Protective effects of Tongxinluo on cerebral ischemia/reperfusion injury related to Connexin 43/Calpain II/Bax/Caspase-3 pathway in rat
Protective effects of Tongxinluo on cerebral ischemia/reperfusion injury related to Connexin 43/Calpain II/Bax/Caspase-3 pathway in rat
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DOI：
10.1016/j.jep.2017.01.004
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发表时间：
2017-02-23
影响因子：
5.4
通讯作者：
Cai, Yefeng
中科院分区：
文献类型：
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作者：
Cheng, Xiao;Hou, Zijun;Cai, Yefeng
民族药理学相关性：通心络（TXL）是一种多功能中药，已广泛用于治疗心脑血管疾病。大量研究表明 TXL 是一种新型神经保护药物，但其机制尚不清楚。研究目的：旨在证明TXL对脑缺血/再灌注（I/R）损伤的保护作用，并为Connexin 43/Calpain II/Bax/Caspase-3通路参与TXL介导的神经保护提供证据。方法：采用短暂性大脑中动脉闭塞（MCAO，持续90分钟）成年雄性Sprague-Dawley大鼠诱导局灶性脑I/R损伤。我们通过TTC染色评估TXL对I/R损伤的影响，包括神经功能缺损评估和脑梗塞体积测量，并通过蛋白质印迹检测Connexin 43（Cx43）的蛋白表达。此外，MCAO术前30 min脑室内注射羧苯索隆（CBX，Cx43的抑制剂）后，通过免疫荧光染色检测缺血半暗区的Calpain II、Bax和cleaved Caspased-3免疫反应性，并通过TUNEL染色检测细胞凋亡。结果：与缓冲液治疗的MCAO相比，TXL治疗大大改善了神经功能缺损，并减少了梗死体积（P < 0.05），TXL 前后处理显示出比 TXL 预处理更好的结果。与使用缓冲液处理的 MCAO 相比，TXL 前后处理显着上调损伤后 3 天、7 天和 14 天的 Cx43 蛋白表达（P < 0.05）。同时，与MCAO组相比，TXL前后治疗后缺血半暗区Calpain II、Bax和cleaved Caspase-3的免疫反应性明显降低（P < 0.05）。然而，随着Cx43抑制剂CBX的治疗，TXL对Calpain II、Bax和cleaved Caspase-3免疫反应性的下调作用被消除（P < 0.05）。此外，TXL对半影区神经元凋亡的保护作用与CBX相抵消（P < 0.05）。结论：TXL可通过Cx43/Calpain II/Bax/Caspase-3通路有效保护缺血再灌注损伤，减少细胞死亡，有助于缺血再灌注损伤的预防和治疗。
Ethnopharmacological relevance: Tongxinluo (TXL) is a multifunctional traditional Chinese medicine and has been widely used in the treatment of cardiovascular and cerebrovascular diseases. Numerous studies demonstrate that TXL is a novel neuroprotective drug, however, the mechanisms are largely unknown. Aim of the study: we aimed to demonstrate the protective effect of TXL on cerebral ischemia/reperfusion (I/R) injury and provide the evidence for the involvement of Connexin 43/Calpain II/ Bax/Caspase-3 pathway in TXL-mediated neuroprotection.Methods: Focal cerebral I/R injury were induced by transient middle cerebral artery occlusion (MCAO, for 90 min) in adult male Sprague-Dawley rats. We estimated the effects of TXL on I/R injury including neurological deficit assessment and cerebral infarct volume measurement via TTC staining, and detected the protein expression of Connexin 43 (Cx43) by western blot. Furthermore, after the intracerebroventricular injection of carbenoxolone (CBX, the inhibitor of Cx43) at 30 min before MCAO surgery, Calpain II, Bax and cleaved Caspased-3 immunoreactivity in ischemic penumbra region was detected by immunofluorescent staining, and cell apoptosis was detected by TUNEL staining.Results: TXL treatment greatly improved neurological deficit and reduced the infarction volume compared to MCAO with buffer treatment (P < 0.05), and TXL pre-post treatment showed better results than TXL pretreatment. TXL pre-post treatment significantly up-regulated Cx43 protein expression at 3d, 7d and 14d post injury compared to MCAO with buffer treatment (P < 0.05). Meanwhile, the immunoreactivity of Calpain II, Bax and cleaved Caspase-3 in ischemic penumbra region was obviously decreased by TXL pre-post treatment compared to MCAO group (P < 0.05). However, with the treatment of the Cx43 inhibitor, CBX, the down regulated effect of TXL on Calpain II, Bax and cleaved Caspase-3 immunoreactivity was abolished (P < 0.05). Moreover, the protective effect of TXL against neuron apoptosis in penumbra region was conteracted by CBX (P < 0.05).Conclusions: TXL could effectively protect against I/R injury and reduced cell death via Cx43/Calpain II/Bax/ Caspase-3 pathway, which contribute to I/R injury prevention and therapy.