Sevoflurane Alleviates Myocardial Ischemia Reperfusion Injury by Inhibiting P2X7-NLRP3 Mediated Pyroptosis

Abstract

Myocardial ischemia is common in aging population. This study investigates the protective effect of Sevoflurane on myocardial ischemia reperfusion injury (MIRI) and its underlying mechanism. A total of 87 patients with a history of myocardial ischemia who underwent abdominal surgery with Sevoflurane general anesthesia were recruited in the study. The clinical data, blood pressure, heart rate, pressure-rate quotient (PRQ) and rate-pressure product (RPP) were recorded. Serum samples were collected and heart-type fatty acid binding protein (H-FABP), ischemia modified albumin (IMA), interleukin-1β (IL-1β), and interleukin-18 (IL-18) were measured to observe whether Sevoflurane anesthesia had protective effect on myocardium. In addition, MIRI rats and hypoxia/reoxygenation (H/R) injury cell model was established using neonatal rat ventricular myocytes (NRVM). Rats or NRVM were pretreated with sevoflurane for 45min before hypoxia. The mRNA expression of purinergic receptor-7 (P2X7) and NLR family pyrin domain containing 3(NLRP3) were examined. The protein expression of P2X7, NLRP3, apoptosis-associated speck-like protein (ASC), cysteine aspartic acid specific protease-1(Caspase-1), Gasdermin-D (GSDMD), Bcl-2 Associated X Protein (Bax), B-cell lymphoma-2 (Bcl-2) in myocardial tissue and cells were evaluated. The serum contents of IL-1β, IL-18, Malondialdehyde (MDA), Superoxide dismutase (SOD), Lactate dehydrogenase (LDH), Creatine kinase (CK), and Creatine kinase isoenzymes (CK-MB) were measured. The cellular localization and fluorescence intensity of NLRP3 and ASC in cells were detected. It was found that the secretion of IL-1β and IL-18 decreased in the patients. After I45 min/R3h in SD rats and H3h/R1h in NRVM, the protein expressions of P2X7, NLRP3, ASC, Caspase-1 and GSDMD were increased, the release of IL-1β, IL-18, CK, CK-MB, LDH and MDA were increased, and SOD activity was decreased. Sevoflurane treatment inhibited the high expression of P2X7, NLRP3, ASC, Caspase-1 and GSDMD, inhibited the release of LDH, CK,CK-MB and MDA in cells, and improved the activity of SOD, indicating that Sevoflurane alleviated the damage of MIRI of rats and H/R of NRVM, and had myocardial protective effect. Taken together, our study suggests that Sevoflurane inhibited the expression of IL-1β, IL-18 and GSDMD by inhibiting the P2X7-NLRP3 signaling pathway. It reduced the H/R injury of cardiomyocytes and protected the cardiac function by regulating inflammatory reaction and pyroptosis.

Keywords: NLRP3; P2X7; hypoxia and reoxygenation; myocardial ischemia reperfusion; pyroptosis; sevoflurane.

FIGURE 1

Rats in each group.In the sham group, the silk suture was passed under LAD without ligation. The other groups were ligated with LAD for 45 min and then reperfused for 180 min. Sevoflurane groups with different concentration of sevoflurane was inhaled with corresponding concentrations for 45 min before ischemia.

FIGURE 2

Changes in vital signs and inflammatory factors in these three groups of patients (A) different general anesthetics on the changes of heart rate, blood pressure, RPP and PRQ in peri-anesthesia in the three groups (B) Changes of IL-18 at different time points at pre- and post-anesthesia.

FIGURE 3

Sevoflurane alleviates MIRI in rats (A) Typical electrocardiogram of normal perfusion, ligation of LAD and reperfusion (B) Cardiac ultrasonography in rats of normal perfusion, MIRI and treatment with different concentrations of sevoflurane (C) Histogram of rat cardiac ultrasonography (D) Myocardial infarction area and histogram of rats treated with different concentrations of sevoflurane (E) Effects of Sevoflurane at different concentrations on the release of LDH, CK and CK-MB in myocardial tissue after MIRI. Data are expressed relative to the mean value of sham group and were presented as mean ± SD (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 vs respective controls.

