CpG-ODN对新生大鼠缺血/缺氧性脑损伤的保护作用研究

摘要

Aim To study the therapeutic effect of CpG-ODN, an agonist of Toll-like receptor 9 (TLR9), on hypoxic/ischemic encephapathy in neonatal rats and investigate the mechanisms.Methods Fifty healthy 7-day-old neonatal Wistar rats (in either gender, weighing 12~17g) were randomly divided into sham operation group, HIBD group, and CpG-ODN low group(0.35 mL·kg-1), CpG-ODN middle group(1.40 mL·kg-1), CpG-ODN high group(5.60 mL·kg-1).The neurological function was scored after 48h operation;ten rats of each group was executed respectively and brains tissue was taken;HE staining was used to observe the brain pathological changes.Western blot assay was used to detect the expressions of TLR9 and phosphor-p38 mitogen-activated protein kinases(p-p38 MAPK), and enzyme linked immunosorbent assay (ELISA) method was adopted to detect TNF-α expression.Results The CpG-ODN low, middle group were improved in impairment significantly compared with the HIBD group, and the brain pathological change was lessened, while the CpG-ODN high group was impaired significantly compared with the HIBD group (P<0.05), and brain pathological change was sharpened.Western blot showed the up-regulation in TLR9 and p-p38 MAPK and a significant increase of the expression of TNF-α in the brain tissue in CpG-ODN group with statistical difference in HIBD group and sham operation group(P<0.05).Conclusions The neuro-behavioral score and nervous system function can be improved and the hypoxic/ischemic brain damage can be reduced in neonatal rats in the CpG-ODN low, middle group.The protective mechanisms may be suitably via activating p38 MAPK signaling pathway to promote p38 MAPK phosphory1ation and up-regulation of the expression of TNF-α in the brain tissue of rats.%目的 探讨Toll 样受体9(Toll-like receptor 9,TLR9) 激动剂——含CpG 基序的寡核苷酸(CpG-ODN)对新生大鼠缺血/缺氧性脑损伤的保护作用机制.方法 50只健康7日龄新生Wistar大鼠,♀♂不限,随机分为5组,假手术组、缺血/缺氧性脑损伤(HIBD)组、CpG-ODN低剂量组(0.35 mL·kg-1)、CpG-ODN中剂量组(1.40 mL·kg-1)、CpG-ODN高剂量组(5.60 mL·kg-1).术后48 h对大鼠神经功能进行评分,光学显微镜下观察脑组织病理学改变.免疫印迹法检测缺血/缺氧侧脑组织中磷酸化p38 MAPK、TLR9表达,酶联免疫吸附法检测TNF-α的表达.结果 CpG-ODN低、中剂量组较模型组神经行为学评分降低,病理学损伤减轻,而高剂量组较模型组神经行为学评分升高(P<0.05),病理学损伤加重;Western blot 分析结果表明,缺血/缺氧侧脑组织中磷酸p38 MAPK、TLR9蛋白表达逐渐上调,TNF-α在脑组织中含量逐渐升高(P<0.05).结论 TLR9激动剂CpG-ODN低、中剂量可改善新生大鼠脑缺血/缺氧损伤神经行为学评分及神经系统功能,减轻新生大鼠缺血/缺氧性脑损伤,其机制可能是通过适度激活p38 MAPK信号通路,并分泌适量TNF-α 发挥作用.