Effect of carbon monoxide postconditioning on pyroptosis induced by oxygen-glucose deprivation and restoration in rat hippocampal neurons: the relationship with mPTP/ROS signaling pathway

Abstract

Objective To evaluate the effect of carbon monoxide(CO) postconditioning on pyroptosis induced by oxygen-glucose deprivation and restoration(OGD/R) in rat hippocampal neurons and the relationship with mitochondrial permeability transition pore(mPTP)/reactive oxygen species(ROS) signaling pathway. Methods Primary hippocampal neurons were cultured in vitro, seed in 6-well or 96-well plates, and divided into 5 groups(n=24 each) using a random number table method: control group(C group), OGD/R group, CO postconditioning group(CO group), specific mPTP opener atractyloside plus CO postconditioning group(ACO group), and specific ROS inducer antimycin A plus CO postconditioning group(KCO group). Neurons were subjected to O2-glucose deprivation(OGD) for 16 h followed by restoration of O2 -glucose supply for 24 h to establish the model of OGD/R injury. In group CO, neurons were exposed to 2% CO-5% CO2 for 3 h at 37 ℃ starting from the end of OGD, followed by normal culture for 21 h. In ACO and KCO groups, atractyloside 20 μmol/L and antimycin A 50 μmol/L were added at the end of OGD, respectively, and the other treatments were similar to those previously described in group CO. Neuronal pyroptosis rate was determined using double immunofluorescent staining cleaved caspase-1-AlexaFluor 568/DAPI after the end of treatments in each group. The neuronal survival rate was determined by MTT, opening of mPTP by Calcein-AM fluorescence, ROS content by DCFH-DA, and expression of interleukin-1beta(IL-1β) and IL-18 by Western blot. Results Compared with C group, neuronal pyroptosis rate, ROS content and opening of mPTP were significantly increased, the neuronal survival rate was decreased, and the expression of IL-1β and IL-18 was up-regulated in the other groups(P<0.05). Compared with OGD/R group, neuronal pyroptosis rate, ROS content and opening of mPTP were significantly decreased, the neuronal survival rate was increased, and the expression of IL-1β and IL-18 was down-regulated in CO, ACO and KCO groups(P<0.05). Compared with CO group, neuronal pyroptosis rate and ROS content were significantly increased, the neuronal survival rate was decreased, and the expression of IL-1β and IL-18 was up-regulated in ACO and KCO groups, and opening of mPTP was significantly inctreased in ACO group(P<0.05). Conclusion CO postconditioning can inhibit OGD/R-induced pyroptosis in rat hippocampal neurons, and the mechanism is related to inhibiting mPTP/ROS signaling pathway. Key words: Carbon monoxide; Neurons; Cell death; Reperfusion injury; Reactive oxygen species; Mitochondrial membrane transport proteins