Currently, the gut microbiota and associated metabolites involved in ACLF pathogenesis remain unclear. Thus, we conducted this study to determine the association between gut microbiota and serum metabolomics in mice with ACLF using high-throughput sequencing of 16S rRNA for comparative analysis of gut microbiology and non-targeted metabolite analyses by GC-MS coupled with LC-MS to provide novel insights for treatment.
Some studies have revealed a serious imbalance in the intestinal microecology of patients with ACLF. Throughout the disease course, the intestinal flora is constantly changing and the immunity of the body may also be altered; changes in the intestinal flora are closely related to the immune function of the body[12, 13]. Intestinal flora translocation, inflammation, and immune disorders play an important role in ACLF pathogenesis. We analyzed the pathogenic bacteria of the genus Bacteroides, which were significantly enriched in the ACLF group. Our findings suggest that the genus Bacteroides is involved in the proinflammatory state of the gut and is positively correlated with intestinal bacterial translocation. A possible mechanism is that Bacteroides competes with Bifidobacterium bifidum for polysaccharide substrates, which is more favorable for bacterial transport; if the growth of Bifidobacterium bifidum is promoted, bacterial transport is improved[14–18]. A study on gut microorganisms in patients with hepatitis B-related hepatocellular carcinoma who developed liver failure following extended hepatectomy revealed that significantly enriched Bacteroides, Enterococcus faecalis spp., Pantoea spp., and Turicibacter could be used as non-invasive characteristic biomarkers of liver failure after extended hepatectomy[19]. In the present study, Bacteroides was highly enriched in the ACLF group, suggesting that after the onset of ACLF, the abundance of intestinal pathogenic bacteria increased and the protection of the intestinal barrier decreased.
Metabolomics is a widely used clinical diagnostic tool for detecting early biomarkers and analyzing hepatic metabolic pathways[20, 21]. This diagnostic tool links metabolites and lipid fractions of microbial origin to the onset and prognosis of ACLF. It was noted that Bacteroidetes abundance significantly increased and taurocholic acid expression was significantly upregulated in CCl4-induced ACLF in rats[22]. In a multicenter study, metabolomic analysis of serum samples from patients with liver cirrhosis and ACLF revealed that elevated levels of microbial-derived compounds (aromatic compounds, secondary or sulfated bile acids, and benzoates) and estrogenic metabolites, along with reduced phospholipid levels, were associated with ACLF occurrence, hospitalization, and 30-day mortality. These metabolites may be linked to pathogens such as Clostridium difficile, Enterococcaceae, Clostridium, and Unicorniaceae[23]. A combined microbiological and metabolomic approach revealed changes in the composition of gut microbiota in patients with ACLF, suggesting that gut microbiota activity may be related to serum metabolism.
Oxylipins are products of polyunsaturated fatty acid (PUFA) oxidation. Examples include eicosanoids such as prostaglandins, leukotrienes (LTs), and HETEs, which play crucial roles in the initiation and reduction of inflammation[24]. Prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and 12-HETE are the primary pro-inflammatory mediators, whereas eicosapentaenoic acid is considered an anti-inflammatory metabolite[25, 26]. The levels of 12-HETE, 12-HEPE, 14(S)-HDHA, PGE2, dihomo-γ-linolenic acid, and 13-HOTrE have been reported to be significantly elevated in the livers of the D-GalN/LPS-induced ALF group. 12-HETE is significantly reduced after oridonin (Ori) treatment, and the protective effect of Ori on ALI is mediated, in part, by the oxidized lipoprotein pathway[27]. In this study, the expression of 12-HETE, screened by LC/MS, was significantly higher in the ACLF group of mice and positively correlated with Parasutterella. It can be observed that 12-HETE is a metabolite of PUFAs and is involved in inflammatory and oxidative stress responses in the liver through lipid metabolic pathways. Qingchangligan formula has been suggested to decrease the intestinal flora and population of ALI mice, specifically with respect to Proteobacteria, Blautia, Romboutsia, Parabacteroides, UCG-008, Parasutterella, Ruminococcus, norank_f: Lachnospiraceae, Eubacterium_xylanophilum_group, Oscillibacter, and Eisenbergiella. It achieves this by participating in carbohydrate and lipid metabolism as well as glycoprotein biosynthesis and metabolic pathways. Furthermore, it aids in the reduction of liver injury caused by D-Gal through the management of lipid and energy metabolism, glycoprotein biosynthesis, and metabolism, as well as other related metabolites[28]. Based on our findings, an increase in 12-HETE levels was linked to lipid metabolism following liver injury, which subsequently affected liver function. Parasutterella was also enriched in the intestines during liver failure, which is consistent with our results. Thus, our research suggests that disturbances in lipid metabolism could be a driving factor in liver disease progression, and that 12-HETE or Parasutterella can serve as potential markers for disease onset.
