右美托咪定通过调节BDNF/NLRP3通路减轻氧糖剥夺诱导的人脑星形胶质细胞炎症和焦亡

Dexmedetomidine alleviates oxygen‑glucose deprivation‑induced inflammation and pyroptosis in human brain astrocytes through regulating the brain‑derived neurotrophic factor/NOD‑like receptor thermal protein domain associated protein 3 pathway

目的本研究采用体外氧糖剥夺(OGD)模型探讨右美托咪定(Dex)在缺血性脑卒中(IS)对人脑星形胶质细胞(HA)中的作用,并揭示其潜在机制。方法以HA为研究对象,按照随机数字表法分为10组(每组3孔):对照组(Control组),OGD组,分别使用0.5、1.0、2.0μmol/L Dex处理OGD模型组(0.5μmol/L Dex+OGD组、1.0μmol/L Dex+OGD组、2.0μmol/L Dex+OGD组),在OGD模型细胞中添加200 nmol/L K⁃252a[脑源性神经营养因子(BDNF)抑制剂]组(OGD+K⁃252a组),在OGD模型细胞中添加1.0μmol/L Dex组(OGD+Dex组),在OGD模型细胞中联合使用1.0μmol/L的Dex和200 nmol/L K⁃252a组(OGD+Dex+K⁃252a组),在正常细胞中添加200 nmol/L K⁃252a组(K⁃252a组),正常细胞中联合使用200 nmol/L K⁃252a和5μmol/L的BAY11⁃7082[NOD样受体热蛋白结构域相关蛋白3(NLRP3)抑制剂]组(K⁃252a+BAY11⁃7082组)。以上细胞处理24 h后,采用蛋白质免疫印迹法(Western blot)检测细胞中BDNF、NLRP3、活性胱天蛋白酶⁃1(caspase⁃1)和消皮素D(GSDMD)蛋白N端(GSDMD⁃N)的蛋白水平,酶联免疫吸附测定(ELISA)法检测白细胞介素(IL)⁃1β和IL⁃18的含量,2',7'⁃二氯荧光素二乙酸盐(DCFDA)法检测细胞中活性氧(ROS)的水平,流式细胞仪检测细胞焦亡情况。结果与Control组比较:OGD组IL⁃1β、IL⁃18含量,ROS荧光强度,细胞焦亡率,NLRP3、活性caspase⁃1、GSDMD⁃N蛋白水平均升高(均P<0.05),BDNF蛋白水平降低(P<0.05);0.5μmol/L Dex+OGD组IL⁃18含量、ROS荧光强度、细胞焦亡率均升高(均P<0.05);1.0μmol/L Dex+OGD组和2.0μmol/L Dex+OGD组ROS荧光强度、细胞焦亡率均升高(均P<0.05);K⁃252a组IL⁃1β、IL⁃18含量,细胞焦亡率,NLRP3、活性caspase⁃1、GSDMD⁃N蛋白水平均升高(均P<0.05)。与OGD组比较:0.5μmol/L Dex+OGD组、1.0μmol/L Dex+OGD组、2.0μmol/L Dex+OGD组IL⁃1β、IL⁃18含量,ROS荧光强度,细胞焦亡率均降低(均P<0.05);1.0μmol/L Dex+OGD组、2.0μmol/L Dex+OGD组BDNF蛋白水平升高(均P<0.05);0.5μmol/L Dex+OGD组、1.0μmol/L Dex+OGD组NLRP3蛋白水平降低(均P<0.05);1.0μmol/L Dex+OGD组、2.0μmol/L Dex+OGD组活性caspase⁃1蛋白水平降低(均P<0.05);0.5μmol/L Dex+OGD组、1.0μmol/L Dex+OGD组、2.0μmol/L Dex+OGD组GSDMD⁃N蛋白水平降低(均P<0.05);OGD+K⁃252a组IL⁃1β、IL⁃18含量,ROS荧光强度,细胞焦亡率,NLRP3、活性caspase⁃1、GSDMD⁃N蛋白水平均升高(均P<0.05),BDNF蛋白水平降低(P<0.05);OGD+Dex组IL⁃1β、IL⁃18含量,ROS荧光强度,细胞焦亡率,NLRP3、活性caspase⁃1、GSDMD⁃N蛋白水平均降低(均P<0.05),BDNF蛋白水平升高(P<0.05)。与OGD+Dex组比较,OGD+Dex+K⁃252a组IL⁃1β、IL⁃18含量,ROS荧光强度,细胞焦亡率,NLRP3、活性caspase⁃1、GSDMD⁃N蛋白水平均升高(均P<0.05),BDNF蛋白水平降低(P<0.05)。与K⁃252a组比较,K⁃252a+BAY11⁃7082组IL⁃1β、IL⁃18含量,细胞焦亡率,NLRP3、活性caspase⁃1、GSDMD⁃N蛋白水平均降低(均P<0.05)。结论Dex通过诱导BDNF的释放,阻断NLRP3通路的过度活化从而减轻由OGD诱导的星形胶质细胞炎症和细胞焦亡。

