Elsevier

Molecular Immunology

Volume 101, September 2018, Pages 409-418
Molecular Immunology

P2X7 receptor is involved in lung injuries induced by ischemia-reperfusion in pulmonary arterial hypertension rats

https://doi.org/10.1016/j.molimm.2018.07.027Get rights and content

Highlights

  • Pulmonary arterial hypertension (PAH) is a progressive deadly disease with unsatisfactory efficacy.

  • Lung injuries caused by ischemia-reperfusion could be complicated by PAH.

  • P2X7 receptor is demonstrated to be involved in lung injuries induced by ischemia-reperfusion in PAH rats.

Abstract

Pulmonary arterial hypertension (PAH) is a progressive disease that ultimately leads to right heart failure and death. Current strategies are ineffective to prevent and cure PAH, especially in those who undergo cardiopulmonary bypass. P2 × 7 receptors (P2 × 7Rs) have been implied to participate in the pathogenesis of PAH and injuries induced by ischemia-reperfusion (IR). In the present study, we aimed to assess the potential therapeutic effects of anti-P2 × 7Rs on PAH and IR-induced lung injuries in rats and explore their underlying cellular and molecular mechanisms. In the present study, we have successfully established rat models with PAH and/or lung IR injuries. Immunohistochemical staining, western blot, and polymerase chain reaction were performed to detect the P2 × 7R expression in these models; P2 × 7R-specific inhibitor, Brilliant Blue G (BBG), was used to antagonize P2 × 7R, and enzyme-linked immunosorbent assay was used to help evaluate the P2 × 7R-mediated function in PAH with or without IR. Moreover, BBG, SB203580 (p38/MAPK inhibitor), and CD39 (adenosine triphosphate hydrolase) were applied to explore the inner signal pathway in vitro and in vivo. Our findings showed that P2 × 7R was involved in the development of PAH. By applying BBG, we have shown that the severity of PAH and IR was ameliorated through reducing the release of proinflammatory cytokines. Moreover, our results in vitro and in vivo indicated that P2 × 7R regulated the release of inflammatory mediators by the p38/MAPK signal pathway. Most important, CD39 showed the most dominant potential in improving inflammation in lung injuries caused by PAH and IR. In conclusion, the inhibition of P2 × 7R could effectively attenuate inflammation in lung injuries caused by PAH and IR in rats by reducing proinflammatory cytokines through regulating the p38/MAPK pathway.

Introduction

Pulmonary arterial hypertension (PAH) is a fast-progressing vascular disorder of the lung vascular system. Vascular remodeling and endothelial dysfunction in small pulmonary arteries are considered to be the causes of elevated pulmonary vascular resistance as well as increased pulmonary arterial pressure (Lai et al., 2014; Liu et al., 2017). Typically, PAH is characterized by uncontrolled proliferation of pulmonary artery smooth muscle cells and dysfunction of endothelial cells. Increasing pulmonary vascular resistance results in right ventricular (RV) failure and, without lung transplantation, ultimately death. When compared with those without PAH, patients with PAH experience a longer postoperative ventilator assist time, intensive care unit stay, and total hospitalization duration and a higher incidence of low cardiac output syndrome, secondary tracheal intubation, pulmonary hypertension crisis, and hospital mortality (Borde et al., 2013; Malik and Chauhan, 2013; Zhang and Wu, 2017), indicating a role of PAH in increasing mortality and perioperative risk.

The etiology of PAH is considered to be related to multiple factors. A variety of reactive molecules, growth factors, and cytokines have been implicated in its pathogenesis (Schlosser et al., 2017; Groth et al., 2014). The activation of nucleotide-binding oligomerization domain-like receptor family protein 3 (NLRP3) inflammatory bodies, caspase-1, and interleukin (IL)-1β has also been found to be increased in PAH. On the other hand, superoxide dismutase analogues can decrease NLRP3 expression (Villegas et al., 2013), and knock out of NLRP3 is able to reduce collagen deposition and muscularization around pulmonary arteries (Cero et al., 2015). Moreover, inhibition of the P2 × 7 receptor (P2 × 7R), which is regarded as an upstream receptor for NLRP3 activation, can attenuate lipopolysaccharide (LPS)–induced acute lung injury (Wang et al., 2015).

It has been reported that adenosine triphosphate (ATP) accumulates at sites of tissue injury and inflammation. Effects of extracellular ATP are mediated by plasma membrane receptors named P2 receptors (P2Rs). The P2R most involved in inflammation and immunity is the P2 × 7R, expressed by virtually all cells of innate and adaptive immunity (Di Virgilio et al., 2017). ATP can trigger the generation of intracellular second messengers in lymphocytes and macrophages. Moreover, it has also been proved that ATP governs some of the most essential responses in immunity, ranging from antigen-driven T lymphocyte proliferation to T helper 1 (Th1) and Th2 cell differentiation, from neutrophil and macrophage chemotaxis to intracellular pathogen killing, and from NADPH-oxidase activation to IL-1β maturation and release (Di Virgilio et al., 2017; Cekic and Linden, 2016).

