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.