ABSTRACT
Objective:To evaluate the role of bilateral superior cervical sympathetic ganglia (SCG) in myocardial ischemia-reperfusion (I/R) injury in mice and the relationship with NOD-like receptor protein 3 (NLRP3) inflammasomes.Methods:Thirty-two healthy SPF male C57BL mice, aged 8-10 weeks, weighing 25-30 g, were divided into 4 groups ( n=8 each) by the random number table method: sham operation group (NS group), myocardial I/R group (NIR group), bilateral SCG excision group (SCGx group) and bilateral SCG excision + myocardial I/R group (SCGx+ IR group). The myocardial I/R injury model was prepared by ligating the anterior descending branch of the left coronary artery for 30 min followed by 24 h reperfusion in isoflurane-anesthetized mice. Bilateral superior cervical sympathectomy was performed at 3 days before reperfusion. Blood samples were collected from the inferior vena cava at 24 h of reperfusion for examination of pathological changes (by HE and WGA staining) and for measurement of serum creatine kinase isoenzymes (CK-MB) activity, cardiac troponin I (cTnI) concentration, norepinephrine (NE) concentration and lactic dehydrogenase (LDH) activity (by enzyme-linked immunosorbent assay), superoxide dismutase (SOD) activity (by colorimetric method), myocardial reactive oxygen species (ROS) level (by DHE method), myocardial infarct size(by TTC method), and expression of interleukin-1beta (IL-1β), IL-6, IL-10, tumor necrosis factor-alpha (TNF-α), NLRP3 mRNA (by quantitativepolymerase chain reaction ), and expression of tyrosine hydroxylase (TH), IL-1β, TNF-α, NLRP3, atrial natriuretic peptide (ANP)and brain natriuretic peptide (BNP) (by Western blot). Results:Compared with NS group, the NE concentration was significantly decreased, and TH expression was down-regulated in SCGx group, and the serum CK-MB activity, concentrations of cTnI and NE, LDH activity and myocardial ROS level were significantly increased, SOD activity was decreased, the expression of IL-1β, TNF-α, NLRP3, ANP and BNP was up-regulated, and the expression of IL-1β, IL-6, TNF-α and NLPR3 mRNA was up-regulated in NIR group ( P<0.05). Compared with SCGx group, the serum CK-MB activity, concentrations of cTnI and NE, LDH activity and myocardial ROS levels were significamtly increased, SOD activity was decreased, the expression of IL-1β, TNF-α, NLRP3, ANP and BNP was up-regulated, and the expression of IL-1β, IL-6, TNF-α and NLPR3 mRNA was up-regulated in SCGx+ NIR group ( P<0.05). Compared with NIR group, the serum CK-MB activity, cTnI concentration, LDH activity and myocardial ROS level were significantly decreased, SOD activity was increased, the expression of IL-1β, TNF-α, NLRP3, ANP and BNP was down-regulated, the expression of IL-1β, IL-6, TNF-α and NLPR3 mRNA was down-regulated, and myocardial infarct size was decreased in SCGx+ NIR group ( P<0.05). Conclusions:The mechanism by which bilateral SCG excision attenuates myocardial I/R injury is associated with decreased NLRP3 inflammatory inflammasome activation and inhibition of inflammatory responses in mice.
ABSTRACT
Objective:To evaluate the role of ferroptosis in hypoxia-reoxygenation (H/R) injury in cardiomyocytes cultured in high-fat high-glucose (HFHG) medium.Methods:Cardiomyocytes H9c2 cells were commonly cultured and divided into 3 groups ( n=20 each) using a random number table method: control group (C group), HFHG-H/R group and Ferrostatin-1 (Fer-1) plus HFHG-H/R group (Fer-1+ HFHG+ H/R group). H9c2 cells were cultured in a HFHG medium for 12 h and then exposed to 1%O 2-5%CO 2-94%N 2 for 4 h, followed by 2 h reoxygenation in a cell incubator.Fer-1 at a final concentration of 10 μmol/L was added while the cells were cultured in the HFHG medium in group Fer-1+ HFHG+ H/R.At 2 h of reoxygenation, the cell viability was measured using CCK-8 assay, the activity of lactate dehydrogenase (LDH) in the supernatant was measured using 2, 4-dinitrophenylhydrazine color method, the activity of reactive oxygen species (ROS) was measured by fluorescent probe DCFH-DA flow cytometry, and the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), nuclear receptor coactivator 4 (NCOA4), and glutathione peroxidase 4 (GPX4) was detected by Western blot. Results:Compared with group C, the cell viability was significantly decreased, the activities of LDH release and ROS were increased, and the expression of ACSL4 and NCOA4 was up-regulated ( P<0.05), and no significant change was found in the expression of GPX4 in group HFHG+ H/R ( P>0.05). Compared with group HFHG+ H/R, the cell activity was significantly increased, the activities of LDH and ROS were decreased, and the expression of ACSL4 and NCOA4 was down-regulated ( P<0.05), and no significant change was found in the expression of GPX4 in Fer-1+ HFHG+ H/R group ( P>0.05). Conclusions:Ferroptosis is involved in the process of H/R injury in cardiomyocytes cultured in HFHG medium.