盐酸右美托咪定通过抑制细胞焦亡发挥脑缺血再灌注损伤保护作用

Protective effect of dexmedetomidine hydrochloride against cerebral ischemia-reperfusion injury by inhibiting pyroptosis

目的探讨盐酸右美托咪定(DEX)减轻脑缺血再灌注损伤(CIRI)与抑制神经细胞焦亡之间的关系。方法①PC12细胞给予氧糖剥夺再复供(OGD/R)处理模拟脑缺血再灌注神经细胞损伤。设细胞对照组、模型组及模型+DEX 2,5和10μmol·L^(-1)组,给药组PC12细胞于OGD/R处理后给予DEX 48 h,采用CCK-8法检测PC12细胞存活率;乳酸脱氢酶(LDH)活性检测试剂盒测定PC12细胞上清LDH释放率;免疫荧光染色法测GSDMD-N蛋白表达水平。②PC12细胞OGD/R处理,设细胞对照组、模型组、模型+VX-7652μmol·L^(-1)组、模型+VX-7652μmol·L^(-1)+DEX 10μmol·L^(-1)组,给药组PC12细胞于OGD/R处理后给予VX-765或VX-765+DEX作用48 h。③通过大脑中动脉阻塞再灌注(MCAO/R)法制备大鼠CIRI模型。将大鼠分为假手术组、模型组及模型+DEX 25,50和100μg·kg^(-1)组,给药组大鼠于再灌注40 min后ip给予DEX,2和24 h后对大鼠进行神经功能评分和脑梗死体积检测;比色法检测大脑皮质胱天蛋白酶1活性。Western印迹法检测分组①和分组②PC12细胞及分组③大鼠脑组织中GSDMD-N、NOD样受体热蛋白结构域相关蛋白3(NLRP3)和胱天蛋白酶1蛋白表达水平;ELISA检测分组①和分组②PC12细胞培养上清液及分组③大鼠脑组织匀浆中白细胞介素18(IL-18)和IL-1β水平。结果①与细胞对照组相比,模型组PC12细胞存活率降低(P<0.01),细胞上清液中LDH释放率升高(P<0.01),GSDMD-N、NLRP3和胱天蛋白酶1蛋白表达及IL-1β和IL-18炎症因子水平明显升高(P<0.01);与模型组相比,模型+DEX 2,5和10μmol·L^(-1)组细胞存活率升高(P<0.01),PC12细胞LDH释放率降低(P<0.05,P<0.01),焦亡相关蛋白GSDMD-N、NLRP3和胱天蛋白酶1蛋白表达及IL-1β和IL-18水平降低(P<0.05,P<0.01)。②与模型组相比,模型+VX-765组PC12细胞GSDMD-N、NLRP3和胱天蛋白酶1蛋白表达及IL-1β和IL-18水平降低(P<0.01),与模型+VX-7652μmol·L^(-1)组相比,模型+VX-7652μmol·L^(-1)+DEX 10μmol·L^(-1)组PC12细胞GSDMD-N、NLRP3和胱天蛋白酶1蛋白表达及IL-1β和IL-18水平无显著变化。③与假手术组相比,模型组大鼠脑组织GSDMD-N、NLRP3和胱天蛋白酶1蛋白表达升高(P<0.01),IL-1β和IL-18水平增加(P<0.01),神经功能评分和脑梗死体积百分比显著增高(P<0.01),大脑皮质胱天蛋白酶1活性显著增高(P<0.05);与模型组相比,模型+DEX组大鼠脑组织GSDMD-N、NLRP3和胱天蛋白酶1蛋白表达及IL-1β和IL-18水平均显著降低(P<0.05,P<0.01),神经功能评分、脑梗死体积百分比和脑皮质胱天蛋白酶1活性显著降低(P<0.05,P<0.01)。结论DEX在体内能降低MCAO/R模型大鼠脑组织GSDMD-N、NLRP3和胱天蛋白酶1蛋白表达及降低IL-1β和IL-18水平,抑制PC12细胞焦亡,从而发挥减轻CIRI作用。

