Western diet contributes to the pathogenesis of non-alcoholic steatohepatitis in male mice via remodeling gut microbiota and increasing production of 2-oleoylglycerol

Abstract

The interplay between western diet and gut microbiota drives the development of non-alcoholic fatty liver disease and its progression to non-alcoholic steatohepatitis. However, the specific microbial and metabolic mediators contributing to non-alcoholic steatohepatitis remain to be identified. Here, a choline-low high-fat and high-sugar diet, representing a typical western diet, named CL-HFS, successfully induces male mouse non-alcoholic steatohepatitis with some features of the human disease, such as hepatic inflammation, steatosis, and fibrosis. Metataxonomic and metabolomic studies identify Blautia producta and 2-oleoylglycerol as clinically relevant bacterial and metabolic mediators contributing to CL-HFS-induced non-alcoholic steatohepatitis. In vivo studies validate that both Blautia producta and 2-oleoylglycerol promote liver inflammation and hepatic fibrosis in normal diet- or CL-HFS-fed mice. Cellular and molecular studies reveal that the GPR119/TAK1/NF-κB/TGF-β1 signaling pathway mediates 2-oleoylglycerol-induced macrophage priming and subsequent hepatic stellate cell activation. These findings advance our understanding of non-alcoholic steatohepatitis pathogenesis and provide targets for developing microbiome/metabolite-based therapeutic strategies against non-alcoholic steatohepatitis.

Fig. 1. Establishment and characterization of a mouse NASH model.

a An outline depicting induction of NASH with a CL-HFS. Six-week-old WT C57BL/6 J mice were fed a CL-HFS or normal diet (ND) for 12 weeks. At week 18, each mouse was euthanized for the following studies. b Representative macroscopic images of livers in CL-HFS- and ND-fed mice. c Representative histological images of liver tissues. Typical hepatic features of NASH were detected in CL-HFS-fed mice in comparison to ND-fed mice: H&E staining showed lipid deposition (L), hepatocytes ballooning (black arrow), and inflammatory cell liver infiltration (red arrow); Sirius red staining showed increased production of collagen (yellow arrows); IHC showed increased production of α-SMA (green arrows). Bar: 50 μm. d qPCR measurement of mRNA expression of extracellular matrix (ECM) genes Col1a1, Col4a1, and Acta2; e qPCR measurement of mRNA expression of proinflammatory cytokine genes IL1b, Tgfb1, and Tnfa in the livers of ND-fed and CL-HFS-fed mice at the indicated time points. For d and e, n = 5, data are presented as mean ± SD. f Representative histological images of liver tissues in healthy individuals and patients with NASH. Compared to healthy individuals, NASH caused lipid deposition (L), hepatocyte ballooning (black arrow), and inflammatory cell liver infiltration (red arrow) assessed by H&E staining; increased production of collagen measured by Sirius red staining (yellow arrows), and enhanced production of α-SMA (green arrows) measured by IHC. Bar: 100 μm. g mRNA expression of ECM genes in human livers. qPCR detected increased gene expression of Col1a1, Col4a1, and Acta2 in the livers of patients with NASH compared to those of healthy individuals. h mRNA expression of proinflammatory cytokines in human livers. qPCR detected the increased gene expression of IL1b, Tgfb1, and Tnfa in the livers of patients with NASH compared to healthy individuals. For g and h, n = 7, data are presented as mean ± SD. Statistical analysis of data was performed by one-way ANOVA with Tukey’s multiple comparison test (≥3 groups) or Mann–Whitney test (two-tailed) using GraphPad Prism 8 software. Source data are provided as a Source Data file.

Fig. 2. ABX treatment slows the development of CL-HFS-induced NASH.

a An outline depicting ABX treatment design. Six-week-old WT C57BL/6 J mice were fed with a CL-HFS for 12 weeks with or without simultaneous ABX treatment, then euthanized for the following studies. ND-fed mice were used for controls. b Effect of ABX on CL-HFS-induced increase in the ratios of liver-to-bodyweight. Compared to ND, CL-HFS caused an increase in the liver-to-bodyweight ratio which was suppressed by ABX treatment. c Effect of ABX on CL-HFS-induced NASH. Compared to untreated mice, ABX treatment led to an obvious reduction in the liver infiltration of inflammatory cells (red arrow) (H&E staining), production of collagen (Sirius red staining), α-SMA (IHC detection), and lipid accumulation (Oil red O staining) in CL-HFS-fed mice. Bar: 100 μm. d Semi-quantification of inflammatory cells infiltratiing into the liver; e Semi-quantification of collagen production, α-SMA protein expression, and lipid accumulation in the livers of three groups of mice shown in c. f NAFLD activity score (NAS). ABX treatment led to reduced NAS in CL-HFS-fed mice. g Hepatic mRNA expression of ECM genes in CL-HFS-fed mice with or without ABX treatment. qPCR measured reduced mRNA expression of Col1a1, Col4a1, and Acta2 in the livers of ABX-treated mice compared to untreated mice. h Hepatic mRNA expression of proinflammatory cytokines, profibrotic cytokines, and chemokine. qPCR measured reduced mRNA expression of IL1b, Tgfb1, Tnfa, Myd88, Nfkb, and Ccl2 in the livers of ABX-treated mice compared to untreated mice. ABX suppressed activation of HSCs. Representative (i) and cumulative results (j) of flow cytometric assays indicated that ABX treatment reduced the frequency of activated HSCs expressing Col1a and α-SMA in CL-HFS-fed mice. n = 5, data are presented as mean ± SD. Statistical analysis of data was performed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test using GraphPad Prism 8 software. Source data are provided as a Source Data file.

