Ginsenoside Rb1 Reduces D-GalN/LPS-induced Acute Liver Injury by Regulating TLR4/NF-κB Signaling and NLRP3 Inflammasome

Abstract

Background and aims: The effect of ginsenoside Rb1 on D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced acute liver injury (ALI) is unknown. The aim of this study was to evaluate the effect of ginsenoside Rb1 on ALI and its underlying mechanisms.

Methods: Mice were pretreated with ginsenoside Rb1 by intraperitoneal injection for 3 days before D-GalN/LPS treatment, to induce ALI. The survival rate was monitored every hour for 24 h, and serum biochemical parameters, hepatic index and histopathological analysis were evaluated to measure the degree of liver injury. ELISA was used to detect oxidative stress and inflammatory cytokines in hepatic tissue and serum. Immunohistochemistry staining, RT-PCR and western blotting were performed to evaluate the expression of toll-like receptor 4 (TLR4), nuclear factor-kappa B (NF-κB), and NLR family, pyrin domain-containing 3 protein (NLRP3) in liver tissue and Kupffer cells (KCs).

Results: Ginsenoside Rb1 improved survival with D-GalN/LPS-induced ALI by up to 80%, significantly ameliorated the increased alanine and aspartate transaminase, restored the hepatic pathological changes and reduced the levels of oxidative stress and inflammatory cytokines altered by D-GalN/LPS. Compared to the control group, the KCs were increased in the D-GalN/LPS groups but did not increase significantly with Rb1 pretreatment. D-GalN/LPS could upregulate while Rb1 pretreatment could downregulate the expression of interleukin (IL)-1β, IL-18, NLRP3, apoptosis associated speck-like protein containing CARD (ASC) and caspase-1 in isolated KCs. Furthermore, ginsenoside Rb1 inhibited activation of the TLR4/NF-κB signaling pathway and NLRP3 inflammasome induced by D-GalN/LPS administration.

Conclusions: Ginsenoside Rb1 protects mice against D-GalN/LPS-induced ALI by attenuating oxidative stress and the inflammatory response through the TLR4/NF-κB signaling pathway and NLRP3 inflammasome activation.

Keywords: Chemical and drug induced liver injury; Ginsenosides; NLR family, pyrin domain-containing 3 protein; Toll-like receptor 4.

Fig. 1. Rb1 protected mice against ALI induced by D-GalN/LPS.

(A) The survival rate of mice was continuously monitored every hour for 24 h. (B-C) The levels of liver damage markers ALT and AST in serum. (D) Hepatic index calculated in different groups. (E) Histological score used to evaluate the severity of liver injury according to the Suzuki’s criteria. (F) Hematoxylin-eosin staining (left, 200× original magnification; right, 400× original magnification). Data are shown as mean±standard error of the mean (n=10). ^#p<0.05 vs. control group, ^*p<0.05 vs. D-GalN/LPS group. ALI, acute liver injury; ALT, alanine transaminase; AST, aspartate transaminase; D-GalN, d-galactosamine; LPS, lipopolysaccharide.

Fig. 2. Effect of Rb1 on oxidative stress in liver tissue of ALI mice.

The pro-oxidation markers such as ROS, MPO and MDA, and antioxidant markers such as SOD and GSH-px were determined by commercial reagent kits in fresh liver tissues. Tests were performed at least three times. Data are shown as mean±standard error of the mean and are expressed as % of normal, U/g of protein, nmol/mg of protein, U/mg of protein and nmol/mg of protein, respectively. ^#p<0.05 vs. control group, ^*p<0.05 vs. D-GalN/LPS group. ALI, acute liver injury; D-GalN, d-galactosamine; GSH-px, glutathione-peroxidase; LPS, lipopolysaccharide; MDA, malondialdehyde; MPO, myeloperoxidase; ROS, reactive oxygen species; SOD, superoxide dismutase.

Fig. 3. Effect of Rb1 on inflammatory cytokine expression in serum and liver tissue of ALI mice.

(A-H) Serum and hepatic levels of IL-6, TNF-α, IL-1β and IL-18 were evaluated by commercial reagent kits. (I) IHC was used to evaluate the expression of IL-6 and TNF-α in liver tissue (400× original magnification). Tests were performed at least three times. Data are shown as mean±standard error of the mean and are expressed as pg/mL and pg/mg, respectively. ^#p<0.05 vs. control group, ^*p<0.05 vs. D-GalN/LPS group. ALI, acute liver injury; ALT, alanine transaminase; D-GalN, d-galactosamine; IHC, immunohistochemistry; IL, interleukin; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-alpha.

Fig. 4. KCs accumulation, and inflammatory cytokine expression and NLRP3 inflammasome activation in KCs among different groups.

(A) F4/80+ KCs were evaluated by IHC staining of hepatic tissues. (B) mRNA expressions of IL-1β. (C) mRNA expressions of IL-18. (D) Western blot analysis of NLRP3, ASC and caspase-1 in KCs. (E) Relative protein content was quantified by densitometry. Tests were performed at least three times. Data are shown as mean±standard error of the mean. ^#p<0.05 vs. control group, *p<0.05 vs. D-GalN/LPS group. ASC, apoptosis associated speck-like protein containing CARD; D-GalN, d-galactosamine; IHC, immunohistochemistry; IL, interleukin; KCs, Kupffer cells; LPS, lipopolysaccharide; NLRP3, NLR family, pyrin domain-containing 3 protein.

Fig. 5. Effect of Rb1 on TLR4/NF-κB signal pathway in D-GalN/LPS-induced ALI mice.

(A) IHC analysis of TLR4 (400× original magnification). (B) mRNA expressions of TLR4 and MyD88. (C) Western blot analysis of TLR4, MyD88, p-IκBα, IκBα, nucleus p65 and cytoplasm p65 expression in liver tissue. (D) Relative protein content was quantified by densitometry. Tests were performed at least three times. Data are shown as mean±standard error of the mean. ^#p<0.05 vs. control group, *p<0.05 vs. D-GalN/LPS group. ALI, acute liver injury; D-GalN, d-galactosamine; IHC, immunohistochemistry; LPS, lipopolysaccharide; MyD88, myeloid differentiation factor 88; TLR4, toll-like receptor 4.

Fig. 6. Effect of Rb1 on the activation of NLRP3 inflammasome in D-GalN/LPS-induced ALI mice.

(A) IHC analysis of NLRP3 (400× original magnificatios). (B) The mRNA expressions of NLRP3, ASC and caspase-1. (C) Western blot analysis of NLRP3, ASC and caspase-1 in liver tissue. (D) Relative protein content was quantified by densitometry. Tests were performed at least three times. Data are shown as mean±standard error of the mean. ^#p<0.05 vs. control group, *p<0.05 vs. D-GalN/LPS group. ALI, acute liver injury; ASC, apoptosis associated speck-like protein containing CARD; D-GalN, d-galactosamine; IHC, immunohistochemistry; LPS, lipopolysaccharide; NLRP3, NLR family, pyrin domain-containing 3 protein.

Fig. 7. Working model of the protective effects of Rb1 against D-GalN/LPS induced ALI: Rb1 inhibition of the TLR4/MyD88/NF-κB signaling pathway and NLRP3 inflammasome activation.

ALI, acute liver injury; D-GalN, d-galactosamine; LPS, lipopolysaccharide; MyD88, myeloid differentiation factor 88; NF-κB, nuclear factor-kappa B; NLRP3, NLR family, pyrin domain-containing 3 protein; TLR4, toll-like receptor 4.