Experimental Study on the Safety of Hypertime Window Thrombolysis for Cerebral Infarction by Inhibiting the Activation of Astrocytes with Acupuncture
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摘要: 目的 观察针刺对提高急性脑梗死大鼠超时间窗溶栓安全性的效应, 并从星形胶质细胞的途径探讨其作用机制。方法 采用改良的大鼠自体血栓栓塞法制备脑梗死模型, 选用“醒脑开窍”针刺法作为介入手段, 尾静脉注射阿替普酶(rt-PA)作为溶栓方法。将66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+4.5 h溶栓组、针刺+6 h溶栓组, 每组11只。Bederson法对各组大鼠在造模后2 h和24 h分别进行神经行为学评分, TTC染色法比较各组大鼠脑梗死体积百分比, EB渗透法比较各组大鼠血脑屏障(BBB)通透性, 干湿质量法测定各组大鼠脑含水量。另取48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12只。qPCR和Western blot法分别检测各组大鼠大脑皮层中星形胶质细胞相关指标胶质纤维酸性蛋白(GFAP)、水通道蛋白-4(AQP-4)的mRNA和蛋白表达。结果 与假手术组相比, 模型组神经行为学评分、脑梗死体积百分比、脑含水量均明显升高(P < 0.01);4.5 h溶栓组、针刺+4.5 h溶栓组神经行为学评分、脑梗死体积百分比、脑含水量均低于模型组(P < 0.01);与模型组和6 h溶栓组相比, 针刺+6 h溶栓组神经行为学评分、脑梗死体积百分比、脑含水量均明显降低(P < 0.05，P < 0.01)。模型组GFAP、AQP-4 mRNA和蛋白表达水平均明显高于假手术组(P < 0.05，P < 0.01);针刺+6 h溶栓组GFAP、AQP-4 mRNA和蛋白表达水平均显著低于模型组和6 h溶栓组(P < 0.05，P < 0.01)。结论 针刺可以通过下调GFAP、AQP-4的表达来抑制星形胶质细胞活化, 从而提高脑梗死超时间窗溶栓安全性。Abstract: OBJECTIVE To observe the effect of acupuncture on the safety of hypertime window thrombolysis in rats with acute cerebral infarction, and to explore its mechanism from the way of astrocytes.METHODS Cerebral infarction model was prepared by improved autoembolism method in rats. "Xingnao Kaiqiao" acupuncture was used as interventional method, and rt-PA was injected through caudal vein as thrombolysis method. 66 SD rats were randomly divided into sham operation group, model group, 4.5 h thrombolysis group, 6 h thrombolysis group, acupuncture+4.5 h thrombolysis group and acupuncture+6 h thrombolysis group, with 11 rats in each group. Bederson method was used to evaluate the neurobehavioral scores of rats at the 2nd and 24th hour after modeling. TTC staining method was used to compare the cerebral infarct volume percentage of rats in each group, EB osmotic method was used to compare the blood-brain barrier (BBB) permeability of rats in each group, and dry-wet weight method was used to measure the brain water content of rats in each group. 48 SD rats were randomly divided into sham operation group, model group, 6 h thrombolysis group and acupuncture+6 h thrombolysis group, with 12 rats in each group. The qPCR and Western blot were used to detect the mRNA and protein expressions of astrocyte-related indexes glial fibrillary acidic protein (GFAP) and aquaporin-4 (AQP-4) in the cerebral cortex of rats in each group.RESULTS Compared with the sham operation group, the neurobehavioral score, percentage of cerebral infarct volume and brain water content in the model group significantly increased (P < 0.01). The neurobehavioral scores, percentage of cerebral infarct volume and brain water content in the 4.5 h thrombolysis group and the acupuncture+4.5 h thrombolysis group were lower than those in the model group (P < 0.01). Compared with the model group and the 6 h thrombolytic group, the neurobehavioral score, the percentage of cerebral infarct volume and brain water content in the acupuncture+6 h thrombolytic group significantly decreased (P < 0.05, P < 0.01). The mRNA and protein expression levels of GFAP and AQP-4 in model group were significantly higher than those in sham group (P < 0.05, P < 0.01). The mRNA and protein expression levels of GFAP and AQP-4 in acupuncture+6 h thrombolysis group were significantly lower than those in model group and 6 h thrombolysis group (P < 0.05, P < 0.01).CONCLUSION Acupuncture can inhibit the activation of astrocytes by down-regulating the expression of GFAP and AQP-4, thus improve the safety of hypertime window thrombolysis in cerebral infarction.
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Key words:
- acupuncture /
- cerebral infarction /
- safety of thrombolysis /
- astrocytes /
- GFAP /
- AQP-4
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图 3 各组大鼠造模后2 h和24 h神经行为学评分比较
注: 与假手术组比较, * *P < 0.01;与模型组比较, #P < 0.05, ##P < 0.01;与6 h溶栓组比较, △△P < 0.01。x±s, n=11。
图 5 各组大鼠脑梗死体积百分比结果比较
注: 与假手术组比较, * *P < 0.01;与模型组比较, #P < 0.05, ##P < 0.01;与6 h溶栓组比较, △P < 0.05。x±s, n=5。
图 7 各组大鼠EB含量结果比较
注: 与假手术组比较, * *P < 0.01;与模型组比较, ##P < 0.01;与6 h溶栓组比较, △△P < 0.01。x±s, n=3。
图 8 各组大鼠脑含水量结果比较
注: 与假手术组比较, * *P < 0.01;与模型组比较, #P < 0.05, ##P < 0.01;与6 h溶栓组比较, △△P < 0.01。x±s, n=3。
图 9 各组大鼠大脑皮层GFAP、AQP-4 mRNA表达结果比较
注: 与假手术组比较, * *P < 0.01;与模型组比较, ##P < 0.01;与6 h溶栓组比较, △△P < 0.01。x±s, n=6。
图 10 各组大鼠大脑皮层GFAP、AQP-4蛋白表达结果比较
注: 与假手术组比较, *P < 0.05, * *P < 0.01;与模型组比较, #P < 0.05;与6 h溶栓组比较, △△P < 0.01。x±s, n=6。
表 1 引物序列
基因 引物(5′→3′) GFAP 上游: ATGACTATCGCCGCCAACTG 下游: CTGGTAACTCGCCGACTCCC AQP-4 上游: CGCCAAGTCCGTCTTCTACA 下游: GCCAGCAGTGAGGTTTCCAT GAPDH 上游: TGGAATTGTGAGGGAGATG 下游: GCCCAGCAAGGATACTGA 
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