HMGB1 Promotes Systemic Lupus Erythematosus by Enhancing Macrophage Inflammatory Response

Figure 4

HMGB1 aggravated macrophage inflammatory response. (a-b) RAW264.7 cells were stimulated with ALD-DNA (0, 25, 50 μg/mL) for 24 h, levels of HMGB1 in the supernatants of RAW264.7 cells were analyzed by ELISA (a) and western blot analysis (b). Data are means ± SD of three independent experiments. (c-d) The efficiency of HMGB1 overexpression (c) and knockdown (d) was monitored by representative immunoblot of three independent experiments in ALD-DNA-stimulated RAW264.7 cells. (e-f) RAW264.7 cells were transfected with pHMGB1 or vector. 72 h after transfection, RAW264.7 cells were stimulated with PBS, UnALD-DNA or ALD-DNA (50 μg/mL) followed by analyzing the levels of TNF-α (e) and IL-6 (f) in the culture supernatants of RAW264.7 cells. Data are means ± SD of three independent experiments. (g-h) RAW264.7 cells were transfected with control siRNA (200 nM) or HMGB1 siRNA (siHMGB1, 200 nM). After 72 h RAW264.7 cells were stimulated with PBS, UnALD-DNA or ALD-DNA (50 μg/mL). ELISA assay was used to analyze the levels of TNF-α (g) and IL-6 (h) in the culture supernatants of RAW264.7 cells. Data are means ± SD of three independent experiments. (i-j) CD11b⁺/F4/ renal macrophages were sorted from nephritic single-cell suspensions from (i) pHMGB1- or empty vector-treated, (j) glycyrrhizin- or PBS-treated SLE mice by flow cytometry. Macrophages (2 × 10⁵/mL) were stimulated with ALD-DNA (50 μg/mL) for 24 h. The supernatants were collected and assayed for the TNF-α and IL-6 concentrations using ELISA. Data are means ± SD from 8 mice in each group. .

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