Fig. 3. ABX treatment alters the profiles of gut microbiota and hepatic metabolites.

Fecal and liver samples were collected from Fig. 2 mice, which were fed with ND or CL-HFS in the absence or presence of ABX for 12 weeks. a Effect of ABX on the relative abundance of operational taxonomic unit (OUT) in CL-HFS-fed mice. 16 S rRNA gene sequencing of fecal samples identified the profiles of gut microbiota in three groups of mice. b ABX treatment changed gut microbiota similarity. PERMANOVA significance test was performed with Principal-coordinate analysis (PCA) to define the Jaccard similarity index. c ABX induced a significant alteration in representative bacterial species. ABX treatment significantly reduced the relative abundance of Blautia and Akkermansia and increased the relative abundance of Alistipes and Muribaculaceae in the fecal samples of CL-HFS-fed mice. n = 5, data are presented as mean ± SD. d ABX changed the hepatic metabolite profile. Hepatic metabolites in three groups of mice were analyzed by non-targeted Gas chromatography-mass spectrometry (GC-MS). Heatmap showed Z-scores of 5 metabolites in 30 liver tissues from 10 ND-fed mice, 10 CL-HFS-fed mice, and 10 CL-HFS-fed mice with ABX treatment. ABX treatment markedly reduced the following metabolite production in CL-HFS-fed mice, L-Phenylalanine (Phe), Pyroglutamic acid (PCA), 2-Oleoylglycerol (2-OG), Cysteine (Cys), and L-Valine (Val). n = 10, data are presented as mean ± SD. Statistical analysis of data was performed by one-way ANOVA with Tukey’s multiple comparison test using GraphPad Prism 8 software. Source data are provided as a Source Data file.

Fig. 4. Discrepancies of liver metabolites in patients with obesity and with or without NASH.

a NAS in patients with obesity and with or without NASH. According to NAS, eight patients with obesity were divided into two groups: patients with obesity and NAS > 4 (high) and individuals with obesity and NAS < 4 (low). b Significantly increased production of hepatic 2-OG and 4-Hydroxybutanoic acid in patients with obesity and high NAS compared to that in patients with obesity and low NAS. n = 4, data are presented as mean ± SD. Statistical analysis of data was performed by Mann–Whitney test (one-tailed) using GraphPad Prism 8 software. Source data are provided as a Source Data file.

Fig. 5. B. producta repopulation promotes CL-HFS-induced liver fibrosis in association with modulation of liver-resident MΦs.

a An outline depicting gut microbiota sterilization and bacterial repopulation. Six-week-old WT C57BL/6 J mice received ABX5 orally in drinking water for two weeks to deplete intestinal bacteria, then received CL-HFS and simultaneous Blautia producta (ATCC 27340) or Alistipes putredinis (ATCC 29800) repopulation by oral gavage twice a week at a dose of 3 × 10⁸ CFU/mouse in 200 µl of PBS. Twelve weeks later, all mice were euthanized to harvest livers and isolate hepatic NPCs for the following studies. b The frequencies of distinct hepatic immune cells. Liver NPCs underwent flow cytometry to define the frequencies of liver-resident CD3 (CD3⁺), CD4 (CD3⁺CD4⁺), CD8 (CD3⁺CD8⁺), NK (CD3^-CD49b⁺), NKT (CD3⁺NK1.1⁺), DCs (CD11b⁺CD11c⁺), and B cells (CD3^-B220⁺). c Representative frequencies of liver-resident MΦs in NPCs. d Mean frequency (left) and absolute number (right) of hepatic MΦs in c. Data showed that repopulation with B. producta, but not A. putredinis, increased the frequency of hepatic MΦs in CL-HFS-fed mice. e Representative frequencies of activated HSCs. f Mean frequency (left) and absolute number (right) of activated HSCs in e. Data indicated that repopulation with B. producta, but not A. putredinis, increased the frequency of activated HSCs expressing Col1α and α-SMA in CL-HFS-fed mice. g Liver infiltration of inflammatory cells (H&E staining), collagen production (Sirius red staining), and α-SMA production (IHC staining). Red arrows point to inflammatory cells. Sirius red staining and IHC staining detected increased production of collagen and α-SMA in the mice with B. producta repopulation versus control mice with or without A. putredinis repopulation. Bar: 100 μm. h Semi-quantification (Mean percentage) of areas positive for Sirius red and α-SMA staining. i Expression of ECM genes. qPCR detected an increased mRNA expression of Col1a1, Col4a1, and Acta2 in the mice with B. producta repopulation versus control mice with or without A. putredinis repopulation. n = 5, data are presented as mean ± SD. Statistical analysis of data was performed by one-way ANOVA with Tukey’s multiple comparison test using GraphPad Prism 8 software. Source data are provided as a Source Data file.

