Narirutin activates TFEB (transcription factor EB) to protect against Acetaminophen-induced liver injury by targeting PPP3/calcineurin

Zhiyuan Fang; Yanyong Xu; Guowen Liu; Qi Shao; Xiaodi Niu; Wenjun Tai; Taiyu Shen; Minghe Fan; Meng Chen; Lin Lei; Wenwen Gao; Yuxiang Song; Zhe Wang; Xiliang Du; Xinwei Li

doi:10.1080/15548627.2023.2179781

Narirutin activates TFEB (transcription factor EB) to protect against Acetaminophen-induced liver injury by targeting PPP3/calcineurin

Autophagy. 2023 Aug;19(8):2240-2256. doi: 10.1080/15548627.2023.2179781. Epub 2023 Feb 24.

Authors

Zhiyuan Fang¹, Yanyong Xu^{2

3}, Guowen Liu¹, Qi Shao¹, Xiaodi Niu⁴, Wenjun Tai¹, Taiyu Shen¹, Minghe Fan⁵, Meng Chen¹, Lin Lei¹, Wenwen Gao¹, Yuxiang Song¹, Zhe Wang¹, Xiliang Du¹, Xinwei Li¹

Affiliations

¹ State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun Jilin, China.
² Key Laboratory of Metabolism and Molecular Medicine of the Ministry of Education, Department of Pathology of School of Basic Medical Sciences, Fudan University, Shanghai, China.
³ Frontier Innovation Center, School of Basic Medical Sciences, Fudan University, Shanghai, China.
⁴ College of Food Science and Engineering, Jilin University, Changchun, China.
⁵ Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China.

Abstract

Acetaminophen (APAP) overdose is the predominant cause of drug-induced liver injury worldwide. The macroautophagy/autophagy-lysosomal pathway (ALP) is involved in the APAP hepatotoxicity. TFEB (transcription factor EB) promotes the expression of genes related to autophagy and lysosomal biogenesis, thus, pharmacological activation of TFEB-mediated ALP may be an effective therapeutic approach for treating APAP-induced liver injury. We aimed to reveal the effects of narirutin (NR), the main bioactive constituents isolated from citrus peels, on APAP hepatotoxicity and to explore its underlying mechanism. Administration of NR enhanced activities of antioxidant enzymes, improved mitochondrial dysfunction and alleviated liver injury in APAP-treated mice, whereas NR did not affect APAP metabolism and MAPK/JNK activation. NR enhanced TFEB transcriptional activity and activated ALP in an MTOR complex 1 (MTORC1)-independent but PPP3/calcineurin-dependent manner. Moreover, knockout of Tfeb or knockdown of PPP3CB/CNA2 (protein phosphatase 3, catalytic subunit, beta isoform) in the liver abolished the beneficial effects of NR on APAP overdose. Mechanistically, NR bound to PPP3CB via PRO31, LYS61 and PRO347 residues and enhanced PPP3/calcineurin activity, thereby eliciting dephosphorylation of TFEB and promoting ALP, which alleviated APAP-induced oxidative stress and liver injury. Together, NR protects against APAP-induced liver injury by activating a PPP3/calcineurin-TFEB-ALP axis, indicating NR may be a potential agent for treating APAP overdose.Abbreviations: ALP: autophagy-lysosomal pathway; APAP: acetaminophen; APAP-AD: APAP-protein adducts; APAP-Cys: acetaminophen-cysteine adducts; CAT: catalase; CETSA: cellular thermal shift assay; CQ: chloroquine; CYP2E1: cytochrome P450, family 2, subfamily e, polypeptide 1; CYCS/Cyt c: cytochrome c, somatic; DARTS: drug affinity responsive target stability assay; ENGASE/NAG: endo-beta-N-acetylglucosaminidase; GOT1/AST: glutamic-oxaloacetic transaminase 1, soluble; GPT/ALT: glutamic pyruvic transaminase, soluble; GSH: glutathione; GPX/GSH-Px: glutathione peroxidase; K_D: dissociation constant; Leu: leupeptin; MCOLN1: mucolipin 1; MTORC1: MTOR complex 1; NAC: N-acetylcysteine; NAPQI: N-acetyl-p-benzoquinoneimine; NFAT: nuclear factor of activated T cells; NR: narirutin; OA: okadaic acid; RRAG: Ras related GTP binding; ROS: reactive oxygen species; PPP3CB/CNA2: protein phosphatase 3, catalytic subunit, beta isoform; PPP3R1/CNB1: protein phosphatase 3, regulatory subunit B, alpha isoform (calcineurin B, type I); SOD: superoxide dismutase; SPR: surface plasmon resonance analysis; TFEB: transcription factor EB.

Keywords: PPP3CB/CNA2; TFEB; autophagy-lysosomal pathway; hepatotoxicity; oxidative stress.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Acetaminophen
Animals
Autophagy / genetics
Calcineurin* / metabolism
Chemical and Drug Induced Liver Injury, Chronic* / metabolism
Glutathione / metabolism
Liver / metabolism
Mechanistic Target of Rapamycin Complex 1 / metabolism
Mice
TOR Serine-Threonine Kinases / metabolism

Substances

Calcineurin
Acetaminophen
narirutin
Glutathione
Mechanistic Target of Rapamycin Complex 1
TOR Serine-Threonine Kinases

Supplementary concepts

Cornea Plana 2

Grants and funding

This work was supported by the Fundamental Research Funds for the Central Universities [2022-JCXK-21]; National Natural Science Foundation of China [32022084]; National Natural Science Foundation of China [32002349]; Science foundation of Education Department of Jilin Province [JJKH20221038KJ].