Ginger essential oil prevents NASH progression by blocking the NLRP3 inflammasome and remodeling the gut microbiota-LPS-TLR4 pathway in mice

Abstract

Background: Diet and gut microbiota contribute to non-alcoholic steatohepatitis (NASH) progression. High-fat diets (HFDs) change gut microbiota compositions, induce gut dysbiosis, and intestinal barrier leakage, which facilitates portal influx of pathogen-associated molecular patterns including lipopolysaccharides (LPS) to the liver and triggers inflammation in NASH. Current therapeutic drugs for NASH have adverse side effects; however, several foods and herbs that exhibit hepatoprotection could be an alternative method to prevent NASH.

Methods: We investigated ginger essential oil (GEO) against palm oil-containing HFDs in LPS-injected murine NASH model.

Results: GEO reduced plasma alanine aminotransferase levels and hepatic pro-inflammatory cytokine levels; and increased antioxidant catalase, glutathione reductase, and glutathione levels to prevent NASH. GEO alleviated hepatic inflammation through mediated NLR family pyrin domain-containing 3 (NLRP3) inflammasome and LPS/Toll-like receptor four (TLR4) signaling pathways. GEO further increased beneficial bacterial abundance and reduced NASH-associated bacterial abundance.

Conclusion: This study demonstrated that GEO prevents NASH progression which is probably associated with the alterations of gut microbiota and inhibition of the LPS/TLR4/NF-κB pathway. Hence, GEO may offer a promising application as a dietary supplement for the prevention of NASH.

Fig. 1. Palm oil-containing HFD with LPS i.p. (PL) resulted in an adverse effect on obesogenic and metabolic biomarkers. Supplementation with GEO trended to prevent obesity and ameliorated lipidemia.

a Experimental design, b body weight change, c area under the curve of body weight, d body weight at 12 weeks, e relative total fat mass, f average energy intake, g plasma glucose, h plasma total triglyceride, i total cholesterol, j high-density lipoprotein (HDL-C), and k low-density lipoprotein (LDL-C) in C57BL/6 mice, which were treated with either control or a palm oil-containing HFD with LPS i.p. (PL) with or without ginger essential oil (GEO) (12.5 (GL), 62.5 (GM), and 125 (GH) mg/kg bw) supplementation by daily oral gavage for 12 weeks. Each value was expressed as the mean ± SD (n = 7–8). Statistical analyses were performed by repeated measures ANOVA or one-way ANOVA with Dunnett’s multiple comparison test for comparing group means against the PL group.

Fig. 2. GEO prevents NASH by improving plasma hepatic damage biomarkers and reversing the NAFLD activity and hepatocyte ballooning scores.

a Plasma aspartate aminotransferase (AST), b alanine aminotransferase (ALT), c liver weight, d representative image of liver images (scale bar: 1 cm) and histopathological changes (400X magnification; scale bar: 100 µm), e NAFLD activity score, f steatosis score, g hepatocyte ballooning score, and h lobular inflammation score and their prevalence in C57BL/6 mice, which were treated with either control or a palm oil-containing HFD with LPS i.p. (PL) with or without ginger essential oil (GEO) (12.5 (GL), 62.5 (GM), and 125 (GH) mg/kg bw) supplementation by daily oral gavage for 12 weeks. Bar plots were expressed as the mean ± SD (n = 7–8). Box plots display median and mean (+), quartiles (boxes), and range (whiskers). Statistical analyses were performed by a one-way ANOVA with Dunnett’s multiple comparison test for comparing group means against the PL group.

Fig. 3. GEO improves hepatic antioxidant enzyme activities.

a Cytochrome P450 2E1 (CYP2E1) protein expression, b hepatic superoxide dismutase (SOD), c catalase (CAT), d glutathione (GSH), e glutathione peroxidase (GPx), and f glutathione reductase (GRd) in C57BL/6 mice, which were treated with either control or a palm oil-containing HFD with LPS i.p. (PL) with or without ginger essential oil (GEO) (12.5 (GL), 62.5 (GM), and 125 (GH) mg/kg bw) supplementation by daily oral gavage for 12 weeks. Each value was expressed as the mean ± SD (n = 7–8). Statistical analyses were performed by a one-way ANOVA with Dunnett’s multiple comparison test for comparing group means against the PL group.

Fig. 4. GEO reduces hepatic inflammation by suppressing inflammatory cytokines and the NLRP3/ASC pathways.

a Liver TNF-α, b IL-1β, c IL-6, d representative image of NLRP3, ASC, caspase-1 protein expression, e NLRP3, f ASC, and g caspase-1 in C57BL/6 mice, which were treated with either control or a palm oil-containing HFD with LPS i.p. (PL) with or without ginger essential oil (GEO) (12.5 (GL), 62.5 (GM), and 125 (GH) mg/kg bw) supplementation by daily oral gavage for 12 weeks. Each value was expressed as the mean ± SD (n = 7–8), protein expression (n = 3). Statistical analyses were performed by a one-way ANOVA with Dunnett’s multiple comparison test for comparing group means against the PL group.

Fig. 5. PL and GEO remodel the composition of fecal microbiota.

a α-diversity indices, observed ASVs, Shannon index, and Simpson diversity index, b principal coordinate analysis (PCoA) plot based on Bray–Curtis dissimilarity with biomarker vector, c heatmap of the relative abundances at genera level, and d Spearman’s correlation analysis between gut microbiota components and NASH-related parameters in C57BL/6 mice, which were treated with either control or a palm oil-containing HFD with LPS i.p. (PL) with or without ginger essential oil (GEO) (12.5 (GL), 62.5 (GM), and 125 (GH) mg/kg bw) supplementation by daily oral gavage for 12 weeks. Each value was expressed as the mean ± SD (n = 7). Statistical analyses were performed by a one-way ANOVA with Dunnett’s multiple comparison test for comparing group means against the PL group. Analysis of variance using distance matrices (Adonis) were calculated to determine the heterogeneity of the feces microbiota among the groups in PCoA. Vectors in the PCoA plot indicated a significant effect of biomarkers (p < 0.05), and its length shows the strength of the correlation.

Fig. 6. GEO reduces liver inflammation by suppressing the gut-derived LPS/TLR4/NF-κB signaling pathway.

a Intestinal permeability based on plasma FITC-dextran intensity, b plasma LPS, c hepatic LPS, d representative image of TLR4 and NF-κB protein expression, e TLR4 protein expression, and f NF-κB protein expression in C57BL/6 mice, which were treated with either control or a palm oil-containing HFD with LPS i.p. (PL) with or without ginger essential oil (GEO) (12.5 (GL), 62.5 (GM), and 125 (GH) mg/kg bw) supplementation by daily oral gavage for 12 weeks. Each value was expressed as the mean ± SD (n = 7–8), protein expression (n = 3). Statistical analyses were performed by a one-way ANOVA with Dunnett’s multiple comparison test for comparing group means against the PL group.