表皮生长因子受体抑制剂对氧糖剥夺/复氧诱导的血脊髓屏障损伤的保护作用研究

Protective Effect of EGFR Inhibitor on Blood Spinal Barrier Damage Induced by Oxygen Glucose Deprivation/Re-Oxygenation

目的:探讨表皮生长因子受体(EGFR)抑制剂PD168393对体外氧糖剥夺/复氧(OGD/R)诱导的血脊髓屏障(BSCB)损伤的保护作用及其可能的机制。方法:体外培养大鼠来源的脊髓微血管内皮细胞和混合胶质细胞,应用transwell培养体系建立体外BSCB模型;将培养的细胞分为3组:对照组(正常培养)、损伤组(OGD/R处理)和治疗组(OGD/R后给予10 nM PD168393干预)。损伤组和治疗组复氧3 h、6 h和12 h后,应用荧光素渗漏实验及内皮细胞跨膜电阻值(TEER)测定来评估各组BSCB通透性变化;应用免疫荧光、Western Blot技术检测各组内皮细胞间紧密连接蛋白ZO-1、Occludin表达的差异;应用ELISA法检测各组细胞分泌致炎因子白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)和诱导型一氧化氮合酶(iNOS)的差异。结果:损伤组在各个时间点的荧光素渗漏量均高于对照组(均P<0.05),而治疗组荧光素渗漏量均低于损伤组(均P<0.05);损伤组TEER值显著低于对照组(均P<0.01),而治疗组TEER值显著高于损伤组(均P<0.05);损伤组紧密连接蛋白ZO-1和Occludin的表达量低于对照组(均P<0.05),而治疗组ZO-1和Occludin的表达量较损伤组显著提高(均P<0.05);损伤组分泌的致炎因子IL-6、iNOS、TNF-α均显著高于对照组,治疗组在各时间点致炎因子IL-6、iNOS、TNF-α的分泌均低于损伤组(均P<0.05)。结论:EGFR抑制剂PD168393有助于维持OGD/R损伤后BSCB的完整性;其机制可能与抑制致炎因子的表达及减少BSCB紧密连接蛋白的破坏有关。

Objective:To investigate the protective effect and possible mechanism of epidemical growth factor receptor(EGFR)inhibitor PD168393 on the blood spinal cord barrier(BSCB)damage induced by oxygen glucose deprivation/re-oxygenation(OGD/R)in vitro.Methods:Primary microvascular endothelial cells and mixed glial cells from the rat spinal cord were cultured using the transwell culture system to establish the in vitro BSCB model.The cultured cells were divided into three groups:control group(untreated),injury group(BSCB damage induced by OGD/R),and treatment group(10 n M PD168393 intervention after OGD/R).The permeability of endothelial cells in the injury and treatment groups was evaluated by the fluorescein leakage test and transepithelial electrical resistance(TEER)at 3 h,6 h,and 12 h after reoxygenation.The expression of tight junction proteins ZO-1 and Occludin in the endothelial cells of each group was determined by immunofluorescence and Western blot.The differences in proinflammatory factors interleukin-6(IL-6),tissue necrosis factor-α(TNF-α),and inducible nitric oxide synthase(iNOS)secreted by each group of cells were measured by ELISA.Results:The fluorescein leakage test showed that the amount of fluorescein leakage in the injury group was higher than that in the control group at every time point(all P<0.05),while the amount of fluorescein leakage in the treatment group was significantly lower than that in the injury group(all P<0.05).TEER value of the injury group was significantly lower than that of the control group(all P<0.01),while TEER value of the treatment group was significantly higher than that of the injury group(all P<0.05).The expression of ZO-1 and Occludin in the injury group was significantly lower than those in the control group(both P<0.05),and the expression of these proteins in the treatment group was significantly higher than those in the injury group(both P<0.05).The expression levels of IL-6,TNF-α,and i NOS in the injury group were significantly higher than those in the control group at every time point,and the levels in the treatment group were lower than those in the injury group at every time point(all P<0.05).Conclusion:The EGFR inhibitor PD168393 can preserve the integrity of the BSCB after OGD/R.The mechanism may involve reducing the expression of proinflammatory factors and suppressing the destruction of tight junction proteins in the BSCB.