Role of NLRP3 in the metabolism of bile acids and gut microbiota in CCl4-induced liver fibrosis.

NOD-like receptor protein 3 (NLRP3) is an important pro-fibrosis factor in the liver. Abnormal bile acid (BA) metabolism and gut dysbiosis are also involved in liver fibrosis. However, the role of NLRP3 in the metabolism of bile acids and gut microbiota in liver fibrosis is unclear, which contributes to the understanding of the mechanism of NLRP3 in liver fibrosis. The male C57BL/6 J mice were divided into the following groups: (i) control group (ii); CCl4 group (iii); NLRP3- + CCl4 group (iv); NLRP3- group. The interventions were delivered three times per week for 12 weeks. Liver tissues and intestinal contents were collected. Targeted metabolomics was performed to determine BAs in liver tissues. The 16S rRNA sequencing was adopted to explore the gut microbiota. Total BAs, primary BAs, and secondary BAs were decreased in the CCl4 group compared to the control group, and NLRP3 knockout restored the CCl4-induced BAs disorder. The increased 7α-hydroxy-4-cholesten-3-one (C4) was observed in the CCl4 group, and NLRP3 knockout decreased the C4 level in CCl4-induced fibrosis. Additionally, the levels of C4 and BAs showed no significant alterations in the NLRP3 group compared with the control group. Compared to the control group, 16S rRNA sequencing revealed the NLRP3 group exhibited increased intestinal alpha diversity and altered beta diversity. Additionally, we observed changes in signaling pathway activity and gut microbiota-derived ECs. 16S rRNA sequencing revealed differential distribution of gut microbiota among the control, CCl4, and NLRP3 +CCl4 mice. The linear discriminant analysis (LDA) coupled with effect size (LEfSe) analysis showed that the abundances of Aeromonas, Akkermansia, and Dehalobacterium increased in the gut of CCl4 group and decreased in NLRP3 +CCl4 mice at the genus level. The abundances of Allobaculum, Odoribacter, and Ruminococcus decreased in the gut of CCl4 group and increased in NLRP3 +CCl4 mice. The abundances of Alistipes, Bacteroides, Clostridium, Lactococcus, Parabacteroides, and Pseudoramibacter_Eubacterium were significantly increased in NLRP3 +CCl4 mice. LPS increased in the CCl4 group and decreased after NLRP3 knockout. Lipopolysaccharides (LPS) had a positive correlation with Bacteroides, Parabacteroides, Aeromonas, and Dehalobacterium and a negative correlation with Allobaculum. Some important signaling pathways (e.g., dopaminergic synapse, serotonergic synapse, non-homologous end-joining, basal transcription factors, and isoflavonoid biosynthesis) and enzymes (e.g., Enzyme Commission (EC) number 5.2.1.4, EC 1.4.3.19, EC 3.4.24.20, EC 1.14.13.1, EC 2.3.3.5, EC 1.14.16.1, EC 1.3.8.7, EC 4.2.1.119 , and EC 5.3.3.17) were upregulated in the CCl4 group and reversed by NLRP3 knockout. Correlation analyses revealed that liver BAs were more closely related to some gut microbiota and LPS. The decreased BA level in CCl4-induced liver fibrosis reflected liver injury. NLRP3 knockout restored the BA level, and NLRP3 knockout protected liver from injury. Increased liver LPS reflected bacterial translocation into the liver. NLRP3 knockout decreased liver LPS level and ameliorated CCl4-induced gut microbiota dysregulation. Taken together, NLRP3 damaged the liver and reduced BAs production and might induce bacterial translocation into the liver to promote liver fibrosis.IMPORTANCENOD-like receptor protein 3 (NLRP3) is an important pro-fibrosis factor in the liver. Our previous study also demonstrated that knockout of NLRP3 can alleviate liver fibrosis. In the current study, we discovered NLRP3 knockout restored the BA level and protected liver from injury. NLRP3 knockout decreased the liver LPS level and ameliorated CCl4-induced gut microbiota dysregulation, and some beneficial bacteria were identified. The current study contributes to the understanding of the mechanism of NLRP3 in liver fibrosis and may provide intervention methods for liver fibrosis caused by damage-associated molecular patterns.