OFO exacerbates the features of ethanol-induced liver injury
The primary and secondary lipid-oxidation products were measured to determine oxidation status in FO and OFO. After air exposure at 65 °C for 2 weeks, OFO showed the higher degree of oxidation as evidence by the greatly elevated levels of peroxide value (POV), p-anisidine value (AV), total oxidation value (Totox), and thiobarbituric acid-reactive substances (TBARS), compared to the unoxidized FO (Table 1). As determined by gas chromatography–mass spectrometry (GC-MS), the contents of n-3 PUFAs, including EPA, DPA and DHA, in OFO were greatly decreased, compared to unoxidized FO. Approximately 50.9% EPA and 58.7% DHA in FO were oxidized during the process (Supplementary Table 1 and Supplementary Fig. 1). Liver injury induced by ethanol exposure was evaluated by biochemical and histological analysis, and oxidative stress in liver. Acute-on-chronic alcohol feeding (Supplementary Fig. 2) elevated plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) by 4.5-fold and 3.0-fold, respectively, in CO-treated mice. These elevations were greatly augmented to 8.1-fold and 4.6-fold, respectively, by replacing a half of CO with OFO, but significantly decreased by supplement with FO (Fig. 1a). OFO-treated mice had a pronounced increase in plasma triglyceride (TG) and hepatic TG levels, compared to the CO-treated mice after alcohol exposure (Fig. 1b). Alcohol feeding caused oxidative stress in the liver from both CO- and OFO-treated mice, as characterized by the decreased antioxidant parameters (e.g., CAT, SOD, GSH), and increased the hepatic malonaldehyde (MDA) level, but the effects were much greater in OFO-treated mice (Fig. 1c). Lipid accumulation in the liver was increased by alcohol exposure in CO-treated mice, and markedly increased in OFO-treated group, but remarkably decreased by FO treatment, as measured by hematoxylin and eosin (H&E) (Fig. 1d) and oil red O staining (Supplementary Fig. 3a). Taken together, these results indicate that hepatic steatosis and oxidative stress induced by alcohol exposure are exacerbated by dietary OFO but reversed by FO.
To determine whether the absorption and metabolism of ethanol is implicated in these deteriorative effects of OFO, the circulating ethanol concentration and hepatic expression of cytochrome P450 2E1 (CYPE21) were measured. After chronic-plus-single-binge ethanol feeding, the ethanol concentrations in plasma were greatly elevated in both CO- and OFO-treated mice. However, plasma ethanol levels were comparable between CO and OFO groups after ethanol exposure (Supplementary Fig. 4a). Similar tendency was observed for hepatic expression of CYPE21 (Supplementary Fig. 4b).
OFO aggravates ethanol-induced hepatic inflammation
Alcohol exposure increased the hepatic inflammation in both CO- and OFO-treated mice, as shown by the increased pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) and the chemokine (MCP-1), and the decreased anti-inflammatory cytokine (IL-10), but the effects were much greater in OFO-treated mice. While FO supplement decreased these pro-inflammatory factors induced by ethanol feeding, but did not rescue hepatic IL-10 (Fig. 2a, b), we speculated that the inhibition of pro-inflammatory factors, but not normalization of anti-inflammatory cytokines, contribute to the protective effects of FO against alcoholic hepatic inflammation in the mouse model of chronic-plus-single-binge ethanol feeding. Consistently, the immunofluorescent staining of F4/80, a specific monocyte/macrophage marker, showed that more F4/80-positive cell number was observed in the section of liver tissue from OFO-treated mice after alcohol feeding, compared to CO group. While FO decreased the macrophage infiltration in the liver (Supplementary Fig. 3b). These data suggest that dietary OFO exacerbates hepatic inflammation upon alcohol challenge.
Activation of Kupffer cell via TLR4/NF-κB signaling plays a vital role in hepatic inflammation induced by ethanol exposure4,30. Next, we examined the protein expressions of the genes related to TLR4/NF-κB signaling in the liver. As shown in Fig. 2c, OFO-treated mice had a remarkable increase in hepatic expressions of TLR4, and its downstream MyD88 and phosphorylated p65 (p-p65) without changing total p65 expression, compared to CO-treated mice after alcohol feeding. These increased protein expressions were reversed by supplement with FO. In addition, OFO alone mice showed slight increases in hepatic expressions of TLR4, MyD88, and p-p65, compared to CON/CO group (Fig. 2c). Consistently, the immunofluorescent staining revealed the higher hepatic expression of p-p65 in OFO-treated group after alcohol intake, compared to CO-treated mice (Fig. 2d). Collectively, our data demonstrate that TLR4/NF-κB signaling is implicated in OFO-exacerbated hepatic inflammation in alcohol-fed mice.
