LXW7 ameliorates focal cerebral ischemia injury and attenuates inflammatory responses in activated microglia in rats

Inflammation plays a pivotal role in ischemic stroke, when activated microglia release excessive pro-inflammatory mediators. The inhibition of integrin αvβ3 improves outcomes in rat focal cerebral ischemia models. However, the mechanisms by which microglia are neuroprotective remain unclear. This study evaluated whether post-ischemic treatment with another integrin αvβ3 inhibitor, the cyclic arginine-glycine-aspartic acid (RGD) peptide-cGRGDdvc (LXW7), alleviates cerebral ischemic injury. The anti-inflammatory effect of LXW7 in activated microglia within rat focal cerebral ischemia models was examined. A total of 108 Sprague-Dawley rats (250–280 g) were subjected to middle cerebral artery occlusion (MCAO). After 2 h, the rats were given an intravenous injection of LXW7 (100 μg/kg) or phosphate-buffered saline (PBS). Neurological scores, infarct volumes, brain water content (BWC) and histology alterations were determined. The expressions of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)], and Iba1-positive activated microglia, within peri-ischemic brain tissue, were assessed with ELISA, western blot and immunofluorescence staining. Infarct volumes and BWC were significantly lower in LXW7-treated rats compared to those in the MCAO + PBS (control) group. The LXW7 treatment lowered the expression of pro-inflammatory cytokines. There was a reduction of Iba1-positive activated microglia, and the TNF-α and IL-1β expressions were attenuated. However, there was no difference in the Zea Longa scores between the ischemia and LXW7 groups. The results suggest that LXW7 protected against focal cerebral ischemia and attenuated inflammation in activated microglia. LXW7 may be neuroprotective during acute MCAO-induced brain damage and microglia-related neurodegenerative diseases.

炎症在缺血性脑卒中中发挥关键作用，活化小胶质细胞（microglia）会释放过量促炎介质。抑制整合素αvβ3（integrin αvβ3）可改善大鼠局灶性脑缺血模型的预后，但小胶质细胞发挥神经保护作用的具体机制仍未明确。本研究旨在探讨缺血后给予另一种整合素αvβ3抑制剂——环精氨酸-甘氨酸-天冬氨酸（RGD）肽cGRGDdvc（LXW7）是否可减轻脑缺血损伤，并检测了LXW7对大鼠局灶性脑缺血模型中活化小胶质细胞的抗炎作用。

共计108只体重250~280g的Sprague-Dawley（SD）大鼠，建立大脑中动脉闭塞（middle cerebral artery occlusion, MCAO）模型。造模2小时后，经静脉注射给予LXW7（100 μg/kg）或磷酸盐缓冲液（phosphate-buffered saline, PBS）。随后检测各组大鼠的神经功能评分、脑梗死体积、脑含水量（brain water content, BWC）及组织学变化；采用酶联免疫吸附试验（ELISA）、蛋白质印迹法（western blot）及免疫荧光染色法，检测缺血周边脑组织中促炎细胞因子[肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）、白细胞介素-1β（interleukin-1β, IL-1β）]的表达水平，以及Iba1阳性活化小胶质细胞的情况。

结果显示，与MCAO+PBS（对照组）大鼠相比，LXW7干预组的脑梗死体积与脑含水量均显著降低；LXW7干预可下调促炎细胞因子的表达水平，Iba1阳性活化小胶质细胞的数量减少，肿瘤坏死因子-α与白细胞介素-1β的表达亦被抑制。但缺血模型组与LXW7干预组的Zea Longa评分无显著差异。

综上，LXW7可对局灶性脑缺血起到保护作用，并抑制活化小胶质细胞的炎症反应，其或许可在急性大脑中动脉闭塞诱导的脑损伤中发挥神经保护作用，亦可用于治疗小胶质细胞相关神经退行性疾病。