Engineered BPBS intervention ameliorates ethanol-induced injury in mice
To explore the effects of BPBS intervention on ethanol-induced injury, we constructed a chronic-binge ethanol feeding mouse model based on a Lieber-DeCarli diet, as described previously (Bertola et al. 2013); the diagrammatic representation of the whole experiment is shown in Figure 1a. During the liquid diet acclimatization period, prophylactic intervention with BS or BPBS was administered by gavage daily until the end of the experiment, and then, the ethanol-containing diet was fed for 10 days. As expected, ethanol consumption significantly lowered the murine body weight after 5 days (Figure 1b) and notably increased the liver/body weight ratio (Figure 1c). However, supplementation with wild-type BS and BPBS significantly alleviated the decline in body weight gain and partially reduced the liver/body weight ratio, and the efficiency of BPBS intervention was greater than that of BS (Figure 1b, 1c). Additionally, the ethanol exposure stimulated the high expression of IL-6 in serum (Figure 1d), however, the serum IL-6 was significantly alleviated via the intervention of BS or BPBS. Collectively, prophylactic BPBS rather than BS supplementation is efficient to alleviate ethanol-induced injury in mice.
BPBS intervention restores the ethanol-induced gut microbiota disorders in mice
Chronic ethanol overconsumption is an important cues of gut microbiota dysbiosis, which may underlie the pathophysiology of ethanol-related morbidity (Bjørkhaug et al. 2019; Veronika B. Dubinkina et al. 2017b). Due to the notable health functions and inherent plasticity, gut microbiota has been suggested as an important target for the prevention of ethanol-related diseases (Bajaj 2019; Bajaj et al. 2018; Aden et al. 2019). In line with prior studies, the influence of ethanol feeding on gut microbiota was confirmed, and ethanol exposure remarkably reduced the gut microbiota abundance and diversity compared with that of the pair-fed group (Figure 2a-c). Meanwhile, the wild-type BS and BPBS intervention significantly increased the microbial diversity (Figure 2a) and obviously restored the microbial composition in the β-diversity analysis (Figure 2c). Interestingly, BPBS supplementation is likely to work more efficiently in a murine ALD model than wild-type BS.
Next, we explored the taxonomic shifts in the bacterial community. At the phylum level, Firmicutes and Bacteroidetes were dominant in the fecal microbiota of the pair-fed group, whereas Firmicutes, Proteobacteria and Bacteroidetes were dominant in the ethanol-fed group (Figure 3a). Interestingly, the proportion of the family Enterobacteriaceae was significantly increased in the ethanol-fed group compared with the pair-fed group (Figure 3c), and more significant alterations were detected in the BS or BPBS intervention groups than the ethanol-fed group (Figure 3b). After the administration of BS or BPBS, the genera Bacillus and Ruminococcaceae were significantly increased (Figure 3d). Specific enrichment of the families Lachnospiraceae and Prevotellaceae was observed in the gut microbiota of the BPBS-fed group (Figure 3c). We further analysed the altered gut microbiota and found that gram-negative bacteria were enriched along with obvious potentially pathogenic phenotypes in the ethanol-fed group (Figure 3e, 3f). In contrast, the administration of BPBS aborted these potentially pathogenic phenotypes and improved the gut microbiota dysbiosis induced by ethanol consumption.
BPBS intervention improves ethanol-disrupted intestinal barrier in mice
Gut microbiota dysbiosis directly influences the physiological status of the intestine, and the improved gut microbiota contributes to facilitate host defence against hazardous substances or unfriend environments. We further investigated how prophylactic BPBS intervention mitigated ethanol-induced intestinal injury. Ethanol exposure notably disrupted intestinal barrier integrity with a high FITC concentration in the EtOH-fed group (Figure 4a). However, significantly reduced serum FITC levels were observed in the mice with BPBS supplementation compared to the EtOH-fed mice, suggesting a recovery of intestinal integrity. In accordance with the intestinal permeability, ethanol feeding resulted in a significant increase in serum LPS compared with that of the pair-fed group, while the BS and BPBS intervention significantly decreased serum LPS levels compared with those of EtOH-fed mice (Figure 4b), and BPBS supplementation worked better than wild-type BS. Overall, these results showed that the administration of BPBS mitigated the translocation of endotoxin from the intestinal lumen to circulatory system.
