Dihydromyricetin attenuates Escherichia coli lipopolysaccharide-induced ileum injury in chickens by inhibiting NLRP3 inflammasome and TLR4/NF-κB signalling pathway

Abstract

Lipopolysaccharide (LPS) as a major component of Escherichia coli cell wall can cause inflammation and cell death. Dihydromyricetin (ampelopsin, DHM) is a natural flavonoid compound with anti-inflammatory, anti-oxidant and anti-bacterial effects. The preventive effects of DHM against ileum injury remain unclear. Here, we explored the protective role of DHM against LPS-induced ileum injury in chickens. In this study, DHM significantly attenuated LPS-induced alteration in diamine oxidase, malondialdehyde, reduced glutathione, glutathione peroxidase and superoxide dismutase levels in chicken plasma and ileum. Histology evaluation showed that the structure of blood vessels in ileum was seriously fragmented and presence of necrotic tissue in the lumen in the LPS group. Scanning electron microscopic observation revealed that the surface of the villi was rough and uneven, the structure was chaotic, and the normal finger shape was lost in the LPS group. In contrast, 0.05% and 0.1% DHM treatment partially alleviated the abnormal morphology. Additionally, DHM maintained the barrier function by restoring the protein expression of occludin, claudin-1 and zonula occludens protein-1. DHM inhibited apoptosis through the reduction of the expression of bax and caspase-3 and restored the expression of bcl-2. Importantly, DHM could reduce ileum NLR family pyrin domain-containing 3 (NLRP3), caspase-1, interleukin (IL)-1β and IL-18 expression to protect tissues from pyroptosis and inhibited toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signalling pathway. In summary, DHM attenuated the ileum mucosal damage, oxidative stress and apoptosis, maintained barrier function, inhibited NLRP3 inflammasome and TLR4/NF-κB signalling pathway activation triggered by Escherichia coli LPS.

Figure 1

Effects of 0.025%, 0.05% and 0.1% DHM on LPS-induced ileum injury and oxidative stress. Changes of DAO activity (A) in plasma, DAO activity (B), MDA content (C), SOD activity (D), GSH content (E) and GSH-Px activity (F) in ileum after 60 mg/kg LPS exposure for 12 h followed by 14 days of 0.025%, 0.05% and 0.1% DHM treatment. Values are expressed as the mean ± SD for each group (n = 5). *p < 0.05 and **p < 0.01 represented all groups compared with the control group. ^#p < 0.05 and ^##p < 0.01 represented all groups compared with the LPS group.

Figure 2

Chickens ileum pathology. Ileum sections were stained by haematoxylin and eosin. All the ileum sections were examined by light microscopy and the images were displayed at 100X the original magnification. (A) Control group; (B) LPS group; (C) 0.025% DHM + LPS group; (D) 0.05% DHM + LPS group; (E) 0.1% DHM + LPS group; (F) 0.1% DHM group.

Figure 3

Scanning electron microscope (SEM) observation. SEM observation for ileum after 60 mg/kg LPS exposure for 12 h followed by 14 days of 0.025%, 0.05% and 0.1% DHM treatment. (A/D) Control group (200X/2000X); (B/E) LPS group (200X/2000X); (C/F) 0.025% DHM + LPS group (200X/2000X); (G/J) 0.05% DHM + LPS group (200X/2000X); (H/K) 0.1% DHM + LPS group (200X/2000X); (I/L) 0.1% DHM group (200X/2000X).

Figure 4

0.025%, 0.05% and 0.1% DHM maintained the barrier function in ileum. Original blots for ZO-1, occluding, claudin-1 and GAPDH (A). Changes of ZO-1 (B), occludin (C) and claudin-1(D) protein expression levels in the ileum after 60 mg/kg LPS exposure for 12 h followed by 14 days of 0.025%, 0.05% and 0.1% DHM treatment. Values are expressed as the mean ± SD for each group (n = 5). *p < 0.05 and **p < 0.01 represented all groups compared with the control group. ^#p < 0.05 and ^##p < 0.01 represented all groups compared with the LPS group.

Figure 5

0.025%, 0.05% and 0.1% DHM inhibited LPS-induced ileum apoptosis. Changes of bcl-2 (A), bax (B) and caspase-3 (C) mRNA expression levels in ileum after 60 mg/kg LPS exposure for 12 h followed by 14 days of 0.025%, 0.05% and 0.1% DHM treatment. Original blots for bcl-2, bax, caspase-3 and GAPDH (D). Changes of bcl-2 (E), bax (F) and caspase-3 (G) protein expression levels in the ileum after 60 mg/kg LPS exposure for 12 h followed by 14 days of 0.025%, 0.05% and 0.1% DHM treatment. Values are expressed as the mean ± SD for each group (n = 5). *p < 0.05 and **p < 0.01 represented all groups compared with the control group. ^#p < 0.05 and ^##p < 0.01 represented all groups compared with the LPS group.

Figure 6

0.025%, 0.05% and 0.1% DHM inhibited inflammasome formation and pyroptosis activation in LPS-induced ileum injury. Changes of NLRP3 (A), caspase-1 (B) IL-1β (C) and IL-18 (D) mRNA expression levels and IL-1β (E) and IL-18 (F) contents in ileum after 60 mg/kg LPS exposure for 12 h followed by 14 days of 0.025%, 0.05% and 0.1% DHM treatment. Values are expressed as the mean ± SD for each group (n = 5). *p < 0.05 and **p < 0.01 represented all groups compared with the control group. ^#p < 0.05 and ^##p < 0.01 represented all groups compared with the LPS group.

Figure 7

DHM inhibited TLR4/NF-κB signalling pathway and inflammation in LPS-induced ileum injury. Original blots for TLR4, p65, p-p65 and GAPDH (A). Changes of TLR4 (B) protein expression levels, p-p65/p65 ratio (C), IL-6 (D), IL-8 (E), TNF-α (F) and IL-10 (G) mRNA expression levels in ileum after 60 mg/kg LPS exposure for 12 h followed by 14 days of 0.025%, 0.05% and 0.1% DHM treatment. Values are expressed as the mean ± SD for each group (n = 5). *p < 0.05 and **p < 0.01 represented all groups compred with thecontrol group. ^#p < 0.05 and ^##p < 0.01 represented all groups compared with the LPS group.