摘要
目的:观察针刺对脑梗死大鼠溶栓治疗后神经元铜死亡的影响,探讨针刺延长脑梗死溶栓时间窗的机制。方法:本实验分为两部分。第一部分实验中,40只SD大鼠随机分为假手术组、模型组、4.5 h溶栓组、6 h溶栓组、针刺+6 h溶栓组,每组8只。采用改良自体血栓栓塞法制备脑梗死大鼠模型。各溶栓治疗组在造模后的4.5 h和6 h予重组人组织型纤溶酶原激活剂(rt-PA)10 mg/kg尾静脉注射;针刺+6 h溶栓组于造模后6 h溶栓的同时予针刺双侧“内关”“水沟”,留针30 min。各组均在造模时采用激光散斑血流成像仪监测脑血流量,采用Bederson法于造模后的2、24 h进行神经行为学评分,氯化三苯基四氮唑染色法比较各组大鼠脑梗死体积,免疫荧光法检测各组大鼠缺血半暗带(IP)区神经元核蛋白(NeuN)的阳性表达。第二部分实验中,24只SD大鼠随机分为假手术组、模型组、6 h溶栓组、针刺+6 h溶栓组,每组6只。采用比色法检测各组大鼠IP区铜离子、丙酮酸、腺苷三磷酸(ATP)的含量,Western blot法及荧光定量PCR法检测铁氧还蛋白1(FDX1)、二氢硫辛酸乙酰基转移酶(DLAT)蛋白及mRNA的表达。结果:与假手术组比较,模型组大鼠神经行为学评分,脑梗死体积百分比,IP区铜离子、丙酮酸含量及FDX1、DLAT蛋白和mRNA表达水平升高(P<0.01),NeuN阳性细胞百分比、ATP含量下降(P<0.01)。与模型组比较,4.5 h溶栓组大鼠24 h神经行为学评分和脑梗死体积百分比下降(P<0.05,P<0.01),NeuN阳性细胞百分比升高(P<0.05);6 h溶栓组大鼠脑梗死体积百分比,IP区铜离子、丙酮酸含量及FDX1、DLAT蛋白和mRNA表达水平升高(P<0.05,P<0.01),NeuN阳性细胞百分比、ATP含量下降(P<0.05);针刺+6 h溶栓组大鼠24 h神经行为学评分,脑梗死体积百分比,IP区铜离子、丙酮酸含量及FDX1、DLAT蛋白和mRNA表达水平降低(P<0.05,P<0.01),NeuN阳性细胞百分比、ATP含量升高(P<0.05)。与4.5 h溶栓组比较,6 h溶栓组大鼠24 h神经行为学评分、脑梗死体积百分比升高(P<0.01),NeuN阳性细胞百分比含量下降(P<0.01)。与6 h溶栓组比较,针刺+6 h溶栓组大鼠24 h神经行为学评分,脑梗死体积百分比,IP区铜离子、丙酮酸含量及FDX1、DLAT蛋白和mRNA表达水平降低(P<0.01),NeuN阳性细胞百分比及ATP含量升高(P<0.01)。结论:针刺及时介入可延长脑梗死大鼠溶栓时间窗,其机制可能与针刺降低IP区FDX1、DLAT蛋白及mRNA表达从而抑制神经元的铜死亡有关。
Objective To observe the effect of acupuncture on neuronal cuproptosis after thrombolytic treatment in rats with cerebral infarction(CI),so as to explore its underlying mechanisms in prolonging the time window of thrombolysis therapy for CI.Methods In the first part of the present study,40 male SD rats were randomly divided into 5 groups:sham operation,model,4.5 h thrombolysis,6 h thrombolysis and acupuncture+6 h thrombolysis groups,with 8 rats in each group.The CI model was prepared by modified autologous thromboembolism method.Rats of the 4.5 h thrombolysis,6 h thrombolysis and acupuncture+6 h thrombolysis groups received thrombolysis with intravenous injection of recombinant human tissue plasminogen activator(rt-PA,10 mg/kg)through tail vein at 4.5 h or 6 h after modeling.Manual acupuncture stimulation was applied to bilateral“Neiguan”(PC6)and“Shuigou”(CV26)at the same time with thrombolysis administration for rats of the acupuncture+6 h thrombolysis group,with the needles retained for 30 min.The cerebral blood flow was monitored using laser diffusion blood flow imaging,and neurological deficit score was evaluated 2 h and 24 h after successful modeling using Bederson’s method.The CI volume was observed after triphenyltetrazole chloride staining.The immunoactivity of neuronal nucle(i NeuN)in the ischemic penumbra(IP)region of the cerebral cortex was detected by immunofluorescence staining.In the second part of the study,24 male SD rats were randomly divided into sham operation group,model group,6 h thrombolysis group and acupuncture+6 h thrombolysis group,with 6 rats in each group.The protein and mRNA relative expression levels of Ferredoxin-1(FDX1)and dihydrolipoamide acetyltransferase(DLAT)in the IP region of the right cerebral cortex were detected by Western blot and