3.3 Compound 270 suppresses the LPS-induced inflammatory response in RAW 264.7 macrophages
Exposure to LPS can trigger inflammatory cells (ie, macrophages and neutrophils) infiltration, contributing to excessive inflammatory reaction via NF-κB and JNK signaling pathways (Chen et al. , 2019). Macrophages are the principal immune cells of biosynthesis of pro-inflammatory cytokines, which act as the first line of defense in the kidney and lung, and exert a vital function in the molecular mechanisms of LPS-induced septic AKI and ALI (Huanget al. , 2019). First, we examined the effect of 270 on LPS-induced inflammatory response in RAW 264.7 macrophages. The activation of NF-κB occurs through phosphorylation of IKK, leading to the degradation of IκB-α (Zhang et al. , 2018a). In addition, JNK can directly activate NF-κB by promoting IκB-α degradation, NF-κB and JNK can coordinate the LPS-induced inflammatory response (Grynberg et al. , 2017). Thus we supposed that 270 may interfere with the NF-κB and JNK signaling pathway proteins. So we examined the effect of 270 on the levels of IκB-α, and phosphorylation of IKK, NF-κB and JNK in LPS-induced RAW 264.7 macrophages via western blotting. Our results proved that 270 decreased LPS-dependent IKK, NF-κB and JNK phosphorylation, alleviated LPS-induced IκB-α degradation in dose-dependently (Figure 3A,B). Upon activation by LPS, NF-κB translocate to the nucleus to promote transcription of inflammation genes. Thus we hypothesized that 270 may down-regulate the expression of pro-inflammation genes by disturbing with NF-κB and JNK signaling pathways. Next, we investigated the expression of inflammatory genes by RT-PCR in LPS-induced RAW 264.7 macrophages in the presence or absence of 270. Our data indicated that the high mRNA levels of TNF-α, IL-1β, IL-6, MCP 1, Nos2 and COX2 caused by LPS were reduced in 270 treated RAW 264.7 cells (Figure 3C), advising that 270 could attenuate LPS-induced inflammation response.