Celastrol ameliorates cholestatic liver injury by promoting a protective iNKT1 polarization via CTSS inhibition and autophagy restoration

Abstract

Cholestatic liver injury involves pathogenic invariant natural killer T17 (iNKT17) cell expansion with limited treatments. While celastrol shows hepato-protective potential, its mechanisms in modulating iNKT cell-driven pathology during cholestasis and fibrosis remain unclear. We aimed to elucidate how celastrol regulates iNKT cell polarization to ameliorate cholestatic liver injury and fibrotic progression. Celastrol alleviated cholestatic liver injury and fibrosis while restoring bile acid homeostasis. It shifted hepatic iNKT cell balance from pathogenic iNKT17 cells to protective iNKT1 cells. Transcriptomic analysis of sorted hepatic iNKT cells identified cathepsin S (CTSS) as a key hub gene, linking celastrol's effects to phagosome/lysosome pathways. Celastrol bound and inhibited CTSS, which in turn enhanced autophagy and mitophagy in iNKT cells. Mechanistically, chloroquine-mediated lysosomal blockade attenuated celastrol-induced p62 degradation, autophagic flux, iNKT1 polarization, and hepatoprotection. CTSS inhibition mimicked celastrol's benefits, whereas CTSS overexpression abolished them. Critically, in CTSS-knockout mice, the hepatoprotective and iNKT1-polarizing effects of celastrol were eliminated, confirming the essential role of CTSS for celastrol. Clinical relevance was confirmed by significantly elevated CTSS mRNA levels in PBMCs from intrahepatic cholestasis of pregnancy (ICP) patients. Furthermore, using CD1d-deficient mice, we established that iNKT cells were the principal cellular source responsible for the pathogenic upregulation of CTSS in cholestasis. Our findings reveal CTSS as a pivotal molecular checkpoint in cholestasis and its fibrotic progression by governing iNKT cell polarization through autophagy/mitophagy regulation, thereby presenting a novel therapeutic target for cholestatic liver injury.

Ethical statements

Animal procedures were approved by the Ethics Committee of China Pharmaceutical University (2021-10-003), and the research follows the 'Replacement, Reduction, and Refinement' principles to minimize harm to animals. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution's human research committee (2021ZDSYLL022-P01, Zhongda Hospital Southeast University).

Authors contributions

The authors confirm contributions to the paper as follows: study conception and design, visualization, software, funding acquisition, draft manuscript preparation: Wang X; investigation, methodology, data collection: Wang X, Wang H, Yang M, Zhang S, Mai M; analysis and interpretation of results: Wang X, Wang H, Zhang S; writing, review and editing: Wang X, Zhang Y; resources, project administration: Zhang L, Liu J, Xing M. All authors reviewed the results and approved the final version of the manuscript.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgements

The present study was supported by the National Natural Science Foundation of China (No. 82073948). We thank Dr. Chuan Su (Nanjing Medical University) for CD1d−/− mice, Dr. Li Bai (University of Science and Technology of China) for DN32.D3 cells, the NIH Tetramer Core Facility for tetramers, Yumeng Shen (Public platform of State Key Laboratory of Natural Medicines) for flow cytometry sorting, Jie Zhao and Zhenglin Hao (Pharmaceutical Animal Experimental Center of China Pharmaceutical University) for animal experiments support.

Conflict of interest

The authors declare no conflicts of interest.

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