Nitro-fatty acids protect against steatosis and fibrosis during development of nonalcoholic fatty liver disease in mice

Abstract

Background: Nonalcoholic fatty liver disease (NAFLD) and resulting nonalcoholic steatohepatitis (NASH) are reaching global epidemic proportions. Lack of non-invasive diagnostic tools and effective therapies constitute two of the major hurdles for a bona fide treatment and a reversal of NASH progression and/or regression of the disease. Nitro-oleic acid (OA-NO₂) has been proven effective in multiple experimental models of inflammation and fibrosis. Thus, the potential benefit of in vivo administration of OA-NO₂ to treat advanced NAFLD was tested herein in a model of long-term NASH diet-induced liver damage.

Methods: Non-invasive imaging (e.g. photoacustic-ultrasound (PA-US)) was pursued to establish advanced experimental model of NASH in mice in which both steatosis and fibrosis were diagnosed prior experimental therapy with OA-NO₂. Experimental controls included equimolar amounts of the non-nitrated oleic acid (OA). CLAMS and NMR-based analysis was used for energy metabolism.

Findings: CLAMS and NMR-based analysis demonstrates that OA-NO₂ improves body composition and energy metabolism and inhibits hepatic triglyceride (TG) accumulation. Photoacoustic-ultrasound imaging revealed a robust inhibition of liver steatosis and fibrosis by OA-NO₂. RNA-sequencing analysis uncovered inflammation and fibrosis as major pathways suppressed by OA-NO₂ administration, as well as regulation of lipogenesis and lipolysis pathways, with a robust inhibition of SREBP1 proteolytic activation and subsequent lipogenesis gene expression by OA-NO₂. These results were further supported by histological analysis and quantification of lipid accumulation, lobular inflammation (F4/80 staining) and fibrosis (collagen deposition, αSMA staining) as well as established parameters of liver damage (ALT). In vitro studies indicate that OA-NO₂ inhibits TG biosynthesis and accumulation in hepatocytes and inhibits fibrogenesis in human stellate cells.

Interpretation: OA-NO₂ improve steatohepatitis and fibrosis and may constitute an effective therapeutic approach against advanced NAFLD that warrants further clinical evaluation.

Keywords: Nitro-fatty acids; Non-alcoholic Steatohepatitis; Non-alcoholic fatty liver disease; Non-invasive liver imaging, liver fibrosis.

Fig. 1

Non-invasive diagnosis reveals OA-NO₂ protection against NASH-diet induced hepatic steatosis and fibrosis. (a) Experimental design: Steatohepatitis was induced in C57BL/6 mice by a NASH diet, rich in saturated fat, trans fat, fructose and cholesterol for 12 weeks. After 12 weeks, high-resolution physio-chemical analysis ultrasound (PCA-US), confirmed coexistence of lipid steatosis and early fibrosis. Then, osmotic minipumps were implanted subcutaneously to deliver PEG, OA or OA-NO₂ (5 mg/kg/d) for additional 12 weeks under chow diet (CD) or NASH-diet feeding (4 groups, n = 10 per group). Established liver damage was confirmed in a subpopulation (n = 3) analyzed after 12 weeks using (b) conventional ultrasound (US) combined with high-resolution PCA high-resolution at 1220 nm optical wavelength to detect hepatic lipids, and (c) at 1370 nm optical wavelength to quantify hepatic collagen content. Two weeks before terminal analysis of liver pathology PA-US was used to quantitatively analyze lipid content and total collagen content in the NASH-diet model. High-resolution PCA demonstrated a marked reduction of total hepatic lipid (d) and collagen content (e) upon OA-NO₂ treatment compared to non-nitrated OA. Quantitative analysis of PA absorption from each experimental group at 1220 nm (f) and 1370 nm fingerprint (g). Size bars = 5 mm Data is plotted as box and whiskers from minimum to maximum values showing all points. *p < .05, **p < .01, ***p < .001 vs. CD; ^{^}p < .05, vs. NASH OA. n = 8.

Fig. 2

OA-NO₂ improves body composition and increases respiratory quotient in NASH-diet induced steatohepatitis. (a) Gross appearance of the peritoneal cavity at the experimental end-point (24 weeks) depicting apparent reduction of liver steatosis upon treatment with OA-NO₂. One week before end-point analysis, NMR-based body composition analysis was conducted (n = 8) revealing a significant reduction of % body fat (b) and subsequent increase in % lean body mass (c), upon OA-NO₂ treatment. No alteration in body composition was observed in mice treated with equimolar amounts of non-nitrated OA. Quantitative data is plotted as box and whiskers from minimum to maximum values showing all points. (d) OA-NO₂ increased the respiratory exchange ratio (RER) calculated as the ratio of CO₂ generation vs. oxygen consumption (VCO₂/VO₂) assessed by CLAMS. Data are shown over a 24 h dark/light period cycle (n = 8). (e) Quantitative analysis of RER in each experimental group shown as box and whiskers from minimum to maximum values. Data is shown as mean ± SEM. *p < .05, **p < .01, ***p < .001 vs CD PEG; ^#p < .05, ^##p < .01, ^###p < .001 vs. NASH PEG ^{^}p < .05 vs. NASH OA.

