Electroacupuncture preconditioning attenuates acute myocardial ischemia injury through inhibiting NLRP3 inflammasome activation in mice

Abstract

Aims

Electro-acupuncture pretreatment (EAP) plays a protective role in myocardial ischemia (MI) injury. However, the underlying mechanism remains unclear. A growing body of evidence suggests postinfarction inflammatory response directly affects the remodeling of ventricular function. The purpose of this study was to investigate whether EAP alleviates MI through NLRP3 inflammasome inhibition.

Materials and methods

We constructed an AMI model by ligating the left anterior descending (LAD) coronary artery after 3 days of EAP with C57BL/6 mice. Echocardiography and TTC staining were employed to evaluate cardiac function and infarct size after 24 h of ischemia. HE staining and immunohistochemistry were employed to determine inflammatory level. Then, inflammasome activation was detected by western blotting, and macrophage polarization and neutrophil infiltration were observed by flow cytometry.

Key findings

Our preliminary findings showed that EAP reduced the infarct area and increased fractional shortening (FS) and ejection fraction (EF) and decreased the degree of inflammation after AMI injury. Meanwhile, EAP inhibited the expression of NLRP3, cleaved caspase-1 and IL-1β in ischemia myocardial tissue, companied by inhibiting the expression of F4/80⁺, CD11b⁺, CD206^low macrophages and activated M2 macrophage, and decreasing Ly-6G⁺CD11b⁺ neutrophils in ischemia myocardial and spleen tissue.

Significance

EAP inhibits the activation of NLRP3 inflammasome, promotes M2 polarization of macrophages and reduces the recruitment of neutrophils in damaged myocardium, thereby decreases the infarct size and improves the cardiac function.

Introduction

Acute myocardial ischemia (AMI) is caused by persistent ischemia and hypoxia of the coronary arteries, which can incur pathological damage such as myocardial infarction and even heart failure, and its morbidity and mortality increase year by year [1]. The damaged myocardium is eventually replaced by scar tissue because of the negligible endogenous regenerative capacity of the heart after myocardial ischemia (MI) [2]. Left ventricle (LV) poor remodeling after MI will eventually lead to heart failure [3]. A growing body of evidence suggests that the aseptic inflammatory response during AMI plays a crucial role in scar formation and myocardial remodeling in damaged myocardium [4,5]. Premature inflammation may increase matrix degradation leading to heart rupture [4]. Simultaneously, accentuation, prolongation, or expansion of the postinfarction inflammatory response results in worse remodeling and dysfunction following MI [6]. Therefore, moderate regulation of inflammatory response after AMI is important for LV myocardial remodeling to prevent the heart failure [7,8], and targeting on the inhibition of overwhelmingly induced inflammatory reaction might be a potential and promising therapy for treating MI.

It is acknowledged that such inflammation is initiated by molecular danger signals (damage-associated molecular patterns, DAMPs) released by necrotic myocardial cells and sensed by pattern recognition receptors including Toll-like receptors (TLR) and NOD-like receptors (NLR) families, which promote intracellular signaling dependent on the formation of the inflammasome [9]. An accumulating body of evidence indicates the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome, results in increased inflammation and cardiac dysfunction and remodeling, aggravates AMI injury in MI animals. In contrast, deficiency of the NLRP3 inflammasome components reduces inflammation and promote cardioprotection [10]. NLRP3 inflammasome is a supramolecular protein complexes that activates caspase-1 and regulates interleukin (IL)-1β maturation, as well as triggering inflammatory cell death and pyroptosis [10,11]. It is reported that the NLRP3 inflammasome can recognize certain non-microbial danger signals and trigger a sterile inflammatory response in many disease conditions [12]. In particular, NLRP3 inflammasome mainly expressed in immune cells (e.g. macrophage and neutrophil) are responsible for detecting and eliminating pathogens or pathogen-associated molecules as well as DAMPs [13,14]. In short, the current evidence supports the therapeutic value of NLRP3 inflammasome-targeted strategies in experimental models of AMI, involving in process of cardiac inflammation and remodeling after MI [10,11].

Acupuncture, the most well-known complementary and alternative medical approach, has been practiced in China for over two thousand years as an effective approach to improve the symptoms of angina and palpitation [[15], [16], [17]]. Electro-acupuncture (EA), a modified therapy method of acupuncture stimulated with a low-voltage electrical current [18], has been widely used as a substitute of classical needle acupuncture [19,20]. More and more studies have shown that EAP has a positive protective effect against myocardial injury in patients [21] and animals [22,23]. Our previous finding also indicated that EAP of Neiguan (PC6) for three days effectively reduced the area of MI [24]. Meanwhile, EAP protects the myocardium from myocardial injury through the activation of endogenous antiapoptotic signaling and reducing the level of serum cardiac troponin I [25,26]. However, the mechanism remains unclear. The therapeutic effect of EA on inflammatory pain has been well recognized clinically [27], increasing evidence also shown that EA can regulate inflammatory response by inhibiting NLRP3 inflammasome activation and promoting pro-inflammatory cytokines production [28]. Therefore, in this study, we investigated whether EAP could alleviates MI injury through NLRP3 inflammasome inhibition.