Fig. 6. 2-OG causes liver fibrosis in ND-fed mice in association with modulation of liver resident MΦs.

a An outline depicting the experimental design of 2-OG treatment. Eight-week-old WT C57BL/6 J mice fed with ND received i.v. injection of 2-OG three times a week for 6 weeks at a dose of 20 μg/mouse in 0.2 mL PBS. PBS injection was used for control. After that, all mice underwent liver perfusion to isolate hepatic NPCs for the following studies. b Liver infiltration of inflammatory cells (H&E staining), collagen production (Sirius red staining), and α-SMA production (IHC staining). Red arrows point to inflammatory cells. Bar: 100 μm. c Semi-quantification of areas positive for Sirius red staining and α-SMA IHC staining. Semi-quantification showed the increased Sirius red staining area and α-SMA IHC staining area in 2-OG-treated mice versus control mice. d Frequencies of different types of immune cells in the liver. Liver NPCs underwent flow cytometry to define mean frequencies of different types of immune cells including CD3 (CD3⁺), CD4 (CD3⁺CD4⁺), CD8 (CD3⁺CD8⁺), NK (CD3^-CD49b⁺), NKT (CD3⁺NK1.1⁺), DCs (CD11b⁺CD11c⁺), and B cells (CD3^-B220⁺). e Representative frequencies of liver-resident MΦs in NPCs. Representative flow cytometry analysis showed that 2-OG injection caused an increased frequency of liver-resident MΦs. f Mean frequency (left) and absolute number (right) of MΦs in NPCs in control and 2-OG-treated mice as shown in e. g Representative frequencies of activated HSCs expressing Col1α and α-SMA in liver NPCs. Representative flow cytometry analysis showed that 2-OG injection caused an increased frequency of activated HSCs expressing Col1α and α-SMA in liver NPCs. h Mean frequency (left) and absolute number (right) of activated HSCs expressing Col1α and α-SMA in NPCs in control and 2-OG-treated mice as shown in g. i mRNA expressions of liver Gpr119 and Acta2. qPCR showed that 2-OG injection led to the increased mRNA expression of Gpr119, Acta2, Col1a1, and Col4a1 in livers. n = 5, data are presented as mean ± SD. Statistical analysis of data was performed by Mann–Whitney test (two-tailed) using GraphPad Prism 8 software. Source data are provided as a Source Data file.

Fig. 7. 2-OG is unable to activate GPR119-knockdown MΦs and their co-cultured HSCs.

a Gpr119-knockdown in MΦs blocks 2-OG-induced activation of the co-cultured HSCs. qPCR did not detect the 2-OG-induced upregulation of genes Acta2, Col1a1, and Col4a1 in HSCs co-cultured with Gpr119-knockdown RAW264.7 cells induced by siRNAs. b 2-OG stimulation induces increased production of GPR119 and proinflammatory cytokines in RAW264.7 cells. qPCR detected increased mRNA expression of Gpr119, Nfkb, Tnfa, IL1b, and Tgfb1 in 2-OG-stimulated RAW264.7 cells for 24 h. TGF-β1 (10 ng/mL) was used as a positive control. c Anti-TGF-β1 inhibits 2-OG-induced HSC activation co-cultured with RAW264.7 cells. qPCR analysis showed that 2-OG stimulation upregulated Acta2, Col1a1, and Col4a1 in HSCs co-cultured with RAW264.7 cells, which was inhibited by antibodies for TGF-β1 (1 µg/mL) but not for IL-1 (1 µg/mL) or TNF-α (1 µg/mL). d Validation of siRNA for Tak1 knockdown. qPCR detected the significantly decreased Tak1 expression in RAW264.7 cells treated with Tak1 siRNA versus scramble siRNA. e 2-OG stimulation is unable to promote Nfkb and Tgfb1 expression in MΦs with Tak1 knockdown. qPCR detection showed that 2-OG stimulation significantly increased expression of Gpr119, but not Nfkb and Tgfb1, in Tak1-knockdown RAW264.7 cells. For a, d and e, n = 6; for b and c, n = 3. Data are presented as mean ± SD. The assay was repeated twice. Statistical analysis of data was performed by one-way ANOVA with Tukey’s multiple comparison test (≥3 groups) or by Mann–Whitney test (two-tailed) using GraphPad Prism 8 software. Source data are provided as a Source Data file.

Fig. 8. Schematic diagram of 2-OG-mediated HSC activation in an MΦ-dependent manner.

CL-HFS and Blautia interplay produces 2-OG, which stimulates MΦ expression of TGF-β1 through GPR119-TAK1-NF-κB signaling. The resultant TGF-β1 activates quiescent HSCs (qHSCs) to activated HSCs (aHSCs) to increase the expression of ECM genes, including Acta2, Col1a1, and Col4a1. This figure was created using Biorender (https://biorender.com).