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

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。 

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

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 zonulaoccludens 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/Rgroup and shRNA-C1QTNF6 group. Before modeling, rats in the first two groups were injectedwith normal saline through tail vein, and rats in the shRNA-C1QTNF6 group were injected withlentivirus vector to silence the messenger RNA (mRNA) level of C1QTNF6. Three days aftertransfection, cerebral ischemia/reperfusion model of rats was established by modified threadembolization method. Neurobehavioral function was evaluated by modified neurological severityscore (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 usedto detect the expression levels of proteins C1QTNF6, IL-1β, occludin and ZO-1 in penumbra ofparietal lobe infarction area. Nissl staining and HE staining were used to observe the pathologicalchanges of penumbra in parietal lobe infarction area. Fluorescence Tunel/Neun double stainingmethod 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 groupwere 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 thatthe 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 lowerthan 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 shamgroup. IL-1β expression in shRNA-C1QTNF6 group (0.47±0.07 vs . 0.76±0.07, P =0.0123) waslower 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 insham 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/Rgroup. 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. Hematoxylineosinstaining showed that the pathological lesion of ischemic penumbra in parietal cortex improvedin shRNA-C1QTNF6 group compared with MCAO/R group. Tunel/Neun double staining showedthat 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 expressionreduced neuronal apoptosis.Conclusions Inhibiting the expression of C1QTNF6 plays a protective role in blood-brainbarrier 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 vascularendothelial tight junction protein.