Objective: To analyze the role of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome in a therapeutic mild hypothermia (34 ℃) treated isolated rat myocardial ischemia-reperfusion model and explore its mechanism.
Methods: Sixty clean grade adult male SD rats, aged 7-10 weeks, weighing 250-300 g. Using a random number table method, the rats were divided into five groups: blank control group (group S), myocardial ischemia-reperfusion group (group IR), 34 ℃ mild hypothermia post-treated myocardial ischemia-reperfusion group (group MH), 34 ℃ mild hypothermia post-treated myocardial ischemia-reperfusion+3-TYP group (group HT), and 34 ℃ mild hypothermia post-treated myocardial ischemia-reperfusion+3-TYP+MCC950 group (group HTM), 12 rats in each group. Group S perfused the rat heart at 37 ℃ with a balanced perfusion solution for 180 minutes. Group IR received balanced perfusion of the rat heart at 37 ℃ for 30 minutes, followed by ischemia for 30 minutes and reperfusion with 37 ℃ perfusion for 120 minutes. Group MH perfused the rat heart at 37 ℃ for 30 minutes, followed by ischemia for 30 minutes and reperfusion with 34 ℃ perfusion solution for 120 minutes. Group HT perfused the hearts of rats at 37 ℃ for 30 minutes, followed by ischemia for 30 minutes, silent mating type information regulation 2 homolog 3 (sirt3) inhibitor 3-TYP was added to the perfusate, and then perfused at 34 ℃ for 120 minutes. Group HTM perfused the hearts of rats at 37 ℃ for 30 minutes, followed by ischemia for 30 minutes, sirt3 inhibitor 3-TYP and NLRP3 inhibitor MCC950 were added to the perfusate, and then perfused at 34 ℃ for 120 minutes. The isolated heart was obtained 120 minutes after reperfusion, and the concentrations of IL-6 and IL-1β in the perfused cardiac fluid was measured using ELISA method, Western blot method for detecting the relative content of NLRP3 and sirt3 proteins in myocardial tissue, 1% triphenyl tetrazolium chloride staining for calculating myocardial infarction area, and HE staining for observing myocardial pathological changes.
Results: Compared with group S, HR were significantly slowed down, LVSP, ±dp/dtmax were significantly decreased, and LVEDP were significantly increased 30, 60, 90, and 120 minutes after reperfusion, the concentrations of IL-6 and IL-1β in cardiac fluid leakage, and the percentage of myocardial infarction area were significantly increased in groups IR, MH, HT, and HTM (P < 0.05), the content of sirt3 protein in myocardial tissue were significantly reduced, while the content of NLRP3 protein were significantly increased in groups IR, HT, and HTM (P < 0.05), the contents of sirt3 and NLRP3 protein in the myocardial tissue were significantly increased in group MH (P < 0.05). Compared with group IR, HR were significantly increased, LVSP, ±dp/dtmax were significantly increased, and LVEDP were significantly decreased 30, 60, 90, and 120 minutes after reperfusion, the concentrations of IL-6 and IL-1β in cardiac fluid leakage and the percentage of myocardial infarction area were significantly decreased in groups MH and HTM (P < 0.05), the content of sirt3 protein in myocardial tissue was significantly increased, while the content of NLRP3 protein was significantly decreased in group MH (P < 0.05), the content of NLRP3 protein in myocardial tissue was significantly reduced in group HTM (P < 0.05). Compared with group MH, HR were significantly slowed down, LVSP, ±dp/dtmax were significantly decreased, and LVEDP were significantly increased 30, 60, 90, and 120 minutes after reperfusion, the concentrations of IL-6 and IL-1β in cardiac fluid leakage, the percentage of myocardial infarction area, and the content of NLRP3 protein in myocardial tissue were significantly increased in group HT (P < 0.05), the content of sirt3 protein in myocardial tissue was significantly reduced in groups HT and HTM (P < 0.05). Compared with group HT, HR were significantly increased, LVSP, ±dp/dtmax were significantly increased, and LVEDP were significantly decreased 30, 60, 90, and 120 minutes after reperfusion, the concentrations of IL-6 and IL-1β in cardiac fluid leakage, the percentage of myocardial infarction area, and the content of NLRP3 protein in myocardial tissue were significantly reduced in group HTM (P < 0.05).
Conclusion: Therapeutic mild hypothermia (34 ℃) can improve hemodynamic parameters of isolated hearts and reduce the concentrations of IL-6 and IL-1β, NLRP3 protein content in myocardial tissue, percentage of myocardial infarction area, improve myocardial pathological changes, and reduce myocardial ischemia-reperfusion injury in rats, the mechanism may be related to the mitochondrial mediated sirt3 pathway inhibiting the high expression of inflammatory corpuscle NLRP3. |
