Non-alcoholic steatohepatitis (NASH), a severe form of non-alcoholic fatty liver disease (NAFLD), is a growing global health concern. NAFLD encompasses a spectrum of liver conditions, with NASH characterized by hepatic steatosis, inflammation, and ballooned hepatocytes, potentially progressing to cirrhosis and hepatocellular carcinoma. The multiple-hit hypothesis posits that genetic and epigenetic factors interact with environmental factors, including diet and gut microbiota, to cause liver injury. High-fat diets (HFDs) contribute to NAFLD pathogenesis through increased hepatic fat accumulation, oxidative stress, and inflammatory responses. HFDs increase free fatty acids (FFAs), stimulating de novo lipogenesis and cholesterol synthesis while decreasing fatty acid oxidation, leading to triglyceride and cholesterol accumulation. Excessive FFAs upregulate CYP2E1, increasing reactive oxygen species (ROS) production, and oxidative stress contributes to chronic hepatic inflammation. Activation of the NLRP3 inflammasome, a key player in inflammation, is observed in HFD-induced hepatic steatosis, leading to IL-1β and IL-18 production and subsequent inflammation and cell death. Diet and gut microbiota interactions significantly impact NASH pathogenesis. Unhealthy diets (low fiber, high fat, high sugar) disrupt gut microbiota, causing dysbiosis and intestinal barrier leakage. This allows pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides (LPS), to enter the liver, triggering inflammation. LPS activates the TLR4 signaling pathway, leading to NF-κB activation and the production of pro-inflammatory cytokines like TNF-α, IL-6, and IL-1β. Rodent models for NASH induction, such as those using trans-fats, have limitations due to trans-fat restrictions. Palm oil-containing HFDs provide a more clinically translatable model. A recent model utilizes a palm oil-containing HFD supplemented with LPS injections to simulate gut dysbiosis and endotoxemia. Current NASH treatments have adverse side effects, prompting investigation into hepatoprotective foods and herbs. Previous studies demonstrated that ginger essential oil (GEO) alleviates hepatic lipid accumulation, oxidative stress, and pro-inflammatory cytokines in HFD-induced NAFLD. GEO also possesses antibacterial and antifungal properties and ameliorates atherosclerosis by modulating trimethylamine-N-oxide and gut microbiota. However, GEO's hepatoprotective effects against NASH and its impact on the gut microbiota-liver axis and NLRP3 inflammasome pathway remain unclear. This study aimed to investigate GEO's hepatoprotective effects via the NLRP3 inflammasome and gut microbiota-LPS-TLR4 pathway using a palm oil-containing HFD with LPS-induced NASH mouse model.

Extensive research highlights the intricate relationship between diet, gut microbiota, and the development of non-alcoholic steatohepatitis (NASH). Studies have shown that high-fat diets (HFDs) lead to gut dysbiosis and increased intestinal permeability, allowing the translocation of lipopolysaccharides (LPS) from the gut to the liver, triggering inflammation and contributing to NASH progression. The NLRP3 inflammasome has been identified as a key mediator of this inflammatory process, with its activation leading to the release of pro-inflammatory cytokines like IL-1β and IL-18. The role of specific gut microbiota members in NASH pathogenesis is also under investigation, with some bacteria showing positive correlations with disease severity, while others exhibit protective effects. Several studies have explored the hepatoprotective properties of various natural compounds, including those found in ginger, showing promising results in preclinical models of NAFLD and other liver diseases. However, the precise mechanisms by which these compounds exert their effects, and their potential clinical applications for NASH, require further investigation. This study builds on the existing literature by exploring the multifaceted effects of ginger essential oil (GEO) on NASH, encompassing its impact on gut microbiota composition, inflammatory pathways, and oxidative stress.

