基于Raf-MEK-ERK信号通路探究梓醇对蛛网膜下腔出血大鼠脑组织损伤的影响

Study on the impact of catalpol on brain tissue damage in rats with subarachnoid hemorrhage based on Raf-MEK-ERK signal pathway

关键词:  梓醇  蛛网膜下腔出血  脑组织损伤  Raf-MEK-ERK信号通路

Key words:  catalpol  subarachnoid hemorrhage  brain tissue damage  Raf-MEK-ERK signal pathway

摘要:

[目的] 通过考察梓醇对蛛网膜下腔出血（SAH）大鼠脑组织丝/苏氨酸蛋白激酶（Raf）-丝裂原活化蛋白激酶（MEK）-细胞外调节蛋白激酶（ERK）信号通路的影响，探讨其抗SAH脑损伤的作用机制。[方法] 将大鼠随机分为假手术组、SAH组、梓醇组、梓醇+U0126组（梓醇+Raf-MEK-ERK信号通路抑制剂U0126）。采用血管内穿孔法构建大鼠SAH模型，评估大鼠神经功能和SAH分级，伊文思蓝（EB）检测血脑屏障通透性，苏木素-伊红（HE）染色观察脑组织病理变化，免疫荧光染色检测神经元细胞凋亡及微管连接蛋白轻链3-Ⅱ（LC3-Ⅱ）、p-ERK阳性细胞表达，蛋白免疫印迹（Western blot）检测脑组织Raf、MEK、磷酸化（p）-MEK、ERK1/2、p-ERK1/2、B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、自噬基因Beclin-1、LC3-Ⅱ表达。[结果] 与假手术组相比，SAH组神经元细胞排列松散，数目减少，SAH分级、脑组织含水量、EB渗出量、原位末端标（TUNEL）阳性细胞数显著增加，凋亡率、LC3-Ⅱ、p-ERK阳性表达、Bax、Beclin-1、LC3-Ⅱ、Raf、p-MEK/MEK、p-ERK1/2/ERK1/2表达显著升高，神经功能评分减少，Bcl-2蛋白表达降低（P<0.05）；与SAH组相比，梓醇组神经元损伤明显减轻，细胞死亡较少，SAH分级、脑组织含水量、EB渗出量、TUNEL阳性细胞数显著减少，凋亡率、Bax显著降低，神经功能评分、Bcl-2表达升高，LC3-Ⅱ、p-ERK阳性表达及Beclin-1、LC3-Ⅱ、Raf、p-MEK/MEK、p-ERK1/2/ERK1/2表达进一步升高（P<0.05）；Raf-MEK-ERK通路抑制剂U0126可逆转梓醇对脑组织损伤的改善作用（P<0.05）。[结论] 梓醇可能通过激活Raf-MEK-ERK信号通路，促进神经细胞自噬，改善SAH大鼠脑损伤。

Abstract:

[Objective] To investigate the effect of catalpol on serine/threonine kinase(Raf)-mitogen activated protein kinase(MEK)-extracellular regulated protein kinase(ERK) signaling pathway in brain tissue of rats with subarachnoid hemorrhage(SAH),and to explore the mechanism of catalpol against SAH brain injury. [Methods] Rats were randomly grouped into sham operation group,SAH group,catalpol group,and catalpol+U0126 group(catalpol+Raf-MEK-ERK signal pathway inhibitor U0126). A rat SAH model was established using intravascular perforations. And evaluate neural function and SAH grading in rats. Evans Blue(EB) was applied to detect blood brain barrier permeability. Hematoxylin eosin(HE) staining was applied to observe pathological changes in brain tissue. Immunofluorescence staining was applied to detect neuronal apoptosis and the expression of LC3-Ⅱ,p-ERK positive cells. Western blot was used to detect the expression of Raf,MEK,phosphorylation(p)-MEK,ERK1/2,p-ERK1/2,B-cell lymphoma 2 (Bcl-2),Bcl-2 related X protein(Bax),autophagy gene Beclin-1,and microtubule associated proteinlight chain 3-Ⅱ(LC3-Ⅱ) in brain tissue. [Results] Compared with the sham operation group,the neuronal cells in the SAH group were loosely arranged and the number decreased,the SAH grading,brain tissue water content,EB exudation,TUNEL positive cells,apoptosis rate,LC3-Ⅱ,p-ERK positive expression,Bax,Beclin-1,LC3-Ⅱ,Raf,p-MEK/MEK,p-ERK1/2/ERK1/2 expression increased obviously,the neurological function score and Bcl-2 expression decreased (P<0.05);compared with the SAH group,the neuronal damage in the catalpol group reduced obviously,and cell death reduced,SAH grade,brain tissue water content,EB exudation,TUNEL positive cell count,apoptosis rate,Bax decreased obviously,and neurological function score,Bcl-2 expression increased,LC3-Ⅱ,p-ERK positive expression,Bax,Beclin-1,LC3-Ⅱ,Raf,p-MEK/MEK,p-ERK1/2/ERK1/2 expression further increased (P<0.05);Raf-MEK-ERK pathway inhibitor U0126 was able to reverse the improvement effect of catalpol on brain tissue damage (P<0.05). [Conclusion] Catalpol may promote autophagy of neural cells and improve brain injury in SAH rats by activating the Raf-MEK-ERK signaling pathway.