Dihydromyricetin alleviates Escherichia coli lipopolysaccharide-induced hepatic injury in chickens by inhibiting the NLRP3 inflammasome

Abstract

Dihydromyricetin (DHM), a flavonoid in vine tea, has many pharmacological activities, including anti-inflammatory and antibacterial effects. Lipopolysaccharide is the key inducer of inflammation in avian pathogenic Escherichia coli (E. coli) infection; however, the effect of DHM on E. coli lipopolysaccharide-induced hepatic injury remains unknown. The present study aimed to explore the role of the NLRP3 inflammasome in hepatic injury and the possible protective mechanisms of DHM against hepatic injury in chickens. The results showed that when chickens were administered lipopolysaccharide, liver damage was observed, accompanied by increased levels of serum transaminases and direct bilirubin. Additionally, hepatic expression levels of NLRP3 and caspase-1 p20, the subunit of caspase-1 that is cleaved after NLRP3 activation, significantly increased in liver injury. We found that treatment with MCC950, a specific NLRP3 inhibitor, significantly decreased serum transaminase activities, direct bilirubin content, and hepatic NLRP3 and caspase-1 p20 expression levels. DHM significantly reduced serum transaminase activities and direct bilirubin content and ameliorated histopathological and ultrastructural changes in the liver. DHM decreased hepatic levels of H₂O₂ and malondialdehyde and increased the activities of superoxide dismutase and glutathione peroxidase. Furthermore, DHM significantly decreased the expression levels of NLRP3, pro-caspase-1 and caspase-1 p20. Moreover, DHM reduced serum lactate dehydrogenase, IL-1β and IL-18 levels and repressed hepatic IL-1β, IL-18 and gasdermin A expression. The results demonstrated that the NLRP3 inflammasome was involved in the mechanism of lipopolysaccharide-induced hepatic injury. Furthermore, DHM could inhibit NLRP3 inflammasome activation and subsequent pyroptosis, eventually ameliorating E. coli lipopolysaccharide-induced liver injury.

Keywords: Dihydromyricetin; Escherichia coli lipopolysaccharide; NLRP3 inflammasome; hepatic injury.

Figure 1

LPS induced hepatotoxicity in chickens. A Changes in AST activity (a), ALT activity (b), and DBILI content (c) in serum after LPS treatment (60 mg/kg). ** P < 0.01 vs. the control group. B Histopathological analysis of chicken liver. Histopathological examination results of liver samples from two experimental groups are shown (scale bar = 50 μm), and the images are displayed at 100 × and 400 × the original magnification. The experimental groups were the control group (a) and LPS-treated group (b). C Ultrastructural analysis of chicken liver. Transmission electron microscopic examination of liver samples from two experimental groups (scale bar = 2 μm). a shows the control group, and b shows the LPS-treated group. ALT: alanine aminotransferase; AST: aspartate aminotransferase; DBILI: direct bilirubin.

Figure 2

MCC950 repressed the activation of the NLRP3 inflammasome. A Western blot analyses of the expression of NLRP3, caspase1-p20, and β-actin (loading control) in total tissue lysates. B, C Changes in the protein expression levels of NLRP3 (fold change in NLRP3/β-actin) and caspase1-p20 (fold change in caspase1-p20/β-actin) in livers from chickens administered MCC950 (5 mg/kg) followed by LPS (60 mg/kg). D, E Changes in the mRNA expression levels of NLRP3 (fold change in NLRP3/β-actin) and pro-caspase-1 (fold change in pro-caspase-1/β-actin) in livers from chickens stimulated by LPS (60 mg/kg) with or without MCC950 administration (5 mg/kg). Values are expressed as the mean ± SD for each group (n = 6). **P < 0.01 vs. the control group. ^##P < 0.01 vs. the LPS-treated group.

Figure 3

NLRP3 participates in LPS-induced hepatotoxicity. A Changes in AST activity (a), ALT activity (b), and DBILI content (c) in serum from chickens administered MCC950 (5 mg/kg) followed by exposure to LPS (60 mg/kg). Values are expressed as the mean ± SD for each group (n = 6). **P < 0.01 vs. the control group. ^##P < 0.01 vs. the LPS-treated group. B Histopathological analysis of chicken liver. The images are displayed at 100 × and 400 × the original magnification (scale bar = 50 μm). The experimental groups were the control group (a), LPS-treated group (b), MCC950 + LPS group (c), and MCC950 alone group (d). ALT: alanine aminotransferase; AST: aspartate aminotransferase; DBILI: direct bilirubin.

