Melatonin Suppresses Macrophage M1 Polarization and ROS-Mediated Pyroptosis via Activating ApoE/LDLR Pathway in Influenza A-Induced Acute Lung Injury

Figure 5

The effects of melatonin on macrophage polarization and oxidative injury in Raw264.7 cells. (a) Representative immunofluorescence images of iNOS (green) and Arg1 (red) expression in Raw264.7 cells infected by influenza A (H3N2) infection (, 12 h) with/without melatonin pretreatment (400 μM, 3 h before H3N2 infection) (original magnification ×630). (b) Western blot analysis of the expression of iNOS and Arg1 to β-actin in Raw264.7 cells. (c) Quantitative RT-PCR measurements of the relative mRNA levels of TNF-α, MCP1, CD86, Arg1, Fizz1, and CD206 in Raw264.7 cells. (d) Representative images of DCF fluorescence (green) in Raw264.7 cells (original magnification ×50); quantitative ROS values were detected using a fluorescence plate reader. (e) The lactic dehydrogenase (LDH) released into the medium was assessed based on OD₄₉₀ values of LDH release in Raw264.7 cells. (f) Quantitative RT-PCR measurement of the relative mRNA level of IL-1β in Raw264.7 cells. (g) Western blot analysis of the expression of NLRP3, Caspase1, and GSDMD-N to β-actin in Raw264.7 cells. Data expressed as (). , , and compared with influenza A- (H3N2-) infected Raw264.7 cells.

(a)

(b)

(c)

(d)

(e)

(f)

(g)