Intestinal barrier integrity is largely dependent on tight junctions, of which Occludin is the major component (Feldman et al. 2005). The protein expression of Occludin in the colon was determined, and ethanol feeding obviously decreased its expression (Figure 4d); however, the administration of BS and BPBS partially restored the protein Occludin expression compared with that of the EtOH-fed group. In line with the protein results, we observed the same restoration of Occludin gene expression in the BPBS group, along with the increased ZO-1 expression (Figure 4c), suggesting that BPBS administration could stimulate the expression of tight junction genes. Furthermore, histological analysis of the colon showed a damaged and thin mucosal layer after ethanol feeding in comparison with those of the pair-fed mice (Figure 4e), whereas supplementation with BPBS significantly restored Muc2 gene expression (Figure 4c) and increased the secretion of mucins in intestine (Figure 4f). Moreover, ethanol feeding significantly cut down the butyrate yield of the gut microbiota and activated the inflammatory reaction in the colon, with increased IL-6, IL-1β and TNF-α gene expression compared with the pair-fed group (Figure 4g,4h); however, the administration of BPBS dramatically restored the butyrate contents in the intestine and decreased the excretion of inflammatory cytokines (Figure 4g,4h). Altogether, our results suggested that BPBS intervention replenished the butyrate yield of the gut microbiota and alleviated the inflammatory reaction in colon, as well as rebuilding intestinal barrier function through restoring the tight junction and mucin components.
BPBS intervention attenuates ethanol-induced hepatic injury
To determine how BPBS protects the liver against ethanol exposure, the biochemical and pathologic changes were further carried out. The development of hepatic injury induced by ethanol feeding was confirmed by obviously increased serum ALT, AST, hepatic triglycerides and MDA levels compared with those of the pair-fed mice (Figure 5a-5d). Daily administration of BPBS remarkedly reduced the serum levels of ALT, AST and hepatic triglycerides (Figure 5a-5c). Moreover, obvious neutrophil infiltration in liver tissues after ethanol feeding was observed through H&E staining and was significantly alleviated by dietary BPBS supplementation (Figure 5e). Next, we quantified hepatic gene expression related to steatosis. Notably, the major liver functions of triglyceride synthesis and fatty acid uptake were disordered, with a significant increase in the expression of peroxisome proliferator activated receptor-γ (PPAR-γ) and transporter CD36 for fatty acids (Figure 5f). Additionally, the decreased expression of Fas, SCD1 and Srebp-1c induced by ethanol feeding were improved via dietary BPBS supplementation, which suggested that BPBS administration likely accelerated fatty acid synthesis in the liver and attenuated the hepatic function injury induced by ethanol consumption.
BPBS intervention ameliorates ethanol-induced liver inflammation
Ethanol consumption seriously damaged the intestinal gut integrity and thus accelerated gut bacterial translocation (especially LPS) in the bloodstream, which greatly triggered hepatic inflammation and contributed to the development of ethanol-induced liver diseases. Additionally, mounting evidence have shown that LPS induces organic inflammation based on a TLR4-dependent mechanism (Kayagaki et al. 2013; Hagar et al. 2013; Park et al. 2009). Interestingly, the increased serum LPS levels significantly stimulated the protein expression of TLR4 and nuclear factor-κB (NF-κB) in the liver (Figure 6a), along with an increased release of critical proinflammatory cytokines in serum, such as tumor necrosis factor alpha (TNF-α) and IL-1β (Figure 6c,6d). Accordingly, hepatic gene expression related to inflammatory cytokines, including TNF-α, IL-1β, NF-κB and monocyte chemoattractant protein-1 (MCP-1), was greatly enhanced in the ethanol-fed group compared to the pair-fed group (Figure 6b). However, after the administration of wild-type BS and BPBS, the expression of hepatic TLR4 protein was remarkably decreased, along with concomitant significant decreases in the TLR4-regulated gene expression of NF-κB, TNF-α, IL-1β, and MCP-1 in the treated group compared to the ethanol-fed group. Notably, BPBS intervention seemed to function better in ameliorating ethanol-induced hepatic inflammatory status via the LPS/TLR4 pathway.