Quercetin attenuates lipopolysaccharide-induced hepatic inflammation by modulating autophagy and necroptosis

Abstract

Lipopolysaccharide (LPS) from Gram-negative bacteria initially induces liver inflammation with proinflammatory cytokines expressions. However, the underlying hepatoprotective mechanism of quercetin on LPS-induced hepatic inflammation remains unclear. Specific pathogen-free chicken embryos (n = 120) were allocated control vehicle, PBS with or without ethanol vehicle, LPS (125 ng/egg) with or without quercetin treatment (10, 20, or 40 nmol/egg, respectively), quercetin groups (10, 20, or 40 nmol/egg). Fifteen-day-old embryonated eggs were inoculated abovementioned solutions via the allantoic cavity. At embryonic d 19, the livers of the embryos were collected for histopathological examination, RNA extraction, real-time polymerase chain reaction, and immunohistochemistry investigation. We found that the liver presented inflammatory response (heterophils infiltration) after LPS induction. The LPS-induced mRNA expressions of inflammation-related factors (TLR4, TNFα, IL-1β, IL-10, IL-6, MYD88, NF-κB1, p38, and MMP3) were upregulated after LPS induction when compared with the PBS group, while quercetin could downregulate these expressions as compared with the LPS group. Quercetin significantly decreased the immunopositivity to TLR4 and MMP3 in the treatment group when compared with the LPS group. Quercetin could significantly downregulate the mRNA expressions of autophagy-related genes (ATG5, ATG7, Beclin-1, LC3A, and LC3B) and necroptosis-related genes (Fas, Bcl-2, Drp1, and RIPK1) after LPS induction. Quercetin significantly decreased the immunopositivity to LC3 in the treatment group when compared with the LPS group; meanwhile, quercetin significantly decreased the protein expressions of LC3-I, LC3-II, and the rate of LC3-II/LC3-I. In conclusions, quercetin can alleviate hepatic inflammation induced by LPS through modulating autophagy and necroptosis.

Keywords: endotoxin; injury; liver; programmed cell death; quercetin.

Figure 1

The histopathologic changes of livers induced by LPS and quercetin in chicken embryos. Hematoxylin and eosin stain (400 ×). (A) Control group; (B) PBS group; (C) PBS + ethanol group; (D) 40 nmol Q, 40 nmol quercetin; (E) LPS group (125 ng LPS/egg); (F) 125 ng LPS + 40 nmol Q, LPS (125ng/egg) and quercetin (Q) at 40 nmol/egg. PV:Portal veins; L: Lipid droplets; H: Heterophils; Cg: Congestion; CL: Caryolysis; KP: Karyopycnosis (arrow). Scale bar: 40 μm.

Figure 2

Quercetin downregulated the hepatic mRNA expressions of inflammation-related genes after LPS induction in the chicken embryos. Data are presented as the mean ± SD. * indicates the significant differences between the PBS group and the LPS group; * < 0.05, ** < 0.01, *** < 0.001; # indicates the significant differences between the LPS group and the LPS + Q group; # < 0.05, ## < 0.01, *** < 0.001; ★ indicates the significant differences between the PBS + ethanol group and the LPS + Q group; ★ < 0.05, ★★ < 0.01, ★★★ < 0.001;● indicates the significant differences between the PBS + ethanol group and the Q (quercetin) group; ● < 0.05, ●● < 0.01, ●●● < 0.001.

Figure 3

Quercetin alleviates the protein expression of TLR4 induced by LPS in the livers of chicken embryos by immunohistochemical investigation. (A) PBS group; (B) LPS group (125 ng /egg); (C) treatment group (125 ng LPS/egg + 40 nmol quercetin /egg); (D) relative immunopositive area of TLR4. Immunopositivity to TLR4 (arrow, brown to yellow). Scale bar: 50 μm. Data were expressed as the mean ± SD. * indicates the significant differences between the PBS group and the LPS group; ***: P < 0.001. # indicates the significant differences between the LPS group and the LPS + Q group; ##: P < 0.01.

Figure 4

Quercetin alleviates the protein expression of MMP3 induced by LPS in the livers of chicken embryos by immunohistochemical investigation. (A) PBS group; (B) LPS group (125 ng /egg); (C) treatment group (125 ng LPS/egg + 40 nmol quercetin /egg); (D) relative immunopositive area of MMP3. Immunopositivity to MMP3 (arrow, brown to yellow). Scale bar: 50 μm. Data were expressed as the mean ± SD. * indicates the significant differences between the PBS group and the LPS group; ***: P < 0.001. # indicates the significant differences between the LPS group and the LPS + Q group; ###: P < 0.001.

Figure 5

Quercetin attenuates LPS-induced hepatic inflammation by modulating the mRNA expressions of autophagy and programmed cell death-related genes in the chicken embryos. Data are presented as the mean ± SD. * indicates the significant differences between the PBS group and the LPS group; * < 0.05, ** < 0.01, *** < 0.001; # indicates the significant differences between the LPS group and the LPS + Q group; # < 0.05, ## < 0.01, *** < 0.001; ★ indicates the significant differences between the PBS + ethanol group and the LPS + Q group; ★ < 0.05, ★★ < 0.01, ★★★ < 0.001;● indicates the significant differences between the PBS + ethanol group and the Q (quercetin) group; ● < 0.05, ●● < 0.01, ●●● < 0.001.

Figure 6

Quercetin alleviates the protein expression of LC3 induced by LPS in the livers of chicken embryos by immunohistochemical investigation. (A) PBS group; (B) LPS group (125 ng /egg); (C) treatment group (125 ng LPS/egg + 40 nmol quercetin /egg); (D) relative immunopositive area of LC3. Immunopositivity to LC3 (arrow, brown to yellow). Scale bar: 50 μm. Data were expressed as the mean ± SD. * indicates the significant differences between the PBS group and the LPS group; **: P < 0.01. # indicates the significant differences between the LPS group and the LPS + Q group; ##: P < 0.01.

Figure 7

Quercetin alleviates protein expression levels of LC3-I and LC3-II after LPS induction in the livers of chicken embryos. PBS group; LPS group:125 ng LPS /egg; quercetin treatment group LPS + Q: (125 ng LPS + 40 nmol Q)/egg. A, the protein expression of LC3-I and LC3-II; B, relative protein expression of LC3-II; C, relative protein expression of LC3-I; D, ratio of relative protein expression of LC3-II/LC3-I. Data were expressed as the mean ± SD. * indicates the significant differences between the PBS group and the LPS group; # indicates the significant differences between the LPS group and the LPS + Q group.