Inhibition of P2Y6 receptor expression in Kupffer cells alleviates alcoholic steatohepatitis in mice

https://doi.org/10.1016/j.intimp.2022.108909Get rights and content

Highlights

  • UDP acts as a danger signal in alcoholic steatohepatitis by activating the P2Y6 receptor.

  • The expression of P2Y6 receptor was significantly increased in EtOH-fed mice and EtOH-induced RAW264.7 cells.

  • P2Y6 receptor may promote inflammatory response by mediating Ca2+ influx and then activating p38 MAPK signaling pathway.

  • Inhibition of P2Y6 receptor alleviates EtOH-induced inflammatory response and reduces AML-12 cell apoptosis in vitro.

Abstract

Inflammation plays an important role in the progression of alcohol-related liver disease (ALD). UDP-P2Y6 signaling is involved in many human diseases. The purinergic P2Y6 receptor, an important regulator of inflammation and phagocytosis, has attracted attention, but its role in alcoholic steatohepatitis remains unclear. Here, we found that P2Y6 levels were significantly elevated in Kupffer cells in the livers of mice with alcoholic steatohepatitis and ethanol (EtOH)-induced RAW264.7 cells. In this study, mice with alcoholic steatohepatitis were intraperitoneally injected with MRS2578, a specific inhibitor of the P2Y6 receptor, and P2Y6 was silenced in EtOH-induced RAW264.7 cells. We found a marked improvement in steatosis and inflammation in the livers of mice with alcoholic steatohepatitis and EtOH-induced RAW264.7 cells. However, P2Y6 activation in vivo and overexpression in vitro showed contrasting results. In addition, the expression of phospho-p38 mitogen-activated protein kinase (p-p38 MAPK), a phosphorylated protein in the p38 MAPK signaling pathway, was significantly altered after P2Y6 silencing or overexpression in vitro. P2Y6 can induce the activation of the p38 MAPK signaling pathway by mediating the calcium influx, whereas inhibition of the expression of P2Y6 can block the inflammatory process to some extent and thus improve the inflammatory response. The results of this study suggested that targeting P2Y6 signaling may be a potentially effective strategy for the treatment of alcoholic steatohepatitis.

Introduction

Alcohol-related liver disease (ALD) is one of the most common liver diseases worldwide, and alcohol abuse is the main cause of its pathogenesis [1]. ALD mainly includes alcoholic fatty liver disease, alcoholic steatohepatitis (ASH), and liver fibrosis, which may develop into cirrhosis or even liver cancer in severe cases [2]. Studies have shown that inflammation plays a central role in the development of ALD [3]. Inflammation is a complex defense response to the stimulation of the body, which can reduce injury and restore normal tissue structure and function [4]. Kupffer cells are resident macrophages in the liver and are an important part of the mononuclear phagocyte system [5]. Activated Kupffer cells release large amounts of pro-inflammatory cytokines and chemokines, which further aggravate liver inflammation and oxidative stress when hepatocytes are damaged, resulting in further deterioration of liver injury [6]. Due to the complexity of ALD mediated by ethanol (EtOH) through various factors, new effective methods must be found for the treatment of ALD [7].

Many studies have reported that extracellular nucleotides (ATP, ADP, UTP, UDP) are rapidly released into the extracellular environment of the inflammatory site during the inflammatory response, which promotes the chemotaxis of immune cells and the release of cytokines by activating corresponding P2Y purine receptors, thus affecting the process of inflammation[8], [9], [10]. P2Y6, a member of the P2Y purine receptor family, is a typical G protein-coupled extracellular nucleotide receptor that is selectively activated by its natural ligand, UDP, to accumulate intracellular Ca2+, thereby influencing a series of signaling pathways [11]. A study found that P2Y6 can affect the development of inflammation by activating macrophages to release chemokines to recruit immune cells to the site of inflammation [12]. For example, Nagai et al. reported the effect of P2Y6 on hypersensitivity pneumonitis in alveolar macrophages [13]. Blackburn et al. demonstrated that inflammation induces the upregulation of endothelial P2Y6 expression to enhance systemic inflammatory responses [14]. Koizumi et al. reported that UDP released by injured nerve cells leads to the upregulation of P2Y6 expression on neuronal microglia to enhance their ability to phagocytose apoptotic cells [15]. However, the role of P2Y6 in ASH and its underlying molecular mechanisms remain unclear. Given that P2Y6 is involved in the inflammatory response in many inflammatory diseases, we hypothesized that P2Y6 may be involved in ASH and the activation of hepatic macrophages. To confirm this hypothesis, we used the Gao-Binge model [16] to establish a mouse model of ASH and observed changes in the levels of P2Y6 in the mouse liver and Kupffer cells. In addition, we used EtOH to stimulate RAW264.7 cells to establish a research model of inflammation in vitro.

Section snippets

Establishment of experimental animals and models

C57BL/6 mice (female, 6–8 weeks old, 18–22 g) required for the establishment of the ASH model were provided by Hangzhou Zi Yuan Laboratory Animal Technology Co., Ltd. (Hangzhou, China). The control liquid diet and Lieber-DeCarli EtOH were provided by TROPHIC Animal Feed High-Tech Co., Ltd. (Nanjing, China). Before the modeling, we gave all C57BL/6 mice seven days to acclimate to the new environment. All mice were randomly divided into six groups, with ten mice in each group. The mice were

Verification of the ASH model in mice

The established mouse model was validated based on the following results. Histologically, HE (Fig. 1A) and BODIPY (Fig. 1B) pathological sections showed that liver injury and steatosis were significantly increased in the EtOH-fed group when compared to the Pair-fed group. Immunohistochemical analysis of F4/80, a marker of macrophages, showed that when compared to the control group, the liver of the model group had more infiltration of macrophages (Fig. 1C). We also found that the body weight

Discussion

ALD is one of the most common chronic liver diseases worldwide. Steatosis is the earliest form of ALD, and liver inflammation can induce the progression of steatosis into a more serious form of inflammatory liver injury known as steatohepatitis[25]. The pathogenesis of alcoholic liver injury is complex, mainly due to the influence of EtOH and its metabolites. Intracellular inflammatory mediators derived from hepatic Kupffer cells caused by EtOH-induced changes in mesenteric permeability are

Conclusion

This study is the first to explore UDP as a “danger signal” in ASH by activating P2Y6 to mediate an EtOH-induced inflammatory response. Our results support the hypothesis that EtOH stimulates the body to induce the release of UDP, which then activates P2Y6 to mediate the development of ASH through the p38 MAPK signaling pathway. We ameliorated the EtOH-induced inflammatory response by inhibiting the expression of P2Y6. Although some aspects are yet to be investigated, this novel mechanism of

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This project was supported by the National Natural Science Foundation of China (Grant 81970518).

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