Lygodium japonicum Herb Ameliorates Nonalcoholic Fatty Liver Disease by Inhibiting Hepatic Lipid Accumulation and Inflammatory Response in ob/ob Mice

Abstract

The ubiquity of nonalcoholic fatty liver disease (NAFLD) as a chronic metabolic disorder across the globe is currently matched by a dearth of efficacious treatment modalities. Lygodium japonicum (Thunb.) Sw. herb (LJ) extensively employed in the management of hepatitis due to its noted anti-inflammatory properties as per the Chinese Materia Medica presents an interesting potential therapeutic agent. However, its applicability and mechanistic action in NAFLD treatment remain underexplored. To investigate the potential role of LJ in mitigating NAFLD and discern whether this effect is interconnected with lipid metabolism and the inflammatory signaling pathway, the HPLC method was employed to identify potential active ingredients in LJ. Subsequently, ob/ob obese mice were used to simulate a NAFLD model and administered oral doses of LJ (300 and 600 mg/kg) over an 8 week period. Multiple measures, including body and liver weights, the liver-to-body weight ratio, lipid metabolism, and inflammatory cytokines, were evaluated. Histological parameters were assessed using HE staining, Masson's trichrome staining, and Oil red O staining. Additionally, the protein expression levels of TLR4/MyD88/NF-κB and the MAPK pathway as well as transcription factors relevant to lipid metabolism and inflammatory transcription levels were investigated. Five principal chemical components of the LJ were identified through HPLC analysis. The animal experiments demonstrated that LJ could significantly curtail weight gain, lower the liver-to-body weight ratio, decrease transaminase release and serum lipid profile, ameliorate liver histopathology alterations, and attenuate hepatocyte inflammatory infiltration and the release of proinflammatory cytokines in obese mice. Further, it was found that LJ could impede the TLR4/MyD88/NF-κB and MAPK pathway and regulate the inflammatory transcription level, thereby moderating liver inflammation. Additionally, LJ was found to inhibit genes associated with lipid synthesis, while augmenting the expression of those associated with lipid oxidation. These findings, for the first time, endorse the protective effects of LJ on obesity-related NAFLD and provide preliminary evidence that the amelioration of hepatic inflammation and restructuring of lipid metabolism play pivotal roles in LJ's intervention against obesity-related NAFLD. Collectively, our research bolsters the case for the potential clinical application of LJ in NAFLD treatment.

1

TIC chromatogram of Lygodium japonicum in both negative and positive scan modes. (A) TIC diagram of Lygodium japonicum in positive ion mode; (B) TIC diagram of Lygodium japonicum in positive ion mode.

2

HPLC fingerprints of LJ as well as its major components including (1) chlorogenic acid; (2) caffeic acid; (3) isoquercetin; (4) rutin; and (5) kaempferol. (A) The HPLC chromatography profiles of LJ. (B) The HPLC chromatography profiles of reference substances.

3

LJ alleviated weight gain and liver function damage in ob/ob mice. (A) Body weight changes; (B) Liver weights; (C) The index of liver to body weight; (D-G) The AST and ALT in serum and TG and TC in the liver. Data were represented as the mean ± SD (n = 10). *P < 0.05, **P < 0.01, and ***P < 0.001 vs the control group; ^# P < 0.05, ^## P < 0.01 vs ob/ob mice group.

4

LJ relieved lipid metabolism and inflammatory factor release in ob/ob mice. The serum levels of FFA (A), TG (B), TC (C), TNF-α (D), IL-6 (E), and IL-1β (F) were measured by kits. Data are shown as mean ± SD (n = 10). **P < 0.01, ***P < 0.001 vs the control group; ^# P < 0.05, ^## P < 0.01 vs ob/ob mice group.

5

LJ relieved hepatic histological abnormalities and inflammatory cell infiltration in ob/ob mice. (A) The liver morphology; (B) Representative micrographs of H&E, Oil red O, Masson’s trichrome stainings, and immunohistochemical analyses with CD68. (C) NAS score; (D) Quantitative analysis of collagen volume fraction in Masson’s trichrome staining; (E) Quantitative analysis of positive area in Oil red O; (F) Quantitative analysis of positive cells of CD68. Data were shown as mean ± SD (n = 4). **P < 0.01, ***P < 0.001 vs the control group; ^# P < 0.05, ^## P < 0.01 vs ob/ob mice group.

6

LJ suppressed the TLR4/MyD88/MAPK/NF-κB signaling pathway in ob/ob mice. (A-B) Protein expression levels and (C-D) Quantitative analysis of TLR4, MyD88, NF-κB, p-p38, p-ERK, and p-JNK. Data were expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 vs the control group; ^# P < 0.05, ^## P < 0.01, and ^### P < 0.001 vs ob/ob mice group.

7

Effects of LJ 600 on gene expressions in the livers of ob/ob mice. (A) Differentially expressed genes regulated by LJ 600 treatment in the livers of ob/ob mice. (B) PCA of each group. n = 3 samples per group. (C) Gene Ontology enrichment analysis of down-regulated genes by LJ 600. (D) Kyoto Encyclopedia of Genes and Genomes pathways analysis. (E-F) The Gene Set Enrichment Analysis of the hepatocyte RNA-Seq data set for signaling associated with fatty acid metabolism and NF-κB signaling pathway.

8

LJ regulated the lipid metabolism and inflammation-related gene expression. (A) Heat map showing the representative gene expression profiles related to inflammatory responses. (B) Heat map showing the representative gene expression profiles related to lipid metabolism. (C) Relative mRNA levels of inflammatory factors in the livers of indicated mice. (D) Relative mRNA levels of lipid metabolism in the livers of indicated mice. Data were expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 vs the control group; ^# P < 0.05 and ^## P < 0.01 vs ob/ob mice group.