NT or TNF‐α plus CaPO 4 under N‐Glc, Man, or H‐Glc for 5 days, and then the culture medium was analyzed. Values are presented as mean±SD for at least 3 replicates; *P<0.05. **P<0.01. Relative expression levels for Western blotting were quantified using the ImageJ program (National Institutes of Health). CaPO 4 indicates calcium phosphate; H‐Glc, hyperglycemic conditions; Man, normoglycemic conditions with high osmotic pressure; Mmp9, matrix metalloproteinase 9; N‐Glc, normoglycemic conditions; NT, no treatment; qPCR, quantitative polymerase chain reaction; TNF‐α, tumor necrosis factor α.