Role of mitochondrial permeability transition pore in cholinergic anti-inflammatory pathway in cardiomyocytes

Abstract

Objective To investigate the role of mitochondrial permeability transition pore (mPTP) in cholinergic anti-inflammatory pathway in cardiomyocytes. Methods After being cultured for 72 h, cardiomyocytes of neonatal Sprague-Dawley rats were randomly divided into 5 groups (n=36 each) using a random number table: control group (group C), anoxia/reoxygenation (A/R) group, specific α7 nicotinic acetylcholine receptor agonist PNU282987 group (PNU282987 group), mPTP opener atractyloside group(ATR group) and PNU282987+ ATR group (P+ A group). The cells were incubated in serum-free and low-glucose DMEM culture medium which was placed in an anaerobic box (O2 < 0.1%) lasting for 6 h, the culture medium was then replaced with high-glucose DMEM culture medium supplemented with 10% fetal bovine serum, and the cells were then re-incubated and exposed to 5% CO2 in normoxia in an incubator at 37 ℃ for 6 h to establish a model of A/R injury. In P and ATR groups, PNU282987 (final concentration 30 μmol/L) and atractyloside (final concentration 20 μmol/L) were added to the culture medium immediately after onset of reoxygenation, respectively, and in P+ A group, the combination of the two drugs previously mentioned was added instead.At 6 h of reoxygenation, lactate dehydrogenase (LDH) release rate, apoptosis, expression of NF-κBp65 and phosphorylated NF-κB p65 Ser536 (p-NF-κB p65 Ser536) in cardiomyocytes, concentrations of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the supernatant, and mitochondrial membrane potential (△Ψm) were detected. Results Compared to group C, the LDH release rate, early and late apoptosis and concentrations of IL-6 and TNF-α in the supernatant were significantly increased, the expression of NF-κB p65 and p-NF-κB p65 Ser536 was up-regulated, and △Ψm was decreased in A/R, P, ATR and P+ A groups.Compared to A/R group, the LDH release rate, early apoptosis and concentrations of IL-6 and TNF-α in the supernatant were significantly decreased, the expression of p-NF-κB p65 Ser536 was down-regulated, and △Ψm was increased in group P, the LDH release rate, early apoptosis and concentrations of IL-6 and TNF-α in the supernatant were increased, the expression of p-NF-κB p65 Ser536 was up-regulated, and △Ψm was decreased in ATR group, and the early apoptosis and concentrations of IL-6 and TNF-α in the supernatant were decreased, the expression of p-NF-κB p65 Ser536 was down-regulated, and there was no significant change in LDH release rate, late apoptosis and Δψ in P+ A group.Compared to group P, the LDH release rate, late apoptosis and concentrations of IL-6 and TNF-α in the supernatant were significantly increased, the expression of p-NF-κB p65 Ser536 was up-regulated, and △Ψm was decreased in P+ A group. Conclusion Cholinergic anti-inflammatory pathway exerts anti-inflammatory effect through inhibiting mPTP opening, thus reducing A/R injury to cardiomyocytes. Key words: Mitochondrial membrane transport protein; Acetylcholine; Vagus nerve; Myocardial reperfusion injury; Myocytes, cardiac