Anti-inflammatory Effects of Hydrogen in LPS-induced RAW264.7 Cells via Inhibiting NF-κB and MAPKs Signaling Pathways

Hydrogen (H 2 ), a new type of medical gas molecule, which has significant preventive effect on numerous diseases and its anti-inflammatory properties has been proven in previous studies. However, the mechanisms of H 2 anti-inflammatory activity in signal transduction pathway or protein level regulation are inadequately inexplicit. In the current study, the effect of H 2 on LPS-induced inflammation in RAW 264.7 cells were assessed and its molecular mechanisms were clarified. The in vitro model of inflammation was induced by lipopolysaccharide (LPS) in RAW264.7 cells. Cell viability was evaluated by MTT assay. Protein expression of inflammatory mediators were analyzed by ELISA and Western blot. mRNA levels were detected by RT-qPCR. In addition, RNA sequencing (RNA-seq) was conducted to explore the molecular targets of H 2 anti-inflammatory. According to the findings, H 2 reversed LPS-induced variety in NO levels and TNF-a production as well as IL-6, IL-10 proteins and related mRNA levels in macrophages. RNA-seq newly discovered that H 2 acted on inflammatory signaling molecule protein kinase C 8 (PKC8) and heterodimer activator protein-1 (AP-1). The WB analysis was then used to determine the key proteins in the inflammatory signaling pathway involved in PKC8 and AP-1, which found that H 2 inhibited the phosphorylation of key proteins in the NF-kB and MAPKs pathways, thereby the expression of mRNA and inflammatory mediators were affected. The findings of this study show that H 2 may serve as a promising anti-inflammatory gas in mitigating inflammatory conditions.