一氧化碳后处理对氧糖剥夺/复氧复糖诱发大鼠神经元焦亡的影响:与mPTP/ROS信号通路的关系

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

目的评价一氧化碳(CO)对氧糖剥夺/复氧复糖诱发大鼠神经元焦亡的影响及其与线粒体通透性转换孔/活性氧(mPTP/ROS)信号通路的关系。方法体外培养大鼠原代海马神经元,接种于6孔板或96孔板,采用随机数字表法分为5组(n=24):对照组(C组)、氧糖剥夺/复氧复糖组(OGD/R组)、CO后处理组(CO组)、mPTP特异性开放剂苍术苷+CO后处理组(ACO组)、ROS特异性诱导剂抗霉素A+CO后处理组(KCO组)。采用无糖培养基低氧(1%O2)孵育16h再正常培养24h的方法制备氧糖剥夺/复氧复糖损伤模型。CO组于低氧处理结束即刻在2%CO-5%CO2混合气体中37℃培养3h,随后正常培养21h。ACO组和KCO组于低氧处理结束分别加入苍术苷20μmol/L和抗霉素A50μmol/L,其余处理同CO组。各组处理结束后,采用免疫荧光双染cleavedcaspase-1-AlexaFluor568/DAPI法确定神经元焦亡率。采用四甲基偶氮唑法确定神经元存活率,采用钙黄绿素法测定mPTP开放程度,采用二氯荧光黄双乙酸盐法测定ROS含量,采用Westernblot法检测IL-1β、IL-18的表达。结果与C组比较,其余各组海马神经元焦亡率、ROS含量和mPTP开放程度升高,存活率降低,IL-1β和IL-18表达上调(P<0.05);与OGD/R组比较,CO组、ACO组和KCO组海马神经元焦亡率、ROS含量和mPTP开放程度降低,存活率升高,IL-1β和IL-18表达下调(P<0.05);与CO组比较,ACO组和KCO组海马神经元焦亡率和ROS含量升高,存活率降低,IL-1β和IL-18表达上调,ACO组mPTP开放程度升高(P<0.05)。结论CO后处理可抑制氧糖剥夺/复氧复糖诱发大鼠神经元焦亡,机制与抑制mPTP/ROS信号通路有关。

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.