Celastrol reduces cholestatic liver injury by inhibiting specific protein

Cholestasis occurs when bile formation, secretion, or flow is impaired, causing toxic bile acids to accumulate and trigger inflammation and liver-cell damage. Without effective treatment, the condition can progress to fibrosis, cirrhosis, or liver cancer. Ursodeoxycholic acid is commonly used as a first-line therapy, but up to 40% of patients do not respond adequately. Previous studies have implicated hepatic nonparenchymal cells, particularly inflammatory iNKT17 cells, in cholestatic injury. Celastrol has shown broad anti-inflammatory and hepatoprotective activity, but how it regulates iNKT-cell behavior during cholestasis has remained unclear. Consequently, researchers needed to identify the molecular mechanism connecting celastrol, iNKT-cell polarization, and protection against cholestatic liver injury.

A study (DOI:10.48130/targetome-0026-0018) published in Targetome on 29 April 2026 by Xinzhi Wang’s team, China Pharmaceutical University, reports that celastrol suppresses CTSS-dependent autophagy impairment, promotes protective iNKT1 polarization, and reduces cholestasis and fibrosis.

The researchers first evaluated celastrol in mouse models of cholestasis induced by ethinylestradiol or α-naphthyl isothiocyanate. Serum biochemical measurements, liver histology, gene-expression analysis, and protein assays showed that celastrol reduced alanine transaminase, aspartate transaminase, and alkaline phosphatase levels; limited necrosis and inflammatory infiltration; and restored regulators of bile-acid synthesis, metabolism, transport, and homeostasis. Flow cytometry revealed that treatment increased protective IFN-γ-producing iNKT1 cells while decreasing pathogenic IL-17-producing iNKT17 cells. Hepatocyte-iNKT-cell co-culture experiments further showed that celastrol altered both CD1d-mediated antigen presentation and IL-12-dependent activation, indicating that communication between hepatocytes and iNKT cells helped determine immune polarization. To identify the underlying target, the team isolated hepatic iNKT cells for transcriptomic sequencing and combined the results with protein-interaction analysis and public single-cell RNA-sequencing datasets. Ctss emerged as a central gene associated with lysosomal, phagosomal, and antigen-presentation pathways. Surface plasmon resonance confirmed direct binding between celastrol and the CTSS protein, while molecular docking predicted a stable interaction involving its His164 residue. Celastrol downregulated Ctss expression, reduced CTSS protein levels, and restored markers of autophagy and mitochondrial quality control. Pharmacological inhibition of CTSS reproduced these protective effects, whereas CTSS overexpression or lysosomal blockade weakened them. In Ctss-knockout mice, celastrol no longer produced its characteristic hepatoprotective or iNKT1-polarizing effects, confirming that CTSS was essential to its mechanism. CD1d-deficient mice demonstrated that iNKT cells were primarily responsible for the pathological upregulation of hepatic Ctss during cholestasis. In additional carbon tetrachloride models, celastrol reduced acute liver injury, collagen deposition, serum fibrosis markers, and the expression of fibrogenic genes and proteins. Finally, peripheral blood mononuclear cells from patients with intrahepatic cholestasis of pregnancy showed significantly elevated human CTSS mRNA levels, supporting the pathway’s potential clinical relevance.

Overall, the study establishes a mechanistic link between CTSS inhibition, restored autophagy and mitophagy, protective iNKT1 polarization, and reduced liver injury. Celastrol acted at multiple levels by improving bile-acid homeostasis, modifying hepatocyte-iNKT communication, and limiting inflammatory and fibrotic responses. Although further safety studies and clinical validation are required, CTSS may provide a promising target for developing treatments that prevent cholestasis from advancing to irreversible liver fibrosis.