Fig. 1

Effects of roflumilast and zatolmilast on nitric oxide (NO) and tumor necrosis factor-α (TNF-α) production in lipopolysaccharide (LPS)-induced BV-2 cells. (A) Roflumilast and zatolmilast cytotoxicity in BV-2 cells was assessed at concentrations up to 100 µM using the WST-8 assay. (B, C) NO and TNF-α production after 1 h pretreatment with roflumilast or zatolmilast with or without LPS (1 µg/mL) exposure for an additional 24 h. Culture supernatant was collected and analyzed for NO and TNF-α levels (n = 6 per group). NO levels were measured using the Griess test, and TNF-α levels were detected via enzyme-linked immunosorbent assay. The NO and TNF-α levels are presented as a percentage of untreated control cell levels. The data are expressed as the mean ± standard deviation. ^###P < 0.001 versus the control group; ^**P < 0.01, ^***P < 0.001 versus the LPS group. (D) Western blotting results of phosphorylated (p-)NF-κB and NF-κB protein expression. Full-length blots are shown in Supplementary Figure S1