靶向 GPR65 通过抑制 JNK 和 NF-κB 通路缓解肝炎症和肝纤维化。

Targeting GPR65 alleviates hepatic inflammation and fibrosis by suppressing the JNK and NF-κB pathways.

机构信息

Department of Histology and Embryology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, 300070, China.

Department of Hepatology and Gastroenterology, Tianjin Union Medical Center, Tianjin Medical University, Tianjin Union Medical Center affiliated to Nankai University, Tianjin, 300000, China.

出版信息

Mil Med Res. 2023 Nov 25;10(1):56. doi: 10.1186/s40779-023-00494-4.

DOI:10.1186/s40779-023-00494-4

PMID:38001521

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10675918/

Abstract

BACKGROUND

G-protein coupled receptors (GPCRs) are recognized as attractive targets for drug therapy. However, it remains poorly understood how GPCRs, except for a few chemokine receptors, regulate the progression of liver fibrosis. Here, we aimed to reveal the role of GPR65, a proton-sensing receptor, in liver fibrosis and to elucidate the underlying mechanism.

METHODS

The expression level of GPR65 was evaluated in both human and mouse fibrotic livers. Furthermore, Gpr65-deficient mice were treated with either bile duct ligation (BDL) for 21 d or carbon tetrachloride (CCl) for 8 weeks to investigate the role of GPR65 in liver fibrosis. A combination of experimental approaches, including Western blotting, quantitative real-time reverse transcription‑polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA), confocal microscopy and rescue studies, were used to explore the underlying mechanisms of GPR65's action in liver fibrosis. Additionally, the therapeutic potential of GPR65 inhibitor in the development of liver fibrosis was investigated.

RESULTS

We found that hepatic macrophages (HMs)-enriched GPR65 was upregulated in both human and mouse fibrotic livers. Moreover, knockout of Gpr65 significantly alleviated BDL- and CCl-induced liver inflammation, injury and fibrosis in vivo, and mouse bone marrow transplantation (BMT) experiments further demonstrated that the protective effect of Gpr65 knockout is primarily mediated by bone marrow-derived macrophages (BMMs). Additionally, in vitro data demonstrated that Gpr65 silencing and GPR65 antagonist inhibited, while GPR65 overexpression and application of GPR65 endogenous and exogenous agonists enhanced the expression and release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and transforming growth factor-β (TGF-β), all of which subsequently promoted the activation of hepatic stellate cells (HSCs) and the damage of hepatocytes (HCs). Mechanistically, GPR65 overexpression, the acidic pH and GPR65 exogenous agonist induced up-regulation of TNF-α and IL-6 via the Gαq-Ca-JNK/NF-κB pathways, while promoted the expression of TGF-β through the Gαq-Ca-MLK3-MKK7-JNK pathway. Notably, pharmacological GPR65 inhibition retarded the development of inflammation, HCs injury and fibrosis in vivo.

CONCLUSIONS

GPR65 is a major regulator that modulates the progression of liver fibrosis. Thus, targeting GPR65 could be an effective therapeutic strategy for the prevention of liver fibrosis.

摘要

背景

G 蛋白偶联受体（GPCRs）被认为是药物治疗的有吸引力的靶点。然而，除了少数趋化因子受体外，GPCR 如何调节肝纤维化的进展仍知之甚少。在这里，我们旨在揭示质子感应受体 GPR65 在肝纤维化中的作用，并阐明其潜在机制。

方法

评估了人源和鼠源纤维化肝脏中 GPR65 的表达水平。此外，用胆管结扎（BDL）或四氯化碳（CCl）处理 Gpr65 缺陷小鼠 21 天或 8 周，以研究 GPR65 在肝纤维化中的作用。使用 Western blot、定量实时逆转录聚合酶链反应（qRT-PCR）和酶联免疫吸附测定（ELISA）、共聚焦显微镜和挽救研究等实验方法，探讨 GPR65 在肝纤维化中的作用机制。此外，还研究了 GPR65 抑制剂在肝纤维化发展中的治疗潜力。

结果

我们发现，富含肝巨噬细胞（HMs）的 GPR65 在人源和鼠源纤维化肝脏中均上调。此外，Gpr65 敲除显著减轻了 BDL 和 CCl 诱导的体内肝炎症、损伤和纤维化，并且小鼠骨髓移植（BMT）实验进一步表明，Gpr65 敲除的保护作用主要是由骨髓来源的巨噬细胞（BMMs）介导的。此外，体外数据表明，Gpr65 沉默和 GPR65 拮抗剂抑制，而 GPR65 过表达和应用 GPR65 内源性和外源性激动剂增强肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）和转化生长因子-β（TGF-β）的表达和释放，所有这些都随后促进了肝星状细胞（HSCs）的激活和肝细胞（HCs）的损伤。在机制上，GPR65 过表达、酸性 pH 和 GPR65 外源性激动剂通过 Gαq-Ca-JNK/NF-κB 途径诱导 TNF-α 和 IL-6 的上调，而通过 Gαq-Ca-MLK3-MKK7-JNK 途径促进 TGF-β的表达。值得注意的是，药理学 GPR65 抑制可延缓体内炎症、HCs 损伤和纤维化的发展。