17β-Estradiol protects female rats from bilateral oophorectomy-induced nonalcoholic fatty liver disease induced by improving linoleic acid metabolism alteration and gut microbiota disturbance

Abstract

After surgical or natural menopause, women face a high risk of nonalcoholic fatty liver disease (NAFLD), which can be diminished by hormone replacement therapy (HRT). The gut microbiota is subject to modulation by various physiological changes and the progression of diseases. This microbial ecosystem coexists symbiotically with the host, playing pivotal roles in immune maturation, microbial defense mechanisms, and metabolic functions essential for nutritional and hormone homeostasis. E₂ supplementation effectively prevented the development of NAFLD after bilateral oophorectomy (OVX) in female rats. The changes in the gut microbiota such as abnormal biosynthetic metabolism of fatty acids caused by OVX were partially restored by E₂ supplementation. The combination of liver transcriptomics and metabolomics analysis revealed that linoleic acid (LA) metabolism, a pivotal pathway in fatty acids metabolism was mainly manipulated during the induction and treatment of NAFLD. Further correlation analysis indicated that the gut microbes were associated with abnormal serum indicators and different LA metabolites. These metabolites are also closely related to serum indicators of NAFLD. An in vitro study verified that LA is an inducer of hepatic steatosis. The changes in transcription in the LA metabolism pathway could be normalized by E₂ treatment. The metabolic perturbations of LA may directly and secondhand impact the development of NAFLD in postmenopausal individuals. This research focused on the sex-specific pathophysiology and treatment of NAFLD, providing more evidence for HRT and calling for the multitiered management of NAFLD.

Keywords: Hormone replacement therapy; Linoleic acid; Nonalcoholic fatty liver disease; Surgical menopause.

Fig. 1

Female rats developed excess weight gain and NAFLD after OVX. (A) Typical estrous cycle changes before and after modeling. (B) Measurement of serum hormones, including LH, FSH and E₂ (n = 6). (C) Weight gain of rats after modeling (n = 6). (D) Representative comparison photographs of liver, perinephric fat, mesenteric adipose and subcutaneous fat. (E) Representative images of Oil Red O staining of liver tissue (scale bar = 50 μm), lipid droplets under TEM of liver tissue (scale bar = 1 μm), H&E staining of perinephric fat, mesenteric adipose and subcutaneous fat (n = 6, scale bar = 20 μm). (F) Weight of the liver, perinephric fat, mesenteric adipose and subcutaneous fat. (G) Adiposity area analysis of the perinephric fat, mesenteric adipose and subcutaneous fat (n = 6). (All error bars, mean values ± SD, p values were determined by unpaired two-tailed Student's t-test of n = 3 independent biological experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Measurement of liver enzymes, blood lipids and lipoproteins (n = 6) (All error bars, mean values ± SD, p values were determined by unpaired two-tailed Student's t-test of n = 3 independent biological experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Fig. 3

Microbiome analysis of fecal samples in the sham group, OVX group and E₂ group. (A) Representative images of HE staining of colon tissue (scale bar = 50 μm), intestinal tight junctions under TEM (scale bar = 50 nm) and IF staining of ZO-1 (green, scale bar = 50 μm). (B) Alpha diversity analysis. (C) Stack diagram of the relative abundance of the top 26 phyla, key phyla and ternary phase diagram in the groups. (D) Stack diagram of the relative abundance of the top 30 genera, key genera and ternary phase diagram in the groups. (E) Stack diagram of the relative abundance of the top 30 species, key species and ternary phase diagram in the groups. (F) Box plots showing bacterial differences at different classifications (n = 6). (G) LEfSe analysis (LDA threshold = 2). (All error bars, mean values ± SD, p values were determined by unpaired two-tailed Student's t-test of n = 3 independent biological experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Disturbance in the intestinal flora was related to clinical abnormalities in OVX-related NAFLD. (A) Correlation heatmap of the intestinal flora and clinical indicators. (B) PICRUSt analysis at level 3. (C) CCA analysis at the family level. (D) CCA analysis at the genus level.

Fig. 5

Transcriptomic analysis of liver tissue in the sham group, OVX group and E₂ group. (A) Double bar plot of KEGG pathway enrichment showing the shared pathways from the OVX group vs. the E₂ group and the OVX group vs. the sham group. (B) Loop graph of GO enrichment showing pathways from the OVX group vs. the E₂ group and the OVX group vs. the sham group.

Fig. 6

Metabonomic analysis of liver tissue in the sham group, OVX group and E₂ group. (A) Coenrichment KEGG pathways of transcriptomic and metabonomic analysis in both the OVX group vs. the E₂ group and the OVX group vs. the sham group comparisons. (B) Differentially expressed metabolites in the linoleic acid metabolism pathway. (C) Correlation heatmap of clinical indicators and the relevant differentially expressed metabolites. (D) Correlation heatmap of the intestinal flora and the relevant differentially expressed metabolites. (All error bars, mean values ± SD, p values were determined by unpaired two-tailed Student's t-test of n = 3 independent biological experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Fig. 7

E₂inhibits LA-induced hepatocyte steatosis in vitro. (A) Relative RNA levels of Cyp1a2, CYP3A9, PLa2g16 and CYP3A18 in liver tissue (n = 3). (B) Cell viability of BRL 3A cells treated with PA + OA or gradient concentrations of LA for 24/48 h (n = 3). (C) TG concentration of BRL 3A cells treated with PA + OA or gradient concentrations of LA for 48 h (n = 3). (D) Representative images of Oil Red O staining (n = 3, scale bar = 50 μm) of BRL 3A cells treated with PA + OA or gradient concentrations of LA for 24 h (upper panel) and 48 h (lower panel). (E) Representative images of Oil Red O staining of the BRL 3A cell line under treatment with LA (200 μM) and/or E₂ (0.1 μM) for 48 h. (F) Cell viability of BRL 3A cells (n = 3). (G) TG concentration of BRL 3A cells (n = 3). (H) Relative RNA levels of Cyp1a2, CYP3A9, PLa2g16 and CYP3A18 in BRL 3A cells (n = 3). (All error bars, mean values ± SD, p values were determined by unpaired two-tailed Student's t-test of n = 3 independent biological experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)