real-time PCR,separately.The copper ion,pyruvate and adenosine triphosphate(ATP)contents in the IP area of the cerebral cortex tissue were detected using colorimetry.Results The neurological deficit scores at both 2 h and 24 h after modeling,percentage of CI volume,the contents of copper ion and pyruvate,and the protein and mRNA expression levels of FDX1 and DLAT were significantly higher in the model group than those in the sham operation group(P<0.01),while the NeuN positive expression and ATP content were evidently lower in the model group than those in the sham operation group(P<0.01).In comparison with the model group,the neurological deficit score and percentage of CI volume in the 4.5 h thrombolysis and acupuncture+6 h thrombolysis groups,the contents of copper ion and pyruvate,and the protein and mRNA expression levels of FDX1 and DLAT in the acupuncture+6 h thrombolysis group,and the NeuN immunoactivity and ATP content in the 6 h thrombolysis group were significantly decreased(P<0.05,P<0.01),while the immunoactivity of NeuN in both 4.5 h thrombolysis and acupuncture+6 h thrombolysis groups,ATP content in the acupuncture+6 h thrombolysis group,the percentage of CI volume,contents of copper ion and pyruvate,and the expression levels of FDX1 and DLAT proteins and mRNAs in the 6 h thrombolysis group were significantly increased(P<0.05,P<0.01).Comparison among the 3 intervention groups showed that the neurological deficit score and percentage of CI volume were considerably lower in both of the 4.5 h thrombolysis and acupuncture+6 h thrombolysis groups than those in the 6 h thrombolysis group(P<0.01),while the NeuN immunoactivity was considerably higher in both of the 4.5 h thrombolysis and acupuncture+6 h thrombolysis groups than in the 6 h thrombolysis group(P<0.01).The contents of copper ion and pyruvate,and the protein and mRNA expression levels of FDX1 and DLAT were considerably lower in the acupuncture+6 h thrombolysis group than in the 6 h thrombolysis group(P<0.01),while the ATP content was considerably higher in the acupuncture+6 h thrombolysis group than in the 6 h thrombolysis group(P<0.01).Conclusion Acupuncture can prolong the time window of thrombolysis therapy for CI rats,which may be related to its function in down-regulating the protein and mRNA expressions of FDX1 and DLAT in ischemic cerebral cortex.
作者
赵艺
宋扬扬
常思琦
黄正
刘欣雨
刘倩倩
倪光夏
ZHAO Yi;SONG Yang-yang;CHANG Si-qi;HUANG Zheng;LIU Xin-yu;LIU Qian-qian;NI Guang-xia(College of Acupuncture-moxibustion and Tuina,College of Health and Rehabilitation,Nanjing University of Chinese Medicine,Nanjing 210023,China;Key Laboratory of Acupuncture and Medicine of Ministry of Education,Nanjing University of Chinese Medicine,Nanjing 210023)
出处
《针刺研究》
北大核心
2025年第7期754-762,共9页
Acupuncture Research
基金
国家自然科学基金项目(No.82274639、82074525、82104993)
江苏省中医药科技发展计划项目(No.QN202204)。
关键词
脑梗死
针刺
溶栓时间窗
铜死亡
Cerebral infarction
Acupuncture
Thrombolysis time window
Cuproptosis
