川芎嗪对大鼠蛛网膜下腔出血早期脑损伤的保护作用及机制研究

Protection and Mechanism of Ligustrazine on Early Brain Injury in Rats with Subarachnoid Hemorrhage

目的探讨川芎嗪(TMP)对大鼠蛛网膜下腔出血早期脑损伤(SAH)的保护作用及机制。方法将80只SPF级SD大鼠随机分为:假手术组、模型组、TMP低剂量组和TMP高剂量组(均n=20),构建SAH大鼠模型。模型建立后,TMP低剂量组(腹腔注射TMP 5 mg·kg^(-1),×14 d)和TMP高剂量组(腹腔注射TMP 10 mg·kg^(-1),×14 d);假手术组和模型组注射等体积0.9%氯化钠溶液。采用Longa’s 5级标准评分法、苏木精-伊红染色、ELISA法和免疫荧光法检测各组大鼠的神经功能缺损症状、脑神经元损伤、脑组织IL-1β、IL-6和TNF-α表达水平、NF-κB核移位;Western blot法检测磷酸化腺苷酸活化蛋白激酶(p-AMPK)、磷酸化内皮型一氧化氮合酶(p-eNOS)和核转录因子κB(NF-κB)蛋白的表达水平。结果与假手术组比较,模型组大鼠神经功能损伤评分、脑组织含水量、脑神经元凋亡率及IL-1β、IL-6和TNF-α表达水平升高,脑组织细胞内NF-κB核移位荧光强度明显增强,且脑组织中p-AMPK、p-eNOS蛋白表达明显降低,NF-κB蛋白表达明显升高,差异均有统计学意义(均P<0.05)。与模型组比较,TMP低剂量组、TMP高剂量组大鼠神经功能损伤评分、脑组织含水量明显增加,脑神经元凋亡率及IL-1β、IL-6和TNF-α表达水平降低,脑组织细胞内NF-κB核移位荧光强度明显减弱,且p-AMPK、p-eNOS蛋白表达水平明显升高,NF-κB蛋白表达水平明显下降,差异均有统计学意义(均P<0.05),且呈剂量依赖性。结论TMP可能通过激活AMPK/eNOS/NF-κB信号通路减轻SAH后的炎症反应,从而发挥对SAH大鼠的脑保护作用。

Aim To investigate the pharmacological effects of tetramethylpyrazine(TMP)on early subarachnoid hemorrhage(SAH)in rats,and explore the mechanism of its cerebral protective effects.Methods Total 80 SPF SD rats were randomly divided into 4 groups(n=20):a sham group,a model group,a low-TMP group and a high-TMP group,construting SAH rat model.After the establishment of the model,the low-TMP group and the high-TMP group were injected with the same volume of normal saline at the concentration of 5 mg·kg^(-1)TMP,10 mg·kg^(-1)TMP,injection dose of 2 m L and model group for 14 d,respectively.Longa’s 5-grade standard score,HE staining,ELISA and immunofluorescence were used to detect the symptoms of neurological impairment,neuron damage in brain tissue,the expression levels of IL-1β,IL-6 and TNF-αin brain tissue,and the nuclear translocations of NF-κB.The expression levels of p-AMPK,p-e NOS and NF-κB were detected by Western blot.Results Compared with the sham group,the nerve function injury score,brain tissue water content,apoptosis rate and the expression levels of IL-1β,IL-6 and TNF-αin the brain tissue were increased in the model group,and the fluorescence intensity of NF-κB nucleus in the brain tissue was significantly increased,and the p-AMPK and p-e NOS in the brain tissue were significantly decreased.The expression of NF-κB protein was significantly increased,and the differences were statistically significant(P<0.05).Compared with model group,the neurological impairment score and brain water content in LowTMP group and High-TMP group were significantly increased,the apoptosis rate and the expression of IL-1β,IL-6 and TNF-αin brain tissue were decreased,the fluorescence intensity of NF-κB nuclear translocation in brain cells was significantly decreased,P-AMPK and p-e NOS in brain tissue were significantly increased,and the expression of NF-κB protein was significantly decreased.The differences were statistically significant(P<0.05),and in a dose-dependent manner.Conclusion TMP may protect the brain of SAH rats by activating AMPK/e NOS/NF-κB signal pathway and relieving inflammation.