C1q肿瘤坏死因子相关蛋白6破坏大鼠脑缺血再灌注损伤后血脑屏障的机制研究

Mechanism of C1q and Tumor Necrosis Factor Related Protein 6 Destroying the Blood-Brain Barrier after Cerebral Ischemia Reperfusion Injury in Rats

目的 探讨C1q肿瘤坏死因子相关蛋白6(C1q and tumor necrosis factor related protein 6,C1QTNF6)对大鼠大脑中动脉缺血再灌注(middle cerebral artery occlusion reperfusion,MCAO/R)后血脑屏障及内皮细胞闭合蛋白(occludin)和闭锁小带蛋白-1(zonula occludens 1,ZO-1)表达的影响。方法 将健康雄性SD大鼠随机分为假手术组、MCAO/R组、shRNA-C1QTNF6组,造模前两组大鼠通过尾静脉注射生理盐水,shRNA-C1QTNF6组大鼠注射慢病毒载体进而沉默C1QTNF6的信使RNA(messenger RNA,mRNA)水平,注射3 d后各组用改良线栓法建立大鼠MCAO/R模型,缺血2 h,再灌注24 h。于造模后和再灌注24 h采用改良神经功能缺损评分(modified neurological severity score,m NSS)法评价各组神经行为功能,TTC染色比较脑梗死体积比值,蛋白质印迹法(western blot,WB)检测梗死区顶叶半暗带脑组织蛋白C1QTNF6、IL-1β、occludin、ZO-1的表达水平,尼氏染色和苏木精-伊红染色观察梗死区顶叶半暗带病理损伤变化,荧光Tunel/Neun双染法计数梗死区顶叶半暗带神经元凋亡。结果 shRNA-C1QTNF6组造模后(10.1±0.6分vs.10.7±1.0分,P=0.0003)与再灌注24 h(7.2±0.4分vs.7.9±0.8分,P=0.0001)m NSS均低于MCAO/R组,脑梗死体积比值(26.32%±5.71%vs.40.56%±7.74%,P=0.0004)低于MCAO/R组。WB检测结果显示,造模后MCAO/R组脑组织(0.66±0.06 vs.0.43±0.05,P=0.0229)C1QTNF6表达高于假手术组;shRNA-C1QTNF6组脑组织C1QTNF6表达均低于假手术组(0.15±0.03 vs.0.43±0.05,P=0.0067)和MCAO/R组(0.15±0.03vs.0.66±0.06,P=0.0001)。MCAO/R组和shRNA-C1QTNF6组IL-1β表达(0.76±0.07 vs.0.18±0.04,P=0.0001;0.47±0.07 vs.0.18±0.04,P=0.0118)均高于假手术组,shRNA-C1QTNF6组IL-1β表达(0.47±0.07 vs.0.76±0.07,P=0.0123)低于MCAO/R组。MCAO/R组和shRNA-C1QTNF6组occludin表达(0.47±0.03 vs.1.07±0.06,P=0.0001;0.84±0.05 vs.1.07±0.06,P=0.0124)和ZO-1表达(0.19±0.02 vs.0.76±0.03,P=0.0001;0.58±0.04 vs.0.76±0.03,P=0.0038)均低于假手术组,shRNA-C1QTNF6组occludin表达(0.84±0.05 vs.0.47±0.03,P=0.0003)和ZO-1表达(0.58±0.04vs.0.19±0.02,P=0.0001)均高于MCAO/R组。尼氏染色显示,shRNA-C1QTNF6组缺血区尼氏小体数量(361.4±18.3个vs.181.6±21.5个,P=0.0001)较MCAO/R组增多,神经元损伤程度更轻。苏木精-伊红染色显示,shRNA-C1QTNF6组缺血半暗带顶叶皮层病理损伤程度较MCAO/R组有所改善。Tunel/Neun双染法显示shRNA-C1QTNF6组(19.67±0.88个vs.45.00±2.89个,P=0.0002)Tunel/Neun双阳性细胞数量比MCAO/R组减少,沉默C1QTNF6表达可减少神经元凋亡。结论 抑制大鼠C1QTNF6表达可能通过下调IL-1β的表达,促进内皮细胞紧密连接蛋白occludin和ZO-1表达上升,减轻脑血管内皮紧密连接蛋白损伤,发挥对脑缺血再灌注后血脑屏障的保护作用。