The activation of ATP by binding of P2 × 7R could induce Ca2+, Na+ influx, K+ outflow, and activate p38/MAPK signal pathway (Svobodova et al., 2018). Moreover, P2 × 7R, one of seven P2X receptors, has been demonstrated to be involved in inflammatory diseases by triggering the release of inflammatory mediators, including IL-1β and tumor necrosis factor (TNF)–α, which are critical for the pathogenesis of PAH (Baudelet et al., 2015). To date, only Yin et al have recently reported that P2 × 7R might contribute to the pathogenesis of PAH. They found a significant increase in the protein levels of P2 × 7R as well as NLRP3 and caspase-1 in diseased lung tissue as compared with normal tissue, indicating that the inhibition of P2 × 7R could reverse NLRP3 inflammasome upregulation and significantly decrease the mean RV pressure and RV hypertrophy (Yin et al., 2017).

Recently, cardiopulmonary bypass (CPB) has become essential in clinical practice because of its advantage of providing a bloodless field during cardiac interventions. CPB incorporated with an extracorporeal circuit can mimic physiological status and ensure the oxygen supply for the whole body when operations are carried out. However, complications of these novel techniques should not be overlooked. CBP can cause or exacerbate postoperative PAH (Aubin et al., 2008). Systemic inflammatory cytokines such as IL-1β and TNF-α caused by PAH and CPBs play a pivotal role in postoperative mortality (Lei et al., 2002; Yi et al., 2015). Moreover, CPB can cause extensive lung ischemia-reperfusion (IR) injury because bronchial arterial blood flow is decreased to approximately 12% during this procedure. In addition, a huge number of free radicals are released during irrigation in CPB (Schlensak et al., 2002). With the advancements in CPB technology and materials, healthy lungs are able to tolerate CPB and usually manifest only a transient and restorable injury, while lung injuries induced by CPB in patients with PAH are always unrecoverable and even fatal. Even though there is still concern that PAH may aggravate lung IR injury, no comprehensive strategy has been proposed for protecting PAH patients from lung IR injury during CPB; thus, it is necessary to further study the mechanism of lung injury and find an ideal target for lung protection, especially for patients with PAH who will be subjected to CPB.

Despite the key fact that IL-1β and TNF-α are common pathogenic factors, and these cytokines could be induced by the activation of P2 × 7R, few studies have explored the potential role and therapeutic value of anti-P2 × 7R in PAH, especially for patients complicated with CBP. Therefore, this study aimed to explore the underlying role and mechanism of P2 × 7R in lung injuries brought by PAH with or without IR (mimic CPB) and to find a therapeutic solution for lung protection. We explored the potential pharmacological effect after application of different inhibitors of P2 × 7R and its related molecules. Eventually, we selected Brilliant Blue G (BBG) as an ideal candidate for clinical application and found that CD39 was able to replenish defected endothelial cells in PAH.

Section snippets

Animals and ethics statement

All animals used in the present study were specific pathogen–free grade and healthy Sprague-Dawley (SD) male rats (200–250 g). They were purchased from Changzhou Carvers Experimental Animal Co., Ltd (Jiang Su, China). All experiments were carried out according to the Declaration of Helsinki and the Guide for the Care and Use of Animals, and the protocols used were approved by the Animal Care and Use Committee of Central South University, China.

Establishment and verification of PAH rat model

SD rats were randomly divided into a model group

Establishment of a rat model with IR-induced lung injuries and/or MCT-PAH

Acute lung injuries were induced by IR procedure. The IR group in Fig. 1A showed that partial alveolar structure was destructed, and widening alveolar septum, inflammatory cell infiltration, lung tissues with edema, and congestion were also frequently found. Damage to tissues 12 h after the intervention were the most serious. As Fig. 1B and C show, the oxygen uptake capacity decreased significantly in rats treated with IR, and the W/D ratio indicated significant exudations in the lungs of IR

Discussion

P2 × 7R has been demonstrated to play a critical role in inflammatory diseases, including intrauterine inflammation, systemic sclerosis fibroblasts, and chronic pancreatitis (Adinolfi et al., 2017; Zhang et al., 2017; Gentile et al., 2017). In the present study, P2 × 7R was found to be involved in the development of PAH with and without IR. By applying the antagonist for P2 × 7R, BBG, the severity of PAH and IR was ameliorated through reducing the release of proinflammatory cytokines. Moreover,

Conflict of interest

None.

Acknowledgements

This work was supported by grants from the Hunan Provincial Natural Science Foundation (13JJ3016) and Hunan Science and Technology Department Sci-Tech Project (2013SK3004).

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