OBJECTIVE To investigate the correlations between dexmedetomidine hydrochloride(DEX)and inhibition of nerve cell pyroptosis in reducing cerebral ischemia-reperfusion injury(CIRI).METHODS①PC12 cells were treated with oxygen-glucose deprivation and resupply(OGD/R)to simulate cerebral ischemia reperfusion injury.PC12 cells were divided into the cell control group,model group(OGD/R),OGD/R+DEX 2,5 and 10μmol·L^(-1) groups.The DEX group was treated with DEX following OGD/R treatment.The survival rate of PC12 cells was measured using the CCK-8 method 48 h after drug administration.The lactate dehydrogenase(LDH)activity assay kit was used to measure the LDH level in the supernatant of PC12 cells.The protein level expression changes of GSDMD-N in PC12 cells were determined by immunofluorescence staining.②PC12 cells were were divided into the cell control group,model group,model+VX-7652μmol·L^(-1) and model+VX-7652μmol·L^(-1)+DEX 10μmol·L^(-1) groups.The drug group of PC12 cells was treated with VX-765 or in combination with DEX after OGD/R treatment,and the experiment was conducted 48 h after drug administration.③A model of cerebral ischemia-reperfusion injury(CIRI)was constructed using the middle cerebral artery occlusion reperfusion(MCAO/R)method.The rats were divided into the sham group,MCAO/R model group,model+DEX 25,50 and 100μg·kg^(-1) groups.The rats in the DEX group were ip given DEX 40 min after reperfusion.Neurological function scores were measured in rats 2 and 24 h after administration.The TTC staining method was used for determination of cerebral infarction volume while the colorimetric method was used to detect the activity of caspase 1 in the cerebral cortex of rats.The expression levels of GSDMD-N,NOD-like receptor thermal protein domain associated protein 3(NLRP3)and caspase 1 in PC12 cells from group①and②as well as in brain tissue from group③were detected by Western blotting.The levels of interleukin-18(IL-18)and IL-1βin the supernate of PC12 cells from group①and②as well as in brain tissue from group③were detected by ELISA.Neural function scores of rats in group③were measured 2 h and 24 h after DEX administration.Caspase 1 activity assay kit(colorimetric method)was used to detect the activity of caspase 1 in the cerebral cortex of rats from group③.RESULTS①Compared with the cell control group,the survival rate of the model group was decreased(P<0.01),LDH levels were increased(P<0.01),expressions of GSDMD-N,NLRP3,caspase 1 protein,IL-1βand IL-18 levels in the supernate of PC12 cells were significantly increased(P<0.01).Compared with the model group,the survival rate of cells increased in model+DEX 2,5 and 10μmol·L^(-1)(P<0.01)groups,while the level of LDH in the supernuant(P<0.05,P<0.01),the expressions of GSDMD-N,NLRP3,caspase 1,and the levels of inflammatory factors IL-1βand IL-18 were significantly decreased(P<0.05,P<0.01).②Compared with the model group,the expressions of GSDMD-N,NLRP3,caspase 1,and the levels of inflammatory factors IL-1βand IL-18 in model+VX-7652μmol·L^(-1) group were significantly decreased(P<0.01).Compared with the model+VX-7652μmol·L^(-1) group,model+VX-7652μmol·L^(-1)+DEX 10μmol·L^(-1) had no significant inhibitory effect on the expressions of GSDMD-N,NLRP3,caspase 1,or the levels of inflammatory factors IL-1βand IL-18 in PC12 cells.③Compared with the sham group,the expressions of GSDMDN,NLRP3 and caspase 1 protein in brain tissue of the model group were increased(P<0.01),so were the levels of IL-1βand IL-18(P<0.01),but the neurobehavioral scores and the percentage of cerebral infarction volume were significantly increased(P<0.01),so was the activity of caspase 1 in cerebral cortex(P<0.05).Compared with the model group,the expressions of GSDMD-N,NLRP3 and caspase 1,IL-1βand IL-18 levels were significantly decreased in the model+DEX group(P<0.05,P<0.01),and the neurobehavioral scores,percentage of the cerebral infarction volume and caspase 1 activity in the cerebral cortex were significantly decreased(P<0.05,P<0.01).CONCLUSION DEX can reduce the protein expressions of GSDMD-N,NLRP3 and caspase 1 as well as the levels of IL-1βand IL-18 in MCAO/R rat brain tissue in vivo,and inhibit PC12 pyroptosis in vitro,thereby alleviating CIRI.