TCA positively correlated with ClostridiavadinBB60_group and negatively correlated with Parasutterella. Bile acids are synthesized in hepatocytes by cytochrome P450 from cholesterol via classical and alternative pathways[29, 30]. It has been reported that the increased serum concentration of TCA in patients with cirrhosis may activate hepatic stellate cells to promote cirrhosis progression by upregulating the expression of toll-like receptor 4, and that TCA may be used as a potential therapeutic target for the prevention and treatment of cirrhosis. In this study, the expression of TCA was significantly increased in mice in the ACLF group and was significantly positively correlated with that of Clostridia vadinBB60. The ethanol extract of Ganoderma lucidum significantly improved intestinal microbial disorders by increasing the relative abundance of Ruminococcus and Clostridia_vadinBB60_group. This extract also alleviates alcohol-induced liver injury by participating in metabolism related to primary bile acid biosynthesis[31].
Our study observed an increase in TCA concomitant with a notable increase in the Clostridia_vadinBB60_group. The elevated presence of ClostridiavadinBB60_group is postulated to be a protective response to suppress inflammation induced by TCA, thereby safeguarding the organism. Consequently, the Clostridia_vadinBB60_group emerged as a potential therapeutic target for ACLF. In summary, our study provides novel insights into the pathogenesis of ACLF and offers valuable indications for future investigations and treatment strategies.
The current positive effects with regard to lipid oxidation in the liver and stimulation of cholesterol synthesis in hepatocytes are accompanied by negative effects, such as increasing glutathione depletion, triggering mitochondrial depletion, increasing oxidative stress in hepatocytes, and promoting the secretion of inflammatory factors by hepatocytes and Kupffer cells[32–36]. Phosphatidylcholine (PC) was found to be negatively correlated with Clostridia_vadinBB60_group. It was reported in the mid-20th century that in CCl4-treated rat controls, the liver had reduced levels of PC and phosphatidylethanolamine (PE) but an elevated PE:PC ratio[37]. In recent years, with advancements in metabolomics, researchers have delved deeper into the role of lipid metabolism in liver injury. In 2016, a study revealed that fatty acid composition was altered in rats with liver failure, with an increase in Lyso PCs and a decrease in certain PEs, which may affect membrane fluidity, inflammation, and mitochondrial dysfunction[38]. A previous study suggested that the plasma levels of PC and PE in mice with drug-induced liver injury peaked at 3 or 6 h and returned to normal baseline levels after 24 h. This may be because acetaminophen interferes with phospholipid metabolism[39]. While no study has specifically reported a correlation between PC and intestinal flora, our study found that PC was negatively correlated with the ClostridiumvadinBB60_ group. We postulated that the decrease in PC concurrent with the elevation of Clostridia_vadinBB60_group after the onset of ACLF might be an organismal response aimed at mitigating membrane fluidity, suppressing inflammation, and alleviating mitochondrial dysfunction. This mechanism likely sustains relative homeostasis by promoting an increase in Clostridia_vadinBB60_group, attenuating the decrease in PC. This speculation provides valuable insights for delving deeper into the regulatory mechanisms of lipid metabolism following ACLF onset.
Isoleucine, a branched-chain amino acid, is an essential amino acid required by the body and is associated with metabolic pathways such as protein digestion and absorption. The intravenous administration of isoleucine has been suggested to improve the amino acid composition in upper gastrointestinal bleeding, thereby promoting hepatic and muscle protein synthesis[40]. In this study, protein digestion and absorption were significantly enriched in metabolic pathways, and the abundance of isoleucine amino acids in this pathway was significantly increased, suggesting that branched-chain amino acids are required as nutritional support to improve the state of liver injury.
This study has some limitations. The murine intestinal microecology macro-genome catalog, which partially overlaps in pairs with that of humans[41], could not completely mimic human intestinal microecology and metabolite changes and is yet to be further verified by human experiments to validate the above experimental methods and results.