Benzoylaconine Modulates LPS-Induced Responses Through Inhibition of Toll-Like Receptor-Mediated NF-κB and MAPK Signaling in RAW264.7 Cells.

Changkai Zhou,Donghua Liu,Fanbo Jing,Qie Guo,Hongyan Ji,Lihua Zhou,Xiaomin Xing,Jing Gao,Wenjing Li

doi:10.1007/s10753-021-01478-z

Changkai Zhou, Donghua Liu + Show 7 more

Open Access

https://doi.org/10.1007/s10753-021-01478-z

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Abstract

Previous studies have shown that benzoylaconine (BAC), a representative monoester alkaloid, has a potential anti-inflammatory effect. This study investigated the underlying molecular mechanisms using the mode of LPS-activated RAW264.7 macrophage cells. Our findings showed that BAC significantly suppressed the release of pro-inflammatory cytokines and mediators, including IL-6, TNF-α, IL-1β, ROS, NO, and PGE2. BAC treatment also effectively downregulated the elevated protein levels of iNOS and COX-2 induced by LPS in a dose-dependent manner. In this study, we found that BAC inhibited LPS-induced NF-κB activation by reducing the phosphorylation and degradation of IκBα by western blotting and blocking the nuclear translocation of p65 using an immunofluorescence assay. The elevated protein levels of JNK, p38, and ERK phosphorylation after LPS stimulation were restored effectively by BAC treatment. The protein expression of Toll-like receptor 4 (TLR4) and LPS-induced phosphorylation of TAK1, which is a crucial upstream regulatory factor of TLR-induced MAPK and NF-κB signaling, were inhibited by BAC in activated RAW264.7 macrophages. Moreover, BAC decreased the levels of TAK1 phosphorylation and pro-inflammatory cytokines and mediators associated with MAPK and NF-κB activation, similar to TLR4 inhibitor TAK-242. These findings demonstrated that BAC exhibited an anti-inflammatory effect by the inhibition of TLR-induced MAPK and NF-κB pathways, indicating that it could potentially be used for treating inflammatory diseases.

Highlights

Inflammation has a protective role when the body is exposed to harmful stimulation, such as tissue injury and infection
transforming growth factor beta-activated kinase 1 (TAK1) activation activates the mitogen-activated protein kinase (MAPK) family, including c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK), which regulates the expression of transcription factor activator protein-1 (AP-1), a protein closely linked to various cellular processes, such as cell survival, differentiation, apoptosis, and pro-inflammatory cytokine production [13]
Classic non-steroidal anti-inflammatory drugs may lead to patient intolerance and cause some side effects when used for inflammatory diseases [20]

Summary

Introduction

Inflammation has a protective role when the body is exposed to harmful stimulation, such as tissue injury and infection. After LPS stimulation, macrophages initiate a series of inflammatory responses by activating specific signaling cascades and releasing both pro-inflammatory cytokines and mediators, including interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β, reactive oxygen species (ROS), nitric oxide (NO), and prostaglandin E2 (PGE2). TAK1 activation activates the MAPK family, including c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK), which regulates the expression of transcription factor activator protein-1 (AP-1), a protein closely linked to various cellular processes, such as cell survival, differentiation, apoptosis, and pro-inflammatory cytokine production [13]. Downstream signaling molecules of TLR involving MAPK and NF-κB pathways are effective therapeutic targets of anti-inflammatory drugs for inflammation treatment

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