依达拉奉预适应延长急性缺血性脑卒中溶栓时间窗的研究及ROS/TXNIP/NLRP3通路参与机制的探讨

Study on edaravone pretreatment prolonging thrombolytic time window by acute ischemic stroke and mechanism of ROS/TXNIP/NLRP3 pathway in rat

目的探讨依达拉奉预处理对延长急性缺血性脑卒中(AIS)大鼠模型溶栓时间窗的影响及作用机制。方法选取健康SD大鼠64只随机分为假手术组、AIS组、重组组织型纤溶酶原激活剂(rt-PA)治疗组及rt-PA+依达拉奉治疗组,采用大鼠自体血栓构建AIS模型。采用Longa评分对各组大鼠在AIS后的神经功能进行评价,采用TTC染色法测定脑梗死体积,原位缺口末端标记观察大鼠脑内细胞凋亡的变化,酶联免疫吸附试验检测脑内活性氧(ROS)水平及超氧化物歧化酶(SOD)水平,硫代巴比妥酸检测脑内丙二醛(MDA)的含量,比色法检测脑内血红蛋白含量,Western blot实验检测ROS/TXNIP/NLRP3通路相关ASC、TXNIP、Caspase-1、IL-18和IL-1β的蛋白表达水平。结果rt-PA+依达拉奉治疗组在造模后6、7 h干预的大鼠的Longa评分较rt-PA治疗组降低,而且不同干预时间点的脑水肿程度和脑梗死体积改善情况均优于rt-PA治疗组,细胞凋亡率明显低于AIS组和rt-PA治疗组,差异具有统计学意义(P<0.05)。与rt-PA治疗组比较,rt-PA+依达拉奉治疗组在造模后7 h干预的大鼠ROS蛋白水平、MDA水平、血红蛋白含量均显著降低,在造模后7、8 h干预的大鼠SOD水平高于rt-PA治疗组,差异均具有统计学意义(P<0.05)。rt-PA治疗组及rt-PA+依达拉奉治疗组在造模后4.5 h干预的大鼠NLRP3、ASC、IL-18和IL-1β蛋白表达水平较AIS组下降,而且rt-PA+依达拉奉治疗组在造模后6、7 h的ASC、TXNIP、Caspase-1、IL-1β蛋白表达水平降低,差异均具有统计学意义(P<0.05)。与rt-PA治疗组比较,rt-PA+依达拉奉治疗组在造模后6 h干预的大鼠NLRP3的蛋白表达水平,造模后7 h的ASC、TXNIP蛋白表达水平,以及在造模后7、8 h的IL-18蛋白表达水平均降低,差异具有统计学意义(P<0.05)。结论依达拉奉预处理可以适当延长AIS大鼠rt-PA治疗的溶栓时间窗,其作用机制可能与ROS/TXNIP/NLRP3通路的参与有关。

Objective To investigate whether edaravone pretreatment can prolong the thrombolytic time window of acute ischemic stroke(AIS)rat model and its mechanism.Methods Sixty-four healthy SD rats were randomly divided into sham operation group,AIS group,recombinant tissue plasminogen activator(rt-PA)treatment group and rt-PA+edaravone treatment group.The AIS model was constructed by autologous thrombosis.The neurological function of rats in each group was evaluated after AIS,and the volume of cerebral infarction was measured by TTC method;Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling was used to observe the changes of cell apoptosis in rats;The levels of ROS and superoxide dismutase(SOD)in brain were detected by enzyme-linked immunosorbent assay;The content of malondialdehyde(MDA)in brain was detected by thiobarbituric acid;The content of hemoglobin in brain was detected by colorimetry;The content of hemoglobin in brain was measured by hemoglobin assay;The protein expression level of ASC,TXNIP,Caspase-1,IL-18 and IL-1βrelated to ROS/TXNIP/NLRP3 pathway were detected by Western blot.Results The Longa score of the rt-PA+edaravone treatment group was lower than that in the rt-PA treatment group after intravenous thrombolysis at 6 and 7 h,and the improvement of cerebral edema degree and cerebral infarction volume at different intervention time points were better than those in the rt-PA treatment group(P<0.05).The apoptosis rate was significantly lower than that in the AIS group and rt-PA treatment group,with a statistically significant difference(P<0.05).Compared with the rt-PA treatment group,the levels of ROS protein,MDA and hemoglobin of the rt-PA+edaravone treatment group after intravenous thrombolysis at 7 h were significantly reduced,and the SOD level after intravenous thrombolysis at 7 and 8 h were higher(P<0.05).The protein expression level of NLRP3,ASC,IL-18 and IL-1βof the rt-PA treatment group and rt-PA+edaravone treatment group after intravenous thrombolysis at 4.5 h were lower than those of AIS group,and the protein expression level of ASC,TXNIP,Caspase-1,IL-1 of rt-PA+edaravone treatment group after intravenous thrombolysis at 6 and 7 h were decreased(P<0.05).Compared with the rt-PA treatment group,the protein expression level of NLRP3 after intravenous thrombolysis at 6 h,the protein expression levels of ASC and TXNIP after intravenous thrombolysis at 7 h,and the protein expression level of IL-18 after intravenous thrombolysis at 7 and 8 h of the rt-PA+edaravone treatment group were decreased,with statistical significance(P<0.05).Conclusion Edaravone pretreatment can appropriately prolong the thrombolytic time window of rt-PA treatment in AIS rats,and its mechanism may be related to the participation of ROS/TXNIP/NLRP3 pathway.