Naturally occurring small molecules with dual effect upon inflammatory signaling pathways and endoplasmic reticulum stress response

Abstract

The endoplasmic reticulum (ER) is determinant to maintain cellular proteostasis. Upon unresolved ER stress, this organelle activates the unfolded protein response (UPR). Sustained UPR activates is known to occur in inflammatory processes, deeming the ER a potential molecular target for the treatment of inflammation. This work characterizes the inflammatory/UPR-related molecular machinery modulated by an in-house library of natural products, aiming to pave the way for the development of new selective drugs that act upon the ER to counter inflammation-related chronic diseases. Starting from a library of 134 compounds of natural occurrence, mostly occurring in medicinal plants, nontoxic molecules were screened for their inhibitory capacity against LPS-induced nuclear factor kappa B (NF-κB) activation in a luciferase-based reporter gene assay. Since several natural products inhibited NF-κB expression in THP-1 macrophages, their effect on reactive oxygen species (ROS) production and inflammasome activation was assessed, as well as their transcriptional outcome regarding ER stress. The bioactivities of several natural products are described herein for the first time. We report the anti-inflammatory potential of guaiazulene and describe 5-deoxykaempferol as a novel inhibitor of inflammasome activation. Furthermore, we describe the dual potential of 5-deoxykaempferol, berberine, guaiazulene, luteolin-4'-O-glucoside, myricetin, quercetagetin and sennoside B to modulate inflammatory signaling ER stress. Our results show that natural products are promising molecules for the discovery and pharmaceutical development of chemical entities able to modulate the inflammatory response, as well as proteostasis and the UPR.

Keywords: ATF4; CHOP; Caspase-1; IL-1β; IL-6; TNF-α.

Fig. 1

Impact of each molecule upon cell viability of THP-1 macrophages after 24 h, as determined by the MTT reduction assay. All molecules were tested at 50 µM. Results are expressed as percentage of the control and correspond to the mean of at least three independent experiments, individually performed in triplicate. The control group is presented in pink, while the positive control (LPS) is presented in purple. The molecules under study are represented in green whenever active, and in black when inactive. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

Effect of nontoxic natural products upon NF-κB gene expression on THP-1 Lucia™ NF-κB cells after 24 h, as determined by the QUANTI-Luc™ luciferase activity assay. The control group is presented in pink, while the positive control (LPS) is presented in purple. The molecules under study are represented in green whenever active, and in black when inactive. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

Effect of anti-inflammatory molecules on ROS production on THP-1 macrophages after 24 h of incubation, as determined with the fluorescent probe DCFH-DA. Results express the relative change against the positive control and represent the mean of at least three independent experiments. The control group is presented in pink, while the positive control (LPS) is presented in purple. The molecules under study are represented in green whenever active, and in black when inactive. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

A: Influence of active molecules on the expression of IL-6, TNF-α and IL-1β, evaluated by ELISA. Results presented as mean ± standard deviation of the mean of at least three independent experiments, each conducted in triplicate. B: Effect of selected molecules on LPS-induced NLRP3 inflammasome activation, as determined by caspase-1 activation. Each column represents the mean ± standard error of the mean of at least three independent experiments, individually performed in duplicate. The control group is presented in pink, while the positive control (LPS) is presented in purple. The molecules under study are represented in green whenever active, and in black when inactive. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5

Effect of LPS on the expression of the UPR-related genes coding for atf4, ddit3 and edem1, as determined by qPCR. Gapdh was the selected reference gene. Results represent the mean ± standard error of the mean of three independent experiments, individually performed in duplicate. The control group is presented in pink, while the positive control (LPS) is presented in purple. The molecules under study are represented in green whenever active, and in black when inactive. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 6

Summary of the obtained results concerning every NF-κB inhibitor discussed herein. Black cells stand for not applicable conditions, yellow cells represent an active molecule and purple cells represent a negative result

Fig. 7

Crosstalk between ER stress and inflammatory signaling