CircN4bp1 Facilitates Sepsis-Induced Acute Respiratory Distress Syndrome through Mediating Macrophage Polarization via the miR-138-5p/EZH2 Axis

Figure 3

CircN4bp1 is involved in promoting M1 macrophage polarization while inhibiting M2 activation in ex vivo raw264.7 and MH-S macrophage cell lines. (a) RAW264.7 and MH-S cells were transfected with small interfering RNAs (siRNAs) targeting the circN4bp1 junction site or a circN4bp1 lentivirus plasmids. The relative expression of circN4bp1 was quantified by qRT-PCR. vs. control of raw264.7 cells; # vs. control of MH-S cells. RAW264.7 was transfected with Si-circN4bp1 (circN4bp1-KD), circN4bp1 lentivirus plasmids (circN4bp1-OE), or scrambled control (NC) and then exposed to either LPS (50 ng/ml) or IL-4 (10 ng/ml) for an additional 24 h. (b) The levels of IL-6, TNF-α, and IL-10 were measured by ELISA in the supernatants of LPS and IL-4 stimulated raw264.7 cells. (c) The relative expression of circN4bp1 was quantified by qRT-PCR. (d) Representative western blot depicting raw264.7 cell lysates probed for iNOS, Arg-1, p-STAT1, PPAR-γ, and GAPDH. Expression levels of iNOS, Arg-1, p-STAT1, and PPAR-γ were quantified by densitometry and normalized using GAPDH. ( vs. NC group, # vs. LPS stimulated group, ^@ vs. circN4bp1-OE + LPS group, & vs. IL-4 stimulated group, and % vs. circN4bp1-OE + IL-4 group determined by one-way ANOVA for multiple group comparisons).

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