硫辛酸对脑缺血再灌注损伤大鼠RAGE/LRP1通路及神经血管单元重塑的影响

目的 探究硫辛酸对脑缺血再灌注损伤大鼠神经血管单元重塑的作用及对晚期糖基化终产物受体（RAGE）/低密度脂蛋白受体相关蛋白1（LRP1）的影响。方法 108只雄性Wistar大鼠，除18只作为假手术组外，其余大鼠采用线栓法制备大脑中动脉短暂缺血再灌注（tMCAO）模型。模型成功大鼠根据神经评分分为模型组、FPS-ZM1 （RAGE抑制剂，1 mg·kg^-1）组和硫辛酸高、中、低剂量组（40、20、10 mg·kg^-1），再灌注2 h后ip给药，假手术组和模型组注射等量生理盐水，每天1次，连续给药14 d。术后1、7、14 d对大鼠进行神经功能缺损程度评分（mNSS）和激光散斑成像仪监测脑血流量（CBF）。术后14 d，2，3，5-氯化三苯基四氮唑（TTC）染色法检测大鼠脑梗死面积；伊文思蓝（EB）检测血脑屏障通透性；免疫荧光染色检测脑微血管密度并采用免疫组化法检测胶质纤维酸性蛋白（GFAP）、神经元核抗原（NeuN）表达；实时荧光定量PCR（qRT-PCR）检测血管内皮生长因子（VEGF）、促血管生成素1（Ang-1）、促血管生成素2（Ang-2）的mRNA表达； Western blotting检测基质金属蛋白酶9（MMP9）、紧密连接相关蛋白5（claudin5）、RAGE、LRP1蛋白表达。结果 与假手术组比较，模型组大鼠mNSS评分、脑梗死百分比、EB含量、GFAP阳性细胞数、脑组织Ang-2 mRNA水平、MMP9和RAGE蛋白表达显著升高（P<0.05），CBF、NeuN阳性细胞数、VEGF和Ang-1 mRNA水平、claudin5和LRP1蛋白表达显著降低（P<0.05）；与模型组比较，硫辛酸高、中剂量组大鼠mNSS评分、脑梗死百分比、EB含量、GFAP阳性细胞数、脑组织Ang-2 mRNA、MMP9和RAGE蛋白表达显著降低（P<0.05），CBF、脑微血管密度、NeuN阳性细胞数、VEGF和Ang-1 mRNA、claudin5和LRP1蛋白表达显著升高（P<0.05）。结论 硫辛酸可降低BBB的通透性，促进血管新生，对神经血管单元起着修复和保护作用；其作用机制可能与下调RAGE的表达，上调LRP1的表达有关。

Effects of lipoic acid on RAGE/LRP1 pathway and neurovascular unit remodeling in rats with cerebral ischemia-reperfusion injury

Objective To investigate the effects of lipoic acid on neurovascular unit remodeling and microvascular regeneration in rats with cerebral infarction and the effect of receptor for advanced glycation end products (RAGE)/low-density lipoprotein receptor related protein 1 (LRP1). Methods A total of 108 male Wistar rats were used to establish tMCAO model by suture method except 18 rats as sham operation group. The successful rats were divided into model group, high, medium and low dose lipoic acid groups (40, 20, 10 mg·kg^-1), RAGE inhibitor group (FPS-ZM1, 1.0 mg·kg^-1), with 18 rats in each group. After two hours of reperfusion, the drug was administered, once a day for 14 consecutive days. On the 1st, 7th and 14th day after operation, neurological deficit score (mNSS) was performed, the cerebral blood flow (CBF), cerebral infarction area (CI) and blood-brain barrier permeability (BBB) were measured by laser speckle imaging, 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and Evans blue (EB) respectively; the number of cerebral microvessels was measured by immunofluorescence staining, and the expression of glial fibrillary acidic protein (GFAP) and neuron nuclear antigen (NeuN) were detected by immunohistochemistry; real-time fluorescent quantitative PCR (RTPCR) was used to detect the mRNA expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2); Western blot was used to detect the expression of MMP9, claudin5, RAGE and LRP1. Results Compared with those in sham operation group, mNSS score, cerebral infarction ratio, EB content, GFAP positive cells number, Ang-2 mRNA, MMP9 and RAGE protein expression in brain tissue of rats in model group were significantly increased (P < 0.05), CBF, NeuN positive cells number, VEGF and Ang-1 mRNA, claudin5 and LRP1 protein expression were significantly decreased (P < 0.05); compared with those in the model group, mNSS score, cerebral infarction ratio, EB content, GFAP positive cells number, Ang-2 mRNA, MMP9 and RAGE protein expression in brain tissue of rats in the high and middle dose lipoic acid groups were significantly decreased (P < 0.05), CBF, microvessel density, NeuN positive cells number, VEGF and Ang-1 mRNA, claudin5 and LRP1 protein expression were significantly increased (P < 0.05). Conclusion Lipoic acid can reduce BBB permeability, promote angiogenesis, repair and protect neurovascular units; its mechanism may be related to down-regulating RAGE expression and up-regulating LRP1 expression.