Objective To explore the effect of dexmedetomidine(Dex)on human astrocytes duing ischemic stroke(IS)using an in vitro oxygen glucose deprivation(OGD)model,and explore the potential mechanisms.Methods According to the random number table method,human astrocytes were divided into ten groups(n=3):a Control group,an OGD group,0.5,1.0μmol/L and 2.0μmol/L Dex treatment plus OGD groups(0.5μmol/L Dex+OGD group,1.0μmol/L Dex+OGD group,and 2.0μmol/L Dex+OGD group),an OGD plus 200 nmol/L K⁃252a[brain derived neurotrophic factor(BDNF)inhibitor](OGD+K⁃252a)group,an OGD plus 1.0μmol/L Dex(OGD+Dex)group,an OGD plus 1.0μmol/L Dex and 200 nmol/L K⁃252a(OGD+Dex+K⁃252a)group,a 200 nmol/L K⁃252(K⁃252a)group using nor⁃mal cells and a 200 nmol/L K⁃252a and 5μmol/L BAY11⁃7082[NOD like receptor thermal protein domain associated protein 3(NLRP3)inhibitor](K⁃252a+BAY11⁃7082)group using normal cells.After treatment for 24 h,the protein levels of BDNF,NLRP3,active cas⁃pase⁃1,and gasdermin D(GSDMD)⁃N were detected by Western blot.The contents of interleukin(IL)⁃1βand IL⁃18 were measured by enzyme⁃linked immunosorbent assay(ELISA).The levels of reactive oxygen species(ROS)were detected by 2',7'⁃dichlorofluorescin diac⁃etate(DCFDA)method.The changes in cell pyroptosis were detected by flow cytometry.Results Compared with the Control group,the OGD group showed increases in the contents of IL⁃1βand IL⁃18,ROS fluorescence intensity,cell pyroptosis rate,and the protein levels of NLRP3,active caspase⁃1 and GSDMD⁃N(all P<0.05),and decreases in BDNF protein levels(P<0.05);the 0.5μmol/L Dex+OGD group presented increases in IL⁃18 content,ROS fluorescence intensity,and cell pyroptosis rate(all P<0.05);the 1.0μmol/L Dex+OGD group and the 2.0μmol/L Dex+OGD group showed increases in ROS fluorescence intensity and cell pyroptosis rate(all P<0.05);and the K⁃252a group presented increases in the contents of IL⁃1βand IL⁃18,cell pyroptosis rate,and the protein levels of NLRP3,active caspase⁃1 and GSDMD⁃N(all P<0.05).Compared with the OGD group,decreases were found as to the contents of IL⁃1βand IL⁃18,ROS fluorescence in⁃tensity,and cell pyroptosis rate in the 0.5μmol/L Dex+OGD group,the 1.0μmol/L Dex+OGD group,and the 2.0μmol/L Dex+OGD group(all P<0.05);the protein levels of BDNF increased in the 1.0μmol/L Dex+OGD group and the 2.0μmol/L Dex+OGD group(all P<0.05);the protein levels of NLRP3 decreased in the 0.5μmol/L Dex+OGD group and the 1.0μmol/L Dex+OGD group(all P<0.05);the protein levels of caspase⁃1 decreased in the 1.0μmol/L Dex+OGD group and the 2.0μmol/L Dex+OGD group(all P<0.05);the protein levels of GSDMD⁃N decreased in the 0.5μmol/L Dex+OGD group,the 1.0μmol/L Dex+OGD group and the 2.0μmol/L Dex+OGD group(all P<0.05);the OGD+K⁃252a group showed increases in the contents of IL⁃1βand IL⁃18,ROS fluorescence intensity,cell pyroptosis rate,and the protein levels of NLRP3,active caspase⁃1 and GSDMD⁃N(all P<0.05),and decreases in BDNF protein levels(P<0.05);and the OGD+Dex group presented decreases in the contents of IL⁃1βand IL⁃18,ROS fluorescence intensity,cell pyroptosis rate,and the protein levels of NLRP3,active caspase⁃1 and GSDMD⁃N(all P<0.05),and increases in BDNF protein levels(P<0.05).Compared with the OGD+Dex group,the OGD+Dex+K⁃252a group showed increases in the contents of IL⁃1βand IL⁃18,ROS fluorescence intensity,cell pyroptosis rate,and the protein levels of NLRP3,active caspase⁃1,and GSDMD⁃N(all P<0.05),and decreases in BDNF protein levels(P<0.05).Compared with the K⁃252a group,the K⁃252a+BAY11⁃7082 presented reduction in the contents of IL⁃1βand IL⁃18,cell pyroptosis rate,and the pro⁃tein levels of NLRP3,activated caspase 1 and GSDMD⁃N(all P<0.05).Conclusions Dex relieves OGD⁃induced astrocyte inflamma⁃tion and pyroptosis through inducing the release of BDNF and suppressing the overactivation of the NLRP3 pathway.