Figure 4

Pyroptosis occurs in LPS-induced hepatotoxicity. A, B Changes in the mRNA expression levels of IL-18 (fold change in IL-18/β-actin) and IL-1β (fold change in IL-1β/β-actin) in livers from chickens stimulated by LPS (60 mg/kg) with or without MCC950 administration (5 mg/kg). C–E Changes in LDH activity, IL-18 content and IL-1β content in serum from chickens administered MCC950 (5 mg/kg) followed by LPS exposure (60 mg/kg). Values are expressed as the mean ± SD for each group (n = 6). **P < 0.01 vs. the control group. ^##P < 0.01 vs. the LPS-treated group. LDH: lactate dehydrogenase.

Figure 5

Protective effects of DHM against LPS-induced hepatotoxicity. Changes in AST activity (A), ALT activity (B), and DBILI content (C) in serum from chickens administered 0.025%, 0.05% or 0.1% DHM for 14 days followed by LPS exposure (60 mg/kg). Values are expressed as the mean ± SD for each group (n = 6). **P < 0.01 vs. the control group. #P < 0.05 and ##P < 0.01 vs. the LPS-treated group. ALT: alanine aminotransferase; AST: aspartate aminotransferase; DBILI: direct bilirubin.

Figure 6

Histopathological analysis of chicken liver. Histopathological examination of liver samples from six experimental groups. The images are displayed at 100 × and 400 × the original magnification (scale bar = 50 μm). The experimental groups were the control group (A), LPS-treated group (B), 0.025% DHM + LPS group (C), 0.05% DHM + LPS group (D), 0.1% DHM + LPS group (E), and 0.1% DHM alone group (F).

Figure 7

Ultrastructural analysis of chicken liver. Transmission electron microscopic images of liver samples from six experimental groups are shown (scale bar = 5 μm). The experimental groups were the control group (A), LPS-treated group (B), 0.025% DHM + LPS group (C), 0.05% DHM + LPS group (D), 0.1% DHM + LPS group (E), and 0.1% DHM alone group (F).

Figure 8

Effects of DHM on oxidative stress in LPS-induced hepatotoxicity. Changes in SOD activity (A), GSH-Px activity (B), MDA content (C), and H₂O₂ content (D) in liver from chickens administered 0.025%, 0.05% or 0.1% DHM for 14 days followed by LPS exposure (60 mg/kg). Values are expressed as the mean ± SD for each group (n = 6). **P < 0.01 vs. the control group. ^#P < 0.05 and ^##P < 0.01 vs. the LPS-treated group. SOD: superoxide dismutase; GSH-Px: glutathione peroxidase; MDA: malondialdehyde.

Figure 9

DHM repressed NLRP3 inflammasome activation in LPS-induced hepatotoxicity. A Western blot analyses of NLRP3, caspase1-p20, and β-actin (loading control) expression in total tissue lysates. B, C Changes in the protein expression levels of NLRP3 (fold change in NLRP3/β-actin) and caspase1-p20 (fold change in caspase1-p20/β-actin) in livers from chickens administered 0.025%, 0.05% or 0.1% DHM for 14 days followed by LPS exposure (60 mg/kg). D, E Changes in the mRNA expression levels of NLRP3 (fold change in NLRP3/β-actin) and pro-caspase-1 (fold change in pro-caspase-1/β-actin) in livers from chickens administered 0.025%, 0.05% or 0.1% DHM for 14 days followed by LPS exposure (60 mg/kg). Values are expressed as the mean ± SD for each group (n = 6). **P < 0.01 vs. the control group. ^#P < 0.05 and ^##P < 0.01 vs. the LPS-treated group.

Figure 10

DHM repressed pyroptosis in LPS-induced hepatotoxicity. A–C Changes in LDH activity, IL-18 content and IL-1β content in serum from chickens administered 0.025%, 0.05% or 0.1% DHM for 14 days followed by LPS exposure (60 mg/kg). D–H Changes in the mRNA expression levels of IL-18 (fold change in IL-18/β-actin), IL-1β (fold change in IL-1β/β-actin), gasdermin A (fold change in gasdermin A/β-actin), gasdermin E (fold change in gasdermin E/β-actin), and DFNB59 (fold change in DFNB59/β-actin) in livers from chickens administered 0.025%, 0.05% or 0.1% DHM for 14 days followed by LPS exposure (60 mg/kg). I Changes in the protein expression level of gasdermin A (fold change in gasdermin A/β-actin) in the livers of chickens administered 0.025%, 0.05% or 0.1% DHM for 14 days followed by LPS exposure (60 mg/kg). J Western blot analyses of for the expression of gasdermin A and β-actin (loading control) in total tissue lysate. Values are expressed as the mean ± SD for each group (n = 6). **P < 0.01 vs. the control group. ^#P < 0.05 and ^##P < 0.01 vs. the LPS-treated group.