Department of Anesthesiology, Dongzhimen Hospital Beijing University of Chinese Medicine, Beijing, China.
Eur Rev Med Pharmacol Sci. 2020 Apr;24(8):4412-4419. doi: 10.26355/eurrev_202004_21023.
The aim of this study was to explore the association between the expression of mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinase (ERK) pathway and neuronal apoptosis in rats with white matter lesions (WML).
Sprague-Dawley (SD) rats were selected as the research objects. Rat models of ischemic WML were established by bilateral common carotid artery ligation. Subsequently, brain tissues were collected from rats in sham operation group and WML group, respectively. Hematoxylin-eosin (HE) staining assay was conducted to observe the pathological changes in white matters (WMs) (callosum, internal capsule, and optic nerve) and apoptotic cells in brain tissues. The protein expression levels of phosphorylated ERK (p-ERK) and ERK, phosphorylated MAPK (p-MAPK), and MAPK in tissues were measured by Western blotting. Immunohistochemistry was employed to detect the expression levels of p-ERK, ERK, p-MAPK, and MAPK in brain tissues of the two groups. Next, nerve cells were isolated from rats with WML as research objects. The phosphorylation of the MAPK/ERK pathway was suppressed using PD03259019 (a chemical drug, hereafter referred to as PD). Then, the changes in the protein expressions of apoptosis proteins B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were determined before and after MAPK/ERK pathway inhibition. Meanwhile, changes in the messenger ribonucleic acid (mRNA) expression levels were detected via real-time fluorescent quantitative polymerase chain reaction (PCR), and changes in apoptosis were observed.
HE staining revealed that in sham operation group, WMs had normal structure and intact morphology. The cells were regularly arranged, with little apoptosis of the nuclei in the center. However, there were abnormally arranged nerve cells, loose cortical structure, swollen cells, aberrant nuclear membrane, pyknosis, signs of cell degeneration and necrosis, apoptotic cells filled most of the field of vision, and relatively evident lesions in WML group. Besides, WML group exhibited significantly up-regulated expressions of p-ERK and p-MAPK, as well as basically unchanged expressions of ERK and MAPK (p<0.05). After PD was added for 1 d, 2 d, and 3 d, the MAPK/ERK pathway was repressed, which was the most significantly at 3 d. Furthermore, the anti-apoptotic phenotype of neurons was detected, which was more pronounced at 3 d (p<0.05).
Rats with WML exhibited elevated MAPK/ERK activity and evident apoptosis. After inhibiting the phosphorylation site of MAPK/ERK in rat neuronal cells, the expression of pro-apoptotic protein decreased, and the apoptosis was relieved. In rats with WML, neuronal apoptosis is promoted by activating the MAPK/ERK pathway, thus worsening the condition.
本研究旨在探讨丝裂原活化蛋白激酶（MAPK）/细胞外调节蛋白激酶（ERK）通路的表达与脑白质病变（WML）大鼠神经元凋亡之间的关系。
选择 SD 大鼠作为研究对象。采用双侧颈总动脉结扎法建立大鼠缺血性 WML 模型。随后，分别采集假手术组和 WML 组大鼠的脑组织。苏木精-伊红（HE）染色观察白质（胼胝体、内囊和视神经）的病理变化和脑组织中的凋亡细胞。采用 Western blot 法检测组织中磷酸化 ERK（p-ERK）和 ERK、磷酸化 MAPK（p-MAPK）和 MAPK 的蛋白表达水平。采用免疫组织化学法检测两组脑组织中 p-ERK、ERK、p-MAPK 和 MAPK 的表达水平。然后，以 WML 大鼠的神经细胞为研究对象，采用 PD03259019（一种化学药物，以下简称 PD）抑制 MAPK/ERK 通路的磷酸化。然后，在抑制 MAPK/ERK 通路前后，检测凋亡蛋白 B 细胞淋巴瘤 2（Bcl-2）和 Bcl-2 相关 X 蛋白（Bax）的蛋白表达变化。同时，采用实时荧光定量聚合酶链反应（PCR）检测 mRNA 表达水平的变化，并观察凋亡情况。
HE 染色显示，假手术组 WMs 结构正常，形态完整。细胞排列整齐，中心核凋亡较少。然而，WML 组的神经细胞排列异常，皮质结构疏松，细胞肿胀，核膜异常，出现固缩、细胞变性坏死的迹象，凋亡细胞充满了大部分视野，病变较为明显。此外，WML 组 p-ERK 和 p-MAPK 的表达明显上调，而 ERK 和 MAPK 的表达基本不变（p<0.05）。加入 PD 后 1 d、2 d、3 d 抑制 MAPK/ERK 通路，3 d 时抑制作用最明显。此外，还检测到神经元的抗凋亡表型，3 d 时更为明显（p<0.05）。
WML 大鼠 MAPK/ERK 活性升高，凋亡明显。抑制大鼠神经元细胞 MAPK/ERK 磷酸化位点后，促凋亡蛋白表达减少，凋亡缓解。在 WML 大鼠中，激活 MAPK/ERK 通路促进神经元凋亡，从而加重病情。
