Activation of autophagy inhibits nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome activation and attenuates myocardial ischemia-reperfusion injury in diabetic rats

Abstract

Aims/introduction: Diabetic hearts are more vulnerable to ischemia-reperfusion injury (I/RI). The activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome can mediate the inflammatory process, and hence might contribute to myocardial I/RI. Activation of autophagy can eliminate excess reactive oxygen species and alleviate myocardial I/RI in diabetes. The present study aimed to investigate whether the activation of autophagy can alleviate diabetic myocardial I/RI through inhibition of NLRP3 inflammasome activation.

Materials and methods: A dose of 65 mg/kg streptozotocin was given by tail vein injection to establish a type 1 diabetes model in the rats. The left anterior descending coronary artery was ligated for 30 min followed by reperfusion for 2 h to establish a myocardial I/RI model. H9C2 cardiomyocytes were exposed to high glucose (33 mmol/L) and subjected to hypoxia-reoxygenation (6 h hypoxia followed by 4 h reoxygenation).

Results: The diabetic rats showed significant inhibition of cardiac autophagy (decreased LC3-II/I and increased p62) that was concomitant with increased activation of NLRP3 inflammasome (increased NLRP3, apoptosis-related spots protein cleaved caspase-1, interleukin-18, interleukin-1β) and more severe myocardial I/RI (elevated creatine kinase myocardial band, lactate dehydrogenase and larger infarct size). However, administration of rapamycin, an inhibitor of the autophagy, to activate autophagy resulted in the inhibition of NLRP3 inflammasome, and finally alleviated myocardial I/RI. In vitro, high glucose inhibited autophagy, while activating NLRP3 inflammasome in H9C2 cardiomyocytes and aggravating hypoxia-reoxygenation injury, but rapamycin reversed these adverse effects of high glucose.

Conclusion: Activation of autophagy can suppress the formation of NLRP3 inflammasome, which in turn attenuates myocardial ischemia-reperfusion injury in diabetic rats.

Keywords: Diabetic myocardium; Myocardial ischemia-reperfusion injury; Nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome.

Figure 1

The (a) myocardial infarct size, (b) lactate dehydrogenase (LDH) and (c) creatine kinase myocardial band (CK‐MB) release in diabetic rats (DM) after ischemia‐reperfusion injury (I/RI) were significantly higher than those in non‐diabetic rats (Ctrl). Compared with the non‐diabetic group, the diabetic group had significant inhibition of autophagy, shown as (d,e) decreased LC3‐II/I and (d,f) increased p62, and activation of NLRP3 inflammasome, (g) shown as increased nucleotide‐binding oligomerization domain‐like receptor protein 3 (NLRP3), (h) apoptosis‐related spots protein (ASC), (i) caspase‐1 p20, (k) interleukin (IL)‐1β and (l) IL‐18, in the myocardium. Also, the diabetic group had more severe myocardial I/RI (elevated creatine kinase myocardial band, LDH and larger infarct size). Data are shown as the mean ± standard error of the mean. *P < 0.05 versus Ctrl sham; ^# P < 0.05 versus DM sham I/RI; and ^& P < 0.05 versus Ctrl I/RI; n = 6/group.

Figure 2

Administration of rapamycin (Rapa) activated autophagy, inhibited domain‐like receptor protein 3 (NLRP3) inflammasome and finally alleviated myocardial after ischemia‐reperfusion injury (I/RI). (a,g) Western blot was used to analyze the expression of (b) LC3‐II/, (c) p62, (d) NLRP3, (e) apoptosis‐related spots protein (ASC), (f) caspase‐1 p20, (h) interleukin (IL)‐1β and (i) IL‐18. (j) Representative images of myocardial infarct size determined by tetrazolium/formazan test and Evans blue staining; post‐ischemic infarct size expressed as the percentage of infarct size (IS) to the area at risk (AAR). (k) Serum lactate dehydrogenase (LDH) level. (l) Serum creatine kinase myocardial band (CK‐MB) level. Data are shown as the mean ± standard error of the mean. *P < 0.05 versus control (Ctrl) sham; ^# P < 0.05 versus Ctrl sham I/R); ^@ P < 0.05 versus diabetes (DM) sham; ^$ P < 0.05 versus Ctrl I/RI + Rapa; ^& P < 0.05 versus DM I/RI; n = 6/group.

Figure 3

H9C2 cells cultured in high glucose for 24 h inhibited autophagy, as evidenced by (b) reduced LC3‐II/I, (c) increased p62 and (d) significantly enhanced nucleotide‐binding oligomerization domain‐like receptor protein 3 (NLRP3) inflammasome after hypoxia–reoxygenation, (e) caspase‐1 p20 and (f) apoptosis‐related spots protein (ASC), and (g,h) increased the expression of inflammatory factors interleukin (IL)‐1β and IL‐18. Rapamycin (Rapa) can activate autophagy and inhibit the further activation of NLRP3 inflammasome induced by hypoxia–reoxygenation. Data are shown as the mean ± standard error of the mean. *P < 0.05 versus normal glucose (NG, 5.5 mmol/L); ^# P < 0.05 versus high glucose (HG, 33 mmol/L); ^& P < 0.05 versus HG + 50 nmol/L Rapa; n = 5/group.

Figure 4

Activated autophagy can attenuate high glucose and aggravate hypoxia–reoxygenation (HR) injury in H9C2 cardiomyocytes by inhibiting nucleotide‐binding oligomerization domain‐like receptor protein 3 (NLRP3) inflammasome. (a) Western blot was used to analyze the expression of (b) LC3‐II/I, (c) p62, (d) NLRP3, (e) ASC, (f) caspase‐1 p20, (g) interleukin (IL)‐1β and (h) IL‐18. Data are shown as the mean ± standard error of the mean. *P < 0.05 versus normal glucose (NG, 5.5 mmol/L); ^& P < 0.05 versus high glucose (HG, 33 mmol/L); ^# P < 0.05 versus NG + HR; ^@ P < 0.05 versus HG + HR, P < 0.05; n = 5/group.

Figure 5

High glucose (HG, 33 mmol/L) can (a) increase lactate dehydrogenase (LDH) release, (b) decrease cell viability and (c,d) increase cell death, as evidenced by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)‐positive apoptotic cells, in H9C2 cells induced by hypoxia–reoxygenation (HR). Rapamycin (Rapa) can alleviate the damage of H9C2 cells induced by hypoxia and reoxygenation. Data are shown as the mean ± standard error of the mean. *P < 0.05 versus normal glucose （NG，5.5 mmol/L); ^& P < 0.05 versus HG; ^# P < 0.05 versus NG + HR; ^@ P < 0.05 versus HG + HR; n = 5/group.