Objective To investigate the effects of C1q and tumor necrosis factor related protein 6(C1QTNF6)on the blood-brain barrier and the expression of occludin and tight junction proteins zonula occludens 1(ZO-1) after middle cerebral artery occlusion reperfusion(MCAO/R) in rats.Methods Healthy male SD rats were randomly divided into sham operation group,MCAO/R group and shRNA-C1QTNF6 group.Before modeling,rats in the first two groups were injected with normal saline through tail vein,and rats in the shRNA-C1QTNF6 group were injected with lentivirus vector to silence the messenger RNA(mRNA) level of C1QTNF6.Three days after transfection,cerebral ischemia/reperfusion model of rats was established by modified thread embolization method.Neurobehavioral function was evaluated by modified neurological severity score(mNSS) after modeling and 24 hours after reperfusion.Ratio of cerebral infarction volume(CIV) was observed by triphenyltetrazole chloride(TTC) staining.Western blot(WB) was used to detect the expression levels of proteins C1QTNF6,IL-1β,occludin and ZO-1 in penumbra of parietal lobe infarction area.Nissl staining and HE staining were used to observe the pathological changes of penumbra in parietal lobe infarction area.Fluorescence Tunel/Neun double staining method was used to count neuronal apoptosis in penumbra of parietal lobe infarction area.Results The mNSS scores after modeling(10.1±0.6 points vs.10.7±1.0 points,P=0.0003) and 24hours after reperfusion(7.2±0.4 points vs.7.9±0.8 points,P=0.0001) in shRNA-C1QTNF6 group were lower than that in MCAO/R group,and the ratio of cerebral infarction volume(26.32%±5.71%vs.40.56%±7.74%,P=0.0004) was lower than that in MCAO/R group.WB results showed that the expression of C1QTNF6 in MCAO/R group was higher than that in sham group(0.66±0.06vs.0.43±0.05,P =0.0229),the expression of C1QTNF6 in shRNA-C1QTNF6 group was lower than that in sham group(0.15±0.03 vs.0.43±0.05,P=0.0067) and MCAO/R group(0.15±0.03 vs.0.66±0.06,P=0.0001).IL-1β expression in MCAO/R group and shRNA-C1QTNF6 group(0.76±0.07vs.0.18±0.04,P=0.0001;0.47±0.07 vs.0.18±0.04,P=0.0118) were both higher than that in sham group.IL-1β expression in shRNA-C1QTNF6 group(0.47±0.07 vs.0.76±0.07,P=0.0123) was lower than that in MCAO/R group.The expression of occludin(0.47±0.03 vs.1.07±0.06,P=0.0001;0.84±0.05 vs.1.07±0.06,P=0.0124) and ZO-1(0.19±0.02 vs.0.76±0.03,P=0.0001;0.58±0.04 vs.0.76±0.03,P=0.0038) in MCAO/R group and shRNA-C1QTNF6 group were lower than those in sham group,the expression of occludin(0.84±0.05 vs.0.47±0.03,P=0.0003) and ZO-1 expression(0.58±0.04 vs.0.19±0.02,P=0.0001) in shRNA-C1QTNF6 group were higher than those in MCAO/R group.Nissl staining showed that shRNA-C1QTNF6 group had more Nissl bodies(361.4±18.3 vs.181.6±21.5,P =0.0001) and less neuron damage,compared with MCAO/R group.Hematoxylineosin staining showed that the pathological lesion of ischemic penumbra in parietal cortex improved in shRNA-C1QTNF6 group compared with MCAO/R group.Tunel/Neun double staining showed that the number of Tunel/Neun double positive cells in shRNA-C1QTNF6 group(19.67±0.88 vs.45.00±2.89,P=0.0002) was lower than that in MCAO/R group,and silencing C1QTNF6 expression reduced neuronal apoptosis.Conclusions Inhibiting the expression of C1QTNF6 plays a protective role in blood-brain barrier after cerebral ischemia reperfusion in rats,may by down-regulating the expression of IL-1β,up-regulating the expression of occludin and ZO-1,to reduce the damage of cerebral vascular endothelial tight junction protein.