mito-KATP在七氟醚后处理抑制氧糖剥夺-复糖复氧原代大鼠心肌细胞焦亡中的作用

Role of mito-KATP channels in sevoflurane postconditioning-induced inhibition of oxygen-glucose deprivation and restoration-induced pyroptosis in primary rat cardiomyocytes

目的 评价线粒体ATP敏感性钾通道（mito-KATP）在七氟醚后处理抑制氧糖剥夺-复糖复氧原代大鼠心肌细胞焦亡中的作用.方法 出生48 h内的SD大鼠,体外培养大鼠心室肌细胞并接种于6孔L板（2 cm）培养皿或96孔板,采用随机数字表法分为6组（n=15）：对照组（C组）、氧糖剥夺-复糖复氧组（O组）、七氟醚后处理组（Sev组）、七氟醚后处理＋5-羟葵酸组（SH组）、5-羟葵酸组（H组）和氧糖剥夺-复糖复氧＋5-羟葵酸组（HO组）.心肌细胞氧糖剥夺4h,复糖复氧24 h进行氧糖剥夺-复糖复氧;复糖复氧后将培养基在2％七氟醚环境下处理1h进行七氟醚后处理;氧糖剥夺前1h培养皿中加入mito-KATP阻断剂5-羟葵酸100 μmol/L;C组心肌细胞正常培养.于复糖复氧24 h时收集心肌细胞,采用流式细胞学AlexaFour488（caspase-1 FLICA标记）和TMR red（DNA标记）双标记法确定心肌细胞焦亡率,采用MTT法确定细胞存活率,采用二氯荧光黄双乙酸盐法测定线粒体活性氧（ROS）含量,采用荧光探针JC-1法检测线粒体膜电位（MMP）,采用Western blot法测定IL-1β表达.结果 与C组比较,O组心肌细胞焦亡率和ROS含量升高,存活率和MMP降低,IL-1β表达上调（P〈0.05）;与0组比较,Sev组心肌细胞焦亡率和ROS含量下降,存活率和MMP升高,IL-1β表达下调（P〈0.05）;与Sev组比较,SH组心肌细胞焦亡率和ROS含量升高,存活率和MMP降低,IL-1β表达上调（P〈0.05）;与SH组比较,HO组心肌细胞焦亡率和ROS含量升高,存活率和MMP降低,IL-1β表达上调（P〈0.05）.结论 七氟醚后处理抑制氧糖剥夺-复糖复氧原代大鼠心肌细胞焦亡的机制可能与增加mito-KATP开放相关.

Objective To evaluate the role of mitochondrial ATP-seusitive potassium （mito-KATP） channels in sevoflurane postconditioning-induced inhibition of oxygen-glucose dcprivation and restoration （OGD/R）-induced pyroptosis in primary rat cardiomyocytes.Methods Cardiomyocytes of newborn Sprague-Dawley rats （〈48 h after birth） were cultured in vitro and seeded in 6-well dishes （2 cm in diameter）or in 96-well plates.The cells were divided into 6 groups （n =15 each） using a random number table：control group （group C）,OGD/R group （group O）,sevoflurane postconditioning group （group Sev）,sevoflurane postconditioning plus 5-hydroxydecanoate （5-HD） group （group SH）,5-HD group （group H） and OGD/R plus 5-HD group （group HO）.The cardiomyocytes were subjected to oxygen-glucose deprivation for 4 h followed by restoration of oxygen-glucose supply for 24 h.After oxygen-glucose restoration,the cardiomyocytes in the culture media were exposed to 2% sevoflurane for 1 h to perform sevoflurane postconditioning.At 1 h before oxygen-glucose deprivation,a specific mito-KATP channel blocker 5-HD 100 μmol/L was added to the culture media.Cardiomyocytes were cultured in normal culture atmosphere in group C.Cardiomyocytes were collected at 24 h of oxygen-glucose restoration.Cell pyroptosis was detected by double flow cytometry AlexaFour488 （caspase-1 FLICA staining） and TMR red （DNA staining） staining.The pyroptosis rate was calculated.The cell survival rate was measured by methyl thiazolyl tetrazolium assay.The content of reactive oxygen species （ROS） in mitochondria was determined by 2＆#39;,7＆#39;-dichlorofluorescin diacetate assay.The mitochondrial membrane potential （MMP） was measured by using JC-I fluorescent probe.The expression of interleukin-1beta （IL-1β） was determined by Western blot.Results Compared with group C,the pyroptosis rate and ROS content were significantly increased,the cell survival rate and MMP were decreased,and the expression of IL-1β was up-regulated in group O （P〈0.05）.Compared with group O,the pyroptosis rate and ROS content were significantly decreased,the cell survival rate and MMP were increased,and the expression of IL-1β was down-regulated in group Sev （P〈0.05）.Compared with group Sev,the pyroptosis rate and ROS content were significantly increased,the cell survival rate and M MP were decreased,and the expression of IL-1β was up-regulated in group SH （P〈0.05）.Compared with group SH,the pyroptosis rate and ROS content were significantly increased,the cell survival rate and MMP were decreased,and the expression of IL-1β was up-regulated in group H O （P〈0.05）.Conclusion The mechanism by which sevoflurane postconditioning inhibits OGD/R-induced pyroptosis in primary rat cardiomyocytes is probably associated with increasing mito-KATP channel opening.