OFO enhances ethanol-induced intestinal barrier dysfunction
We next examined whether intestinal barrier integrity is influenced by dietary OFO. TJ proteins, the markers of intestinal integrity, play the crucial role in maintaining barrier function of intestinal epithelium31,32. Immunofluorescent and western blot analysis clearly showed the decreased expression of TJ proteins, including ZO-1, occludin, and claudin-4, in jejunum tissue from CO-treated mice after alcohol exposure, along with the increased expression of claudin-2, a TJ protein involving in the formation of paracellular water channel that is typically expressed in leaky epithelial tissues. But these changes were more severe in OFO-treated mice, while were reversed by supplement with FO (Fig. 3a, b). Importantly, OFO alone did not affect the expressions of all TJ protein investigated, compared to CO alone group (Fig. 3a, b). The impaired intestinal barrier integrity leads to the increased intestinal permeability to bacterial products, such as LPS, and the greater bacteria translocation. After chronic-plus-single-binge ethanol feeding, plasma LPS level and the hepatic expression of gram-negative bacterial 16S rRNA, a marker of bacterial translocation, were increased in both CO- and OFO-treated mice, much greater increase was observed in OFO group, but significantly decreased by FO supplement. Although OFO alone group has the slightly increased hepatic expression of 16S rRNA, compared to CO alone group, but their plasma LPS levels were comparable (Fig. 3c, d). While fecal LPS contents were increased in both CO- and OFO-treated mice, OFO alone mice showed the significantly increased LPS level in feces, compared to CO alone group (Fig. 3e). A most plausible explanation is that OFO alone cause the great increased fecal LPS, which may not be diffused into the circulation, because OFO alone cannot greatly disrupt the intestinal epithelial barrier.
An increasing evidence has demonstrated that intestinal inflammation contributed to intestinal barrier dysfunction33. To further investigate the role of OFO in intestinal inflammation in ethanol-fed mice, immunofluorescent staining of F4/80 and TNF-α in jejunum tissue was examined. As shown in Fig. 3f, the intestinal expressions of F4/80 and TNF-α were slightly increased in CO-treated group after alcohol exposure, but remarkably increased in OFO-treated mice. OFO alone group showed the higher expressions, compared to CO alone mice, even CO plus ethanol group (Fig. 3f).
OFO deteriorates alcohol-induced intestinal dysbiosis
Intestinal dysbiosis and bacterial products play the crucial role in pathogenesis of ALD5. To whether intestinal dysbiosis contributes to OFO-aggravated alcoholic liver injury, intestinal microbiome was profiled by the analysis of bacterial 16S rRNA V3-V4 region. Rank abundance curve showed the sequencing depth was enough sufficient to characterize the bacterial communities in the samples (Fig. 4a). Principal coordinate analysis (PCoA) with unweighted UniFrac distance showed the tight clusters of the samples from each group. OFO-treated mice clustered closely regardless ethanol and pair feeding, which were far apart from other groups (Fig. 4b). Furthermore, ethanol-fed group, including ETH/CO and ETH/FO mice but except ETH/OFO, had similar microbiota profiles, and clustered separately with CON/CO. These results were confirmed by unweighted pair group method with arithmetic mean (UPGMA) tree (Fig. 4c), which indicated that the diversity of intestinal microbiota was slightly changed by ethanol feeding, but strongly influenced by dietary OFO. We next identified a specific intestinal microbiota related to OFO treatment. As shown in Fig. 4d, e, Phyla analysis showed that Bacteroidetes, Proteobacteria, and Firmicutes are dominant microbiota in all groups of mice, both ETH and OFO-fed mice had the increased Bacteroidetes and Proteobacteria, and the decreased Firmicutes, compared to CON/CO group. Furthermore, the proportion of Proteobacteria was slightly higher in CON/OFO mice (22%) over CON/CO mice (9.1%), and significantly increased in ETH/OFO mice (34%), while, FO treatment exhibited the moderate decrease in Proteobacteria (16%). Cladogram analysis showed that the OFO- and ETH-fed mice mainly clustered in Proteobacteria phylum, but exhibited at different taxonomic levels. The beta-proteobacteria and delta-proteobacteria family of Proteobacteria phylum was dominant in ETH/CO mice, while grama-proteobacteria family was dominant in OFO-fed mice. Moreover, Enterobacter and Klebsiella genus was specifically clustered in ETH/OFO mice, but bacteria Desulfovibrio and Parasutterela were dominated in ETH/CO mice (Fig. 4f). These data indicated that OFO-exacerbated alcoholic liver injury might be associated with intestinal dysbiosis, especially the specific microbiota profile of Proteobacteria.
