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SEW2871 attenuates ANIT-induced hepatotoxicity by protecting liver barrier function via sphingosine 1-phosphate receptor-1–mediated AMPK signaling pathway

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Abstract

Cholestatic liver injury, a group of diseases characterized with dysregulated bile acid (BA) homeostasis, was partly resulted from BA circulation disorders, which is commonly associated with the damage of hepatocyte barrier function. However, the underlying hepatocyte barrier-protective molecular mechanisms of cholestatic liver injury remain poorly understood. Interestingly, recent studies have shown that sphingosine-1-phosphate (S1P) participated in the process of cholestasis by activating its G protein–coupled receptors S1PRs, regaining the integrity of hepatocyte tight junctions (TJs). Here, we showed that SEW2871, a selective agonist of sphingosine-1-phosphate receptor 1(S1PR1), alleviated ANIT–induced TJs damage in 3D-cultured mice primary hepatocytes. Molecular mechanism studies indicated that AMPK signaling pathways was involved in TJs protection of SEW2871 in ANIT-induced hepatobiliary barrier function deficiency. AMPK antagonist compound C (CC) and agonist AICAR were all used to further identify the important role of AMPK signaling pathway in SEW2871’s TJs protection of ANIT-treated mice primary hepatocytes. The in vivo data showed that SEW2871 ameliorated ANIT-induced cholestatic hepatotoxicity. Further protection mechanism research demonstrated that SEW2871 not only regained hepatocyte TJs by the upregulated S1PR1 via AMPK signaling pathway, but also recovered hepatobiliary barrier function deficiency, which was verified by the restored BA homeostasis by using of high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). These results revealed that the increased expression of S1PR1 induced by SEW2871 could ameliorate ANIT–induced cholestatic liver injury through improving liver barrier function via AMPK signaling and subsequently reversed the disrupted BA homeostasis. Our study provided strong evidence that S1PR1 may be a promising therapeutic approach for treating intrahepatic cholestatic liver injury.

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Abbreviations

ANIT:

α-Naphthylisothiocyanate

TJs:

Tight junctions

ZO-1:

Zonula occludens 1

S1P:

Sphingosine-1-phosphate

S1PR1:

sphingosine-1-phosphate receptor 1

AMPK:

AMP-activated protein kinase

LKB1:

Liver kinase B1

ERK1/2:

Extracellular signal-regulated kinases

CC:

Compound C

MPO:

Myeloperoxidase

IL-6:

Interleukin-6

IL-1β:

Interleukin-1β

TNF-α:

Tumor necrosis factor-α

SCMHs:

Sandwich cultured primary mice hepatocytes

LC-MS/MS:

High-performance liquid chromatography-tandem mass spectrometry

PCA:

Principal component analysis

G-BAs:

Glycine-conjugated bile acids

T-BAs:

Taurine conjugated bile acids

UDCA:

Ursodeoxycholic acid

HDCA:

Hyodeoxycholic acid

CDCA:

Chenodeoxycholic acid

DCA:

Deoxycholic acid

CA:

Cholic acid

Beta-MCA:

Beta-muricholic acid

LCA:

Lithocholic acid

TUDCA:

Tauroursodeoxycholic acid

THDCA:

Taurohyodeoxycholic acid

TCDCA:

Taurochenodeoxycholic acid

TDCA:

Taurodeoxycholic acid

TCA:

Taurocholic acid

TLCA:

Taurolithocholic acid

GUDCA:

Glycoursodeoxycholic acid

GHDCA:

Glycohyodeoxycholic acid

GCDCA:

Glycochenodeoxycholic acid

GDCA:

Glycodeoxycholic acid

GCA:

Glycocholic acid

GLCA:

Glycolithocholic acid

dhCA:

Dehydrocholic acid

NorUDCA:

24-norursodeoxycholic acid

OCA:

Obecholic acid

EE:

17α-Ethinylestradiol

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Author notes

  1. Zhenzhou Jiang and Luyong Zhang contributed equally to this work.

Authors and Affiliations

  1. Center for Drug Research and Development, Guangdong Pharmaceutical University, Guangzhou, 510006, China

    Luyong Zhang

  2. New Drug Screening Center, Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical University, Nanjing, 210009, China

    Xue Wang, Qiongna Yu, Zihang Yuan, Yingying Miao, Yuanyuan Chai, Ziteng Wu, Lixin Sun, Xin Huang, Zhenzhou Jiang & Luyong Zhang

  3. Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou, 221004, China

    Tingting Yang, Yi Zhou, Cai Heng & Hao Yang

  4. Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, Nanjing, 210009, China

    Xin Huang & Zhenzhou Jiang

  5. Department of Pharmacology, School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, China

    Bing Liu

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Contributions

Zhenzhou Jiang, Luyong Zhang, and Tingting Yang conceived the original ideas and designed the study. Tingting Yang did immunofluorescence, hierarchical clustered heat map analysis and PCA analysis. Xue Wang carried out the HPLC-MS/MS analysis, quantitative real-time-PCR, and measurement of inflammatory cytokines. Yi Zhou and Cai Heng helped by doing MPO activity analysis and western blot analysis of animals and cells. Qiongna Yu carried out the isolation and sandwich culture of mice primary hepatocyte. Hao Yang did hematoxylin-eosin staining and immunohistochemistry. Tingting Yang, Xue Wang, Yi Zhou, and Cai Heng analyzed the data. Tingting Yang and Hao Yang edited of the figures and tables. Tingting Yang and Zhenzhou Jiang wrote the manuscript. Zhenzhou Jiang, Luyong Zhang, and Tingting Yang reviewed the manuscript. The rest of the authors either supplied reagent or helped in treatments to mice and cells.

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Correspondence to Zhenzhou Jiang or Luyong Zhang.

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Yang, T., Wang, X., Zhou, Y. et al. SEW2871 attenuates ANIT-induced hepatotoxicity by protecting liver barrier function via sphingosine 1-phosphate receptor-1–mediated AMPK signaling pathway. Cell Biol Toxicol 37, 595–609 (2021). https://doi.org/10.1007/s10565-020-09567-9

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