Department of Neurosurgery, The Third Medical Centre Chinese PLA (People's Liberation Army) General Hospital, Beijing.
Eur J Histochem. 2022 Jan 12;66(1):3350. doi: 10.4081/ejh.2022.3350.
The purpose of this study was to investigate the effect of Ghrelin on the polarization of microglia/ macrophages after cerebral ischemia (CI) in rats. 60 wild-type SD rats were randomly divided into sham group, CI group, CI+Ghrelin group, 20 rats in each group. The modified Longa suture method was used to establish the middle cerebral artery occlusion (MCAO) model in rats. Before surgery, Ghrelin was injected subcutaneously (100μg/kg, twice a day) for 4 consecutive weeks. After modeling, neurological function scores were performed with three behavioral experiments: mNSS score, Corner test, and Rotarod test, to evaluate the recovery of neurological function after Ghrelin treatment. At the same time, the brain tissues were collected and stained with 2,3,5-triphenyltetrazolium chloride (TTC) to detect the cerebral infarct volume. RT-qPCR was used to detect the expression of TNF-α and IL-1β in the ischemic brain tissue, and the TUNEL staining was used to detect the apoptosis of brain tissue. Flow cytometry was used to detect the percentage of M1 type microglia/macrophages which were isolated by trypsin digestion of fresh cerebral cortex. Then, the Western blotting and immunofluorescence method were used to detect the phosphorylation level of AKT (P-AKT) and AKT. Compared with the CI group, the neurological function of the rats in the CI+Ghrelin group was dramatically improved, and the cerebral infarction area was dramatically reduced. At the same time, the expression of TNF-α and IL-1β in the ischemic brain tissue of rats in the CI+Ghrelin group decreased, and the apoptotic cells in the brain tissue also decreased. Compared with the CI treatment group, the activation of M1 microglia/macrophages in the cortex of the ischemic side of the infarct and the peri-infarct area in the CI+Ghrelin group was dramatically inhibited. At the same time, the ratio of P-AKT/AKT of the brain tissue in the CI+Ghrelin group was dramatically higher than that of the CI group. In the rat cerebral ischemia model, Ghrelin can promote the repair of brain damage and the recovery of neurological function after ischemia. Its mechanism may be related to activating AKT to selectively reduce M1 microglia/macrophages, reducing inflammation and cell apoptosis in brain tissue.
本研究旨在探讨 Ghrelin 对大鼠脑缺血（CI）后小胶质细胞/巨噬细胞极化的影响。60 只野生型 SD 大鼠随机分为假手术组、CI 组、CI+Ghrelin 组，每组 20 只。采用改良的 Longa 缝线法建立大鼠大脑中动脉闭塞（MCAO）模型。术前连续 4 周皮下注射 Ghrelin（100μg/kg，每天 2 次）。造模后，采用 mNSS 评分、Corner 试验和转棒试验进行 3 项行为学实验，评估 Ghrelin 治疗后神经功能恢复情况。同时取脑组织，用 2,3,5-三苯基氯化四氮唑（TTC）染色检测脑梗死体积。采用 RT-qPCR 检测缺血脑组织中 TNF-α 和 IL-1β 的表达，TUNEL 染色检测脑组织凋亡情况。采用胰酶消化新鲜大脑皮质分离 M1 型小胶质细胞/巨噬细胞，流式细胞术检测其百分比。然后采用 Western blot 和免疫荧光法检测磷酸化 AKT（P-AKT）和 AKT 的表达水平。与 CI 组相比，CI+Ghrelin 组大鼠的神经功能明显改善，脑梗死面积明显减小。同时，CI+Ghrelin 组大鼠缺血脑组织中 TNF-α 和 IL-1β 的表达降低，脑组织中凋亡细胞也减少。与 CI 治疗组相比，CI+Ghrelin 组梗死灶及梗死灶周围皮质区 M1 型小胶质细胞/巨噬细胞的激活明显受到抑制。同时，CI+Ghrelin 组脑组织中 P-AKT/AKT 的比值明显高于 CI 组。在大鼠脑缺血模型中，Ghrelin 可促进脑损伤修复和缺血后神经功能恢复。其机制可能与激活 AKT 选择性减少 M1 型小胶质细胞/巨噬细胞，减少脑组织炎症和细胞凋亡有关。