ABx abolishes OFO-aggravated alcoholic liver injury
To reveal the role of gut bacteria in OFO-aggravated liver injury in ethanol-fed mice, nonabsorbable antibiotics (ABx), comprising of polymyxin B and neomycin, were orally administered to the mice daily during liquid-diet adaptation and subsequent alcohol-feeding period. Nonabsorbable antibiotics did not alter the intestinal absorption and metabolism of ethanol, as measured by plasma ethanol level and hepatic expression of CYPE21 (Supplementary Fig. 5a, b). Gut sterilization by ABx significantly decreased LPS levels in feces and plasma, and bacterial translocation in both ETH/CO and ETH/OFO groups, but there is no difference between ETH/CO and ETH/OFO after ABx treatment (Fig. 5a–c). Immunofluorescent (Fig. 5d) and western blot (Fig. 5e) analysis indicated that the expressions of TJ proteins, ZO-1, occludin, claudin-4, and claudin-2 in jejunum tissue were reversed by ABx treatment in ethanol-fed mice, and OFO-aggravated intestinal barrier dysfunction was completely abolished by ABx treatment. Similar tendency was observed in immunofluorescent staining of F4/80 and TNF-α in jejunum tissue (Supplementary Fig. 6a).
Consistently, nonabsorbable antibiotics remarkably inhibited the hepatic inflammation in ethanol-fed mice, as evidenced by the reduced hepatic inflammatory cytokines TNF-α, IL-6, IL-1β (Fig. 6a), and MCP-1 (Fig. 6b), the less F4/80-positive monocytes/macrophage (Fig. 6c) in the liver, and the decreased hepatic expressions of TLR4, MyD88, and p-p65 (Fig. 6d and Supplementary Fig. 6b). These results demonstrated that OFO-exacerbated hepatic inflammatory response was blunted by ABx treatment. Undoubtedly, intestinal sterilization with nonabsorbable antibiotics prevented hepatic steatosis and injury in alcohol-fed mice, as determined by plasma biochemical analysis (Fig. 7a), hepatic TG content assay (Fig. 7b), and histological staining of H&E and oil red O (Fig. 7c). These data suggest that the worse effects of OFO on alcoholic liver injury are resorted after ABx treatment.
OCA blunts OFO-aggravated liver injury in alcohol-fed mice
Alcohol and its metabolites disrupt intestinal barrier integrity, resulting in the increased permeability of intestinal barrier. Our data also indicated that OFO-induced intestinal inflammation may be involved in intestinal barrier dysfunction. Obeticholic acid (OCA), a potent agonist of farnesoid X receptor (FXR), is effective in the treatment of nonalcoholic steatohepatitis with liver fibrosis in animal models34 and in patients35. OCA has been also shown to ameliorate gut barrier dysfunction and bacterial translocation in cholestatic36 and in cirrhotic rats37. To further elucidate the role of intestinal barrier in OFO-aggravated alcoholic liver injury, OCA was gavaged daily (30 mg kg−1) to the mice during alcohol-feeding period. As expected, FXR agonist OCA upregulated the expressions of TJ proteins, ZO-1, occludin and claudin-4 in jejunum tissue, and suppressed the intestinal inflammation in ethanol-fed mice (Supplementary Fig. 7). OCA treatment remarkably decreased LPS levels in plasma, but not changed in feces (Fig. 8a). Consistently, OCA inhibited the hepatic inflammation in ethanol-fed mice, as manifested by the reduced hepatic inflammatory cytokines TNF-α and IL-6 (Fig. 8b), and less F4/80-positive monocytes/macrophage in the section of liver tissue (Fig. 8c). Eventually, OCA treatment prevented hepatic steatosis and injury in alcohol-fed mice, as examined by histological analysis (Fig. 8d), plasma levels of AST and ALT (Fig. 8e), and hepatic TG quantification (Fig. 8f). These results demonstrate that stabilization of intestinal barrier by the treatment of FXR agonist OCA blunts OFO-aggravated liver injury in alcohol-fed mice.