This study utilized a murine model of NASH induced by a palm oil-containing high-fat diet (P-HFD) supplemented with intraperitoneal lipopolysaccharide (LPS) injections (PL). Six-week-old male C57BL/6J mice were randomly assigned to five groups: a control diet group (CON); the PL group; and three groups receiving the PL diet with varying doses of GEO (12.5 mg/kg/day (PL+GL), 62.5 mg/kg/day (PL+GM), and 125 mg/kg/day (PL+GH)) administered via oral gavage for 12 weeks. GEO was extracted from aged ginger via steam distillation, and its chemical composition was analyzed using gas chromatography (GC). Citral was identified as the major component. After 12 weeks, mice were sacrificed, and various parameters were assessed. Body weight and food intake were monitored weekly. Liver, adipose tissue, and fecal samples were collected. Plasma biochemical analyses were conducted to measure total cholesterol, triglycerides, LDL, HDL, AST, ALT, and glucose. Liver homogenates were analyzed for glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GRd), superoxide dismutase (SOD), catalase (CAT), and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) using ELISA. Western blot analysis was performed to assess the expression of proteins involved in lipid metabolism (SREBP-1c, HMGCR, PPARα, CYP2E1) and the NLRP3 inflammasome pathway (NLRP3, ASC, caspase-1, TLR4, NF-κB). Fecal DNA was extracted, and 16S rRNA amplicon sequencing was performed to analyze gut microbiota composition. Plasma and hepatic LPS levels were quantified using a cell-based colorimetric assay. Intestinal permeability was assessed using FITC-dextran. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparison test, Wilcoxon signed-rank test, Kruskal-Wallis test with Dunn’s multiple comparison test, and ANOVA with Dunnett’s multiple comparison test, depending on the data type.

The palm oil-containing HFD with LPS injections (PL) resulted in increased body weight, total fat mass, plasma glucose, total cholesterol, and LDL-C compared to the control group. GEO supplementation, particularly at medium and high doses, significantly reduced body weight gain and improved lipidemia by decreasing plasma glucose and triglycerides and increasing HDL-C. The PL group exhibited significantly higher plasma ALT levels and increased liver weight compared to the control group. GEO supplementation, especially at medium and high doses, significantly reduced plasma ALT levels. Histopathological analysis revealed that the PL group had increased hepatic steatosis, hepatocyte ballooning, and lobular inflammation. GEO supplementation, especially at higher doses, significantly reduced hepatocyte ballooning scores. GEO significantly improved hepatic antioxidant enzyme activities, increasing CAT, GSH, and GRd levels and reducing CYP2E1 expression. The PL group showed increased levels of hepatic TNF-α, IL-1β, and IL-6. GEO supplementation at medium and high doses significantly reduced hepatic IL-1β and IL-6 levels, and high dose GEO also significantly reduced TNF-α levels. Western blot analysis showed increased NLRP3 and ASC expression in the PL group, which were significantly reduced by GEO supplementation. Caspase-1 expression also trended lower with GEO. 16S rRNA sequencing revealed that the PL group had altered gut microbiota α-diversity and β-diversity. GEO supplementation did not significantly affect α-diversity but caused a shift in β-diversity, particularly at high doses. GEO reduced the abundance of NASH-associated bacteria (*Blautia*, *Tyzzerella*) and increased the abundance of beneficial bacteria (*Alistipes*, *Lactobacillus*, *Olsenella*). While intestinal permeability and plasma LPS levels were not significantly altered by GEO, hepatic LPS levels were significantly reduced by GEO supplementation. Hepatic TLR4 and NF-κB expression was also significantly reduced with GEO, particularly at higher doses. These data suggest GEO may exert its effect by reducing hepatic LPS levels and subsequently, TLR4 and NF-κB expression.

This study demonstrates that GEO effectively prevents NASH progression in a clinically relevant mouse model. The observed improvements in body weight, lipid profile, and liver function parameters support GEO's hepatoprotective effects. The reduction in hepatic inflammation, as evidenced by decreased pro-inflammatory cytokine levels and NLRP3 inflammasome activation, suggests a crucial mechanism of action. The significant impact of GEO on gut microbiota composition, particularly the reduction of harmful bacteria and increase in beneficial bacteria, strongly supports a role for the gut-liver axis in mediating GEO's effects. The reduction in hepatic LPS levels and the subsequent downregulation of the TLR4/NF-κB pathway further strengthens this conclusion. The study's findings highlight GEO's potential as a dietary supplement for NASH prevention and warrants further investigation.

This study provides strong evidence supporting the use of ginger essential oil (GEO) as a potential dietary supplement for NASH prevention. GEO's multifaceted action, including its impact on lipid metabolism, oxidative stress, inflammation (via NLRP3 inflammasome inhibition), and gut microbiota remodeling (via modulation of the LPS/TLR4/NF-κB pathway), contributes to its hepatoprotective effects. Future studies should investigate the clinical efficacy of GEO in human subjects and explore the optimal dosage and administration strategies.

This study utilized a mouse model, which may not fully recapitulate the complexities of human NASH. While the palm oil-containing HFD with LPS injections provides a more clinically relevant model than previous models, interspecies differences remain. The study also housed mice separately to prevent coprophagy, which might have influenced the gut microbiome composition and reduced the generalizability of the gut microbiota findings. Future studies should address these limitations.