5-Nitro-2-(3-phenylpropylamino) Benzoic Acid Promotes Lipopolysaccharide-induced Inflammation via p38 MAPK Pathway in RAW264.7 Macrophages

Youwei Huang, Xueqin Chen, Guopeng Pan, Fang Wang, Chuangye Chen and Xi Lin

Background and Objectives: 5-nitro-2-(3-phenylpropylamino) benzoic acid is a significant chlorine channel blocker and an agonist of G protein-coupled receptor 35 but its function in macrophage inflammatory response has not been reported yet. Therefore, this study aims to reveal the unrecognized role of 5-Nitro-2-(3-phenylpropylamino) benzoic acid in macrophage inflammation activation and its mechanism, which is helpful for further understanding of macrophage inflammation. Materials and Methods: For treatments, cells were pre-treated respectively with different drugs and then treated with Lipopolysaccharide for 4 hrs. Proteins were resolved by the SDS page and analyzed by immunoblotting using antibodies. The qRT-PCR was conducted with the SuperReal Pre Mix×Plus kit. One-Way ANOVA analysis of variance and t-test were using SPSS 13.0 statistical software package. Results: Silencing voltage-gated chloride channel 3 inhibited the expression of tumour necrosis factor-alpha in Lipopolysaccharide (100 ng mL^–1)-induced murine macrophages RAW264.7. 5-Nitro-2-(3-phenylpropylamino) benzoic acid (100 μM) downregulates the Lipopolysaccharide-induced nuclear factor kappa-light-chain-enhancer of activated B cells phosphorylation level via inhibiting the chlorine channel. But G protein-coupled receptor 35 agonists up-regulate tumour necrosis factor-alpha in macrophages induced by Lipopolysaccharide. 5-Nitro-2-(3-phenylpropylamino) benzoic acid enhances LPS-induced macrophages inflammation by promoting the associated p38 mitogen-activated protein kinase after activating G protein-coupled receptor 35. Conclusion: Current work indicated that not a chlorine channel blocker but a G protein-coupled receptor 35 agonists 5-Nitro-2-(3-phenylpropylamino) benzoic acid acts to enhance the inflammatory response of macrophages, which suggested that G protein-coupled receptor 35 activations may promote inflammatory response and aggravate the clinical diseases including bacterial infection.

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