ATP-Sensitive Potassium Channels Mediate the Cardioprotective Effect of Panax notoginseng Saponins against Myocardial Ischaemia-Reperfusion Injury and Inflammatory Reaction

Abstract

Inflammatory response during myocardial ischemia reperfusion injury (MIRI) is essential for cardiac healing, while excessive inflammation extends the infarction and promotes adverse cardiac remodeling. Understanding the mechanism of these uncontrolled inflammatory processes has a significant impact during the MIRI therapy. Here, we found a critical role of ATP-sensitive potassium channels (K_ATP) in the inflammatory response of MIRI and its potential mechanism and explored the effects of Panax Notoginseng Saponins (PNS) during this possess. Rats underwent 40 min ischemia by occlusion of the left anterior descending (LAD) coronary artery and 60 min of reperfusion. PNS was treated at the corresponding time point before operation; 5-hydroxydecanoate (5-HD) and glybenclamide (Gly) (or Nicorandil (Nic)) were used as pharmacological blocker (or nonselective opener) of K_ATP. Cardiac function and pathomorphology were evaluated and a set of molecular signaling experiments was tested. K_ATP current density was measured by patch-clamp. Results revealed that in MIRI, PNS pretreatment restored cardiac function, reduced infarct size, and ameliorated inflammation through K_ATP. However, inhibiting K_ATP by 5-HD and Gly significantly reversed the effects, including NLRP3 inflammasome and inflammatory mediators IL-6, MPO, TNF-α, and MCP-1. Moreover, PNS inhibited the phosphorylation and nuclear translocation of NF-κB in I/R myocardium when the K_ATP was activated. Importantly, PNS promoted the expression of subunits and activation of K_ATP. The study uncovered K_ATP served as a new potential mechanism during PNS modulating MIRI-induced inflammation and promoting injured heart recovery. The manipulation of K_ATP could be a potential therapeutic approach for MIRI and other inflammatory diseases.

Figure 1

The operation process of animals and the time of each drug. Schematic illustration of the experimental process regarding I/R model protocol and drug treatment.

Figure 2

PNS pretreatment could improve hemodynamics and myocardial morphology in rats with MIRI. (a) Maximum rise rate of left ventricular pressure (+dp/dt_max). (b) Maximum fall rate of left ventricular pressure (−dp/dt_max). (c) Mean left ventricular diastolic pressure (mLVDP). (d) Mean left ventricular systolic pressure (mLVSP) (n = 12 − 15). (e–j) Myocardial histology according to H&E staining in different groups (n = 3). ^∗p < 0.05 vs. Sham, #p < 0.05 vs. I/R, ^▲p < 0.05 vs. I/R+PNS.

Figure 3

PNS reduced myocardial infarct size. The effect of with or without PNS (Sham, I/R, and I/R+PNS) on myocardial infarct size (n = 5). ^∗p < 0.05 vs. Sham, #p < 0.05 vs. I/R; Scale bar = 5 mm.

Figure 4

PNS pretreatment improved the expression and opening of K_ATP in rats with myocardial reperfusion injury. (a) Subunits of K_ATP in rats with myocardial reperfusion injury were measured using western blotting. (b–e) Quantitative analysis results for Kir6.1, Kir6.2, SUR1, and SUR2, respectively. (f–i) Quantitative analysis results for mRNA of Kir6.1, Kir6.2, SUR1, and SUR2 from real-time PCR, respectively. (j) Representative traces of whole-cell currents at voltage-clamp pulses at voltages ranging from −80 to 80 mV. (k) Pretreatment with PNS (50, 100 mg/L) on left ventricular cardiomyocytes at 0 mV after I/R (n = 5–7). ^∗p < 0.05 vs. Sham, #p < 0.05 vs. I/R, ^▲p < 0.05 vs. I/R+PNS.

Figure 5

PNS inhibited the inflammatory response of reperfused myocardium. (a–c) Protein expression levels of NLRP3 and IL-6. (d) MPO activity level in the myocardium. (e–h) mRNA and protein expression levels of TNF-α and MCP-1. (n = 5–7). ^∗p < 0.05 vs. Sham, #p < 0.05 vs. I/R, ^▲p < 0.05 vs. I/R+PNS.

Figure 6

PNS inhibited NF-κB phosphorylation and nuclear translocation in reperfused myocardia. (a) Protein levels of p-NF-κB in cytoplasm; total NF-κB level was used as a control. (b) Quantitative analysis of the ratios of p-P65/P65. (c) Immunofluorescence results indicating changes in the expression and nuclear translocation of p65 under different treatments (p65 was labelled with red fluorescence, original magnification ×400). The yellow arrow indicates that p-NF-κB was colocalized with the nucleus. (d) Quantitative analysis of the fluorescence intensity of p65 in each group (n = 5). ^∗p < 0.05, #p < 0.05 vs. I/R, ^▲p < 0.05 vs. I/R+PNS. The primers encoding rat TNF-α, MCP-1, Kir6.1, Kir6.2, SUR1, SUR2, and GAPDH are presented in the following table.