FIGURE 4

Sevoflurane alleviates inflammatory cell infiltration in MIRI rats (A) The expression of CD11b in rat myocardial tissue determined by immunofluorescence staining (n = 6,Scale bars 20 um) (B) Effects of Sevoflurane at different concentrations on the release of IL-1β and IL-18 in myocardial tissue after MIRI. All values are expressed as means ± SD (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 vs respective controls (C) Expression of NLRP3 in rat myocardial tissue by immunohistochemical staining of normal perfusion, MIRI and sevoflurane treatment with different concentrations in rats (n = 6,Scale bars 20 um) (D) The Expression of GSDMD in rat myocardial tissue by immunohistochemical staining of normal perfusion, MIRI and sevoflurane treatment with different concentrations in rats (n = 6,Scale bars 20 um).Data are expressed relative to the mean value for sham group and were presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs respective controls.

FIGURE 5

Sevoflurane alleviates oxidative stress and pyroptosis in MIRI rats (A) Effects of different concentrations of Sevoflurane on the expression of MDA and SOD in myocardial tissue after ischemia reperfusion. All values are expressed as means ± SD (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 vs respective controls (B) ROS Fluorescent Probe-DHE. Sevoflurane increased ATP content, stabilized membrane potential, and reduced ROS production (n = 6,Scale bars 50 um) (C) The Expression of apoptosis-related proteins Bax and Bcl-2.Data are expressed relative to the mean value for sham group and were presented as mean ± SD (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 vs respective controls (D) Effects of different concentrations of Sevoflurane on the TUNEL staining in myocardial tissue after MIRI. All values are expressed as means ± SD (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 vs respective controls. Scale bars 50 um (E) Histogram of TUNEL and the protein expression of P2X7, NLRP3, ASC, Caspase-1 and GSDMD (F) Immunoblotting measured the levels of P2X7, NLRP3, ASC, Caspase-1 and GSDMD protein expression. Data are expressed relative to the mean value for control group and were presented as mean ± SD (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 vs respective controls.

FIGURE 6

Sevoflurane inhibited the activation of inflammasome and pyroptosis induced by hypoxia and reoxygenation of NRVM (A) The expression of P2X7 and NLRP3 mRNA detected by real-time PCR. Data are expressed relative to the mean value for control group and were presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs respective controls (B) Effects of different concentrations of Sevoflurane on the content of MDA and SOD. All values are expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs respective controls (C) Effects of Sevoflurane at different concentrations on the release of IL-1β, IL-18 and LDH in NRVM after H/R. All values are expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs respective controls (D) Effects of Sevoflurane at different concentrations on the protein expression of P2X7,NLRP3,ASC,Caspase-1 and GSDMD in NRVM after H/R by immunoblotting analysis (E) Histogram of the protein expression of P2X7,NLRP3,ASC,Caspase-1 and GSDMD. Data are expressed relative to the mean value for control group and were presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs respective controls (F) Colocalization of NLRP3 and ASC in NRVM was observed by the laser scanning confocal microscope(n = 3,Scale bars five um).

FIGURE 7

Effects of NLRP3 inhibitor and agonist on NRVM after Sevoflurane pretreatment (A) Effects of NLRP3 inhibitor and agonist on the protein expression of P2X7, NLRP3, Caspase-1 and GSDMD in NRVM after Sevoflurane pretreatment by immunoblotting. Data are expressed relative to the mean value for control group and were presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs respective controls (B) Effects of NLRP3 agonists and inhibitors on the levels of IL-1β, IL-18 and LDH after H/R of cardiomyocytes. All values are expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs respective controls.