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.

胆汁淤积性肝损伤是一组以胆汁酸（BA）稳态失调为特征的疾病，部分是由胆汁酸循环障碍引起的，这通常与肝细胞屏障功能的损害有关。然而，胆汁淤积性肝损伤的潜在肝细胞屏障保护分子机制仍然知之甚少。有趣的是，最近的研究表明，1-磷酸鞘氨醇 (S1P) 通过激活其 G 蛋白偶联受体 S1PRs 参与胆汁淤积过程，恢复肝细胞紧密连接 (TJs) 的完整性。在这里，我们发现 SEW2871 是一种选择性的 1-磷酸鞘氨醇受体 1 (S1PR1) 激动剂，可减轻 3D 培养的小鼠原代肝细胞中 ANIT 诱导的 TJ 损伤。分子机制研究表明AMPK信号通路参与了TJs对ANIT诱导的肝胆屏障功能障碍SEW2871的保护作用。AMPK 拮抗剂化合物 C (CC) 和激动剂 AICAR 均用于进一步确定 AMPK 信号通路在 SEW2871 对 ANIT 处理的小鼠原代肝细胞的 TJs 保护中的重要作用。体内数据显示 SEW2871 改善了 ANIT 诱导的胆汁淤积性肝毒性。进一步的保护机制研究表明，SEW2871不仅通过AMPK信号通路上调的S1PR1恢复肝细胞TJs，而且恢复了肝胆屏障功能缺陷，通过高效液相色谱-串联质谱法恢复BA稳态得到证实。 LC-MS/MS）。这些结果表明，SEW2871 诱导的 S1PR1 表达增加可以通过 AMPK 信号传导改善肝屏障功能来改善 ANIT 诱导的胆汁淤积性肝损伤，并随后逆转被破坏的 BA 稳态。我们的研究提供了强有力的证据，表明 S1PR1 可能是治疗肝内胆汁淤积性肝损伤的一种有前途的治疗方法。