Effect of sevoflurane on necroptosis in isolated hippocampal neurons in rats: relationship with ryanodine receptor / 中华麻醉学杂志

ABSTRACT

Objective:To evaluate the effect of sevoflurane on necroptosis in isolated hippocampal neurons and the relationship with ryanodine receptor.Methods:Primarily cultured hippocampal neurons of fetal rats of Sprague-Dawley rats were inoculated in culture wells (100 μl/well) or culture flasks (3 ml/bottle) at a density of 5×10 5 cells/ml at 7 days of culture and divided into 3 groups ( n=24 each) using a random number table method: control group (group C), sevoflurane group (group S) and ryanodine receptor antagonist group (group R). Group C received routine culture.Ryanodine receptor antagonist Dantrolene at a final concentration of 3 μmol/L was added in group R. Thirty minutes later, the cells were placed in the incubator containing 2% sevoflurane and cultured for 5 h at 37 ℃ in S and R groups.Then cells were collected, the morphology of neurons was observed with an inverted microscope, the concentrations of free calcium ion ([Ca 2+ ] i) in cytoplasm were determined by flow cytometry, the expression of ryanodine receptor and phosphorylated MLKL protein (p-MLKL) was detected by Western blot, the expression of RIP3 was measured by immunofluorescence, and necroptosis rate was calculated. Results:Compared with group C, the [Ca 2+ ] i were significantly increased, the expression of ryanodine receptor and p-MLKL was up-regulated, and the necroptosis rate was increased in S and R groups ( P<0.05). Compared with group S, the expression of ryanodine receptor and p-MLKL was significantly down-regulated, and the [Ca 2+ ] i and necroptosis rate were decreased in group R ( P<0.05). There was no obvious abnormality in the morphology of neurons in group C. The cell body of neurons were shrunk, the processes were broken, and the network between processes was sparse in group S. The cell body was round, and the morphology was close to normal in group R. Conclusion:Sevoflurane can cause necroptosis in isolated hippocampal neurons of rats, and the mechanism is related to up-regulating the expression of ryanodine receptors and leading to calcium overload.