Fig. 3

OA-NO₂ prevents NASH diet-induced hepatomegaly and liver damage, and improves NAFLD activity scores. (a) Gross liver morphology, H&E and Oil Red O histology from each experimental group. H&E histology was used for quantitative NAFLD scoring as described and summarized in Table 1. (b) Quantitative analysis of hepatomegaly was determined as of liver to body weight ratio. (c) Fasting plasma analysis of alanine aminotransferase (ALT) and (d), aspartate aminotransferase (AST). (e) Hepatic triglyceride mass and (f) cholesterol mass from each experimental group. Quantitative data is plotted as box and whiskers from minimum to maximum values showing all points. *p < .05, **p < .01, ***p < .001 vs CD/PEG; ^#p < .05, ^##p < .01, ^###p < .001 vs. NASH PEG ^{^}p < .05, ^{^^}p < .01, ^{^^^}p < .001, vs. NASH OA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Global hepatic transcriptomic profile in response to OA-NO₂ analyzed by RNA-sequencing. Volcano plots of differentially regulated genes (DEGs) in (a) NASH (PEG) vs. chow diet group (CD); (b) OA vs. CD and (c) OA-NO₂ vs. CD. Principal component analysis (PCA) plot and additional volcano plot comparisons between groups are depicted in Supplementary Fig. 5a and b. X axis represents the log2FoldChange of each gene, and Y axis represents the -log10 transformation of the p values. Genes with p values <.05 and log2FoldChange larger than 1 were considered significant DEGs. Up-regulated DEGs are depicted in red, down-regulated DEGs in green, and the non-significant genes in grey. (d) Venn diagrams showing common DEGs in the NASH-diet (PEG) and OA vs. chow diet (CD). (e) Venn diagrams showing DEGs dissimilarity in the OA-NO₂ group vs. OA. (f) Heatmap depicting the top 50 DEGs among all experimental groups as determined by log2FoldChage compared with CD group. Each row represents one gene, and each column represents one comparison to CD group. The log2FoldChange was row scaled and depicted by colors. (g) Heatmap of 50 NASH-related genes. The classification of the genes based on their function in inflammation, fibrosis, lipogenesis and lipolysis was labeled by different colors as shown at the left side of the heatmap. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

OA-NO₂ inhibits hepatocyte triglyceride accumulation. (a) qPCR analyses of hepatic expression of genes regulating lipid biosynthesis. Data presented in bars are means ± SEM (n = 7–10). (b) Total liver lysates from each experimental group were subjected to western blot analysis of precursor, cleaved SREBP1 and GAPDH as loading control. Quantitative densitometry analysis is shown as box and whiskers from minimum to maximum values showing all points (n = 6). ^⁎p < .05, ^⁎⁎p < .01, ^⁎⁎⁎p < .001 vs CD PEG; ^###p < .001 vs. NASH PEG ^{^}p < .05 vs. NASH OA. (c) Triglyceride (TG) content in primary hepatocytes and HepG2 cells treated with or without palmitic acid (200 μM), OA or OA-NO₂ (1 μM) for 20 h. ^#p < .05 vs. palmitic acid (n = 3). (b) TG biosynthetic rate determined using [³H]-acetate incorporation into TG in HepG2 cells treated with either OA or OA-NO₂ (1 μM) for 6 h (n = 3). ^#p < .05 vs. vehicle control (EtOH).

Fig. 6

OA-NO₂ inhibits NASH-diet induced hepatic and systemic inflammation. (a) Representative F4/80 immunohistochemistry from each experimental group. Positive areas were calculated using ImageJ to determine F4/80 immunohistochemistry. Scale bar: 50 μm. (b) qPCR validation of inflammatory gene expression in the liver including TREM2, TLRs and adhesion molecules ICAM1, VCAM1 and E-cadherin. (c) Common cytokine mRNA expression levels and (d) plasma pro-inflammatory biomarkers Mcp-1 and IL6. Quantitative data is plotted as box and whiskers from minimum to maximum values showing all points. *p < .05, **p < .01, ***p < .001 vs CD PEG; ^#p < .05, vs. NASH PEG; ^p < .05, ^^^p < .001 vs. NASH OA.

Fig. 7

OA-NO₂ regulates hepatic fibrogenic signaling in vivo and in human stellate cells in vitro and inhibits liver fibrosis in NASH. (a) Representative Sirius Red histology and αSMA immunohistochemistry from each experimental group. Scale bar: 50 μm. Positive areas were calculated using ImageJ to determine fibrosis scoring and quantification of αSMA immunohistochemistry and summarized in Table 2. (b) Total hepatic collagen content in each experimental group analyzed by acidic conversion to hydroxyproline. Box and whiskers plot indicate mean and range (n = 10). (c) Total liver lysates from each experimental group were subjected to western blot analysis of pro-fibrotic SMAD signaling using phosphorylated SMAD2 (p-SMAD2) antibody vs. total SMAD2. Quantitative densitometry analysis is shown as box and whiskers from minimum to maximum values showing all points (d) Fibrogenic gene expression in the livers. mRNA levels were quantified by qPCR. Relative expression levels were normalized against GAPDH. Data presented in bars are means ± SEM (n = 7–9). *p < .05, **p < .01, ***p < .001 vs. CD; ^#p < .05, ^###p < .001 vs. NASH PEG; ^{^}p < .05, ^{^^}p < .01, ^{^^^}p < .001, vs. NASH OA. (e) Western blot analysis of CTGF and αSMA protein levels in human stellate cells stimulated with TGFβ (20 ng/ml) and increasing concentrations of OA-NO2 (0.1-2 μM) or OA control (2 μM) for 24 h. (f) Human stellate cells were stimulated with or without TGFβ (20 ng/ml), OA or OA-NO₂ (1 μM). 6 h after TGFβ stimulation fibrogenic gene expression (e.g. ACTA2, CTGF, COL1A1, COL1A2) was analyzed by qPCR. Data shown as mean ± SEM, n = 3. ^#p < .05, ^###p < .001 vs. TGFβ; ^^ p < .01 ^^^ p < .001 vs. TGFβ + OA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)