烟酰胺通过抑制TLR4/NF-κB通路改善蛛网膜下腔出血大鼠早期脑损伤的研究

Nicotinamide improves early brain injury in rats with subarachnoid hemorrhage by inhibiting TLR4/NF-κB pathway

目的探讨聚(ADP-核糖)聚合酶-1(poly ADP-ribose polymerase-1,PARP-1)抑制剂烟酰胺(nicotinamide,NA)对蛛网膜下腔出血(subarachnoid hemorrhage,SAH)大鼠早期脑损伤的影响及其可能的作用机制。方法采用刺破大鼠颈内动脉构建蛛网膜下腔出血模型。实验分为假手术组、SAH组和SAH+NA组。造模48 h后,酶联免疫吸附实验(enzyme-linked immuno sorbent assay,ELISA)检测丙二醛(malondialdehyde,MDA)和烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinu-cleotide,NAD+)含量。免疫荧光检测8-OHdG的表达。Western Blotting检测Toll样受体4(Toll like receptor 4,TLR-4)、核转录因子-κB(nuclear transcription factor-κB,NF-κB)、白细胞介素-1β(in-terleukin-1β,IL-1β)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、PARP-1、聚腺苷二磷酸-核糖(pol ADP-ribose,PAR)及凋亡诱导因子(apoptosis inducing factor,AIF)的蛋白表达变化。通过测定脑水肿和伊文思蓝考察NA对血脑屏障的影响。SAH分级评分和神经功能评分考察NA对脑损伤的影响。结果大鼠SAH 48 h后MDA和8-OHdG表达增加,NAD+含量降低,NA能够抑制其变化。NA能够抑制SAH诱导的PARP-1、PAR及AIF的蛋白表达。NA能够显著减少TLR4、NF-κB、IL-1β、iNOS的表达。NA能够减少SAH后的脑水肿和伊文思蓝渗出,提高神经功能评分。结论NA可能通过抑制TLR4/NF-κB炎症通路改善蛛网膜下腔出血后的早期脑损伤。

Objective To investigate the effects of nicotinamide(NA),an inhibitor of poly ADP-ribose polymerase-1(PARP-1),on early brain injury in rats with subarachnoid hemorrhage(SAH)and its possible mechanism.Methods The SAH model was constructed by puncturing the internal carotid artery of rats.The rats were divided into sham group,SAH group and SAH+NA group.After 48 h,the contents of malondialdehyde(MDA)and nicotinamide adenine dinucleotide(NAD+)were detected by enzyme-linked immunosorbent assay(ELISA).Immunofluorescence examined the 8-OHdG expression.The expression of Toll like receptor 4(TLR-4),nuclear transcription factor-κB(NF-κB),interleukin-1β(IL-1β),inducible nitric oxide synthase(iNOS),PARP-1,poly(ADP-ribose)(PAR)and apoptosis-inducing factor(AIF)were detected by western Blotting.Cerebral edema and Evans Blue were measured to investigate the effect of NA on the blood-brain barrier.SAH grade and neurological score were used to investigate the effect of NA on brain injury.Results After SAH for 48 h,the expression of MDA and 8-OHdG increased,while the content of NAD+decreased.NA could inhibit the changes.NA could inhibit the protein expression of PARP-1,PAR and AIF induced by SAH.The expression of TLR4,NF-κB,IL-1βand iNOS could be significantly decreased by NA.NA could reduce brain edema and Evans blue exudation after SAH,and improve neurological score.Conclusion NA may improve early brain injury after SAH by inhibiting the TLR4/NF-κB inflammatory pathway.