Sub-chronic realgar exposure causes liver inflammatory injury in mice by inducing bile acid-mediated NLRP3 inflammasome activation through down-regulation of ileal FXR

Ethnopharmacological relevance

Realgar is extensively utilized in both modern medicine and traditional Chinese medicine. Although it has therapeutic uses, realgar exhibits hepatotoxicity, and improper use can result in liver inflammation and injury. The disruption of bile acid (BA) homeostasis, identified as an initiating event in liver injury, has been observed in the livers of realgar-exposed mice. However, the relationship between realgar-induced BA homeostasis imbalance and liver inflammation remains to be elucidated.

Aim of the study

To investigate the relationship between hepatic BA homeostasis disorder and liver inflammation caused by realgar exposure in mice, as well as the role and potential mechanism of intestinal farnesoid X receptor (FXR) in realgar-induced liver inflammation.

Methods

A sub-chronic realgar exposure mouse model was established and subjected to interventions with an intestinal-restricted FXR agonist or inhibitor. Plasma hepatic enzyme activities and total bile acid (TBA) levels were quantified using spectrophotometry. Hepatic inflammatory cytokine levels were analyzed by ELISA or RT-qPCR. Histopathological evaluation of liver injury was performed using HE staining. F4/80 expression was assessed via immunohistochemistry (IHC). Western blot (WB) and IHC were employed to examine the expression of proteins involved in the FXR-FGF15 axis, and plasma FGF15 concentrations were determined by ELISA. The expression of proteins related to enterohepatic circulation and the NLRP3 inflammasome pathway were evaluated using WB. Ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC-TOF-MS) was utilized to analyze BA profiles in both ileal and hepatic tissues.

Results

Our findings demonstrate that realgar exposure induces changes in ileal BA composition and subsequent suppression of the FXR-FGF15 signaling axis in mice. This axis suppression promotes hepatic BA overproduction and disrupts BA homeostasis in the livers of realgar-exposed mice, particularly elevates the levels of taurochenodeoxycholic acid (TCDCA) and taurolithocholic acid (TLCA). In vitro studies revealed that both TCDCA and TLCA can directly activate the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome in AML-12 cells, triggering pro-inflammatory cascades. Pharmacological activation of intestinal FXR effectively ameliorated realgar-induced hepatic inflammatory damage via restoring BA homeostasis and suppressing NLRP3 inflammasome activation.

Conclusion

Realgar exposure causes liver inflammatory injury by inducing BA-mediated NLRP3 inflammasome activation through down-regulating ileal FXR. Pharmacological activation of intestinal FXR alleviates realgar-induced liver inflammatory injury by restoring BA homeostasis and inhibiting BA-mediated NLRP3 inflammasome activation. These findings suggest that intestinal FXR may serve as a potential target for the prevention and treatment of realgar-induced liver injury.