通窍活血汤调节Nrf2通路对新生大鼠缺血缺氧性脑损伤血管保护及神经功能改善作用研究

Effects of Tongqiao Huoxue Decoction regulating Nrf2 pathway on vascular protection and neurological improvement of newborn rats with ischemic hypoxic brain injury

[目的]研究通窍活血汤对新生大鼠缺血缺氧性脑损伤血管保护和神经功能改善作用及对核转录因子2(Nrf2)通路的调节作用。[方法]新生大鼠随机分为正常对照组(12只)及造模组(60只),采用颈总动脉结扎法建立缺血缺氧性脑损伤大鼠模型,并随机分为脑损伤组、通窍活血汤低、中、高剂量组及神经节苷脂组,每组12只,通窍活血汤低、中、高剂量组分别按照4、8、16 g/kg的剂量灌胃给予通窍活血汤,神经节苷脂组按照10 mg/kg的剂量腹腔注射神经节苷脂,每日1次,连续14 d,Morris水迷宫实验测定大鼠90 s内靶象限停留时间及穿越平台次数,酶联免疫吸附法(ELISA)测定血清可溶性内皮细胞蛋白C受体(sEPCR)、可溶性血栓调节蛋白(sTM)、内皮素(ET)-1含量、血管组织超氧化物歧化酶(SOD)、丙二醛(MDA)及肿瘤坏死因子(TNF)-α表达水平、脑组织TNF-α、白介素(IL)-1β及IL-6表达水平,苏木精-伊红(HE)染色法测定海马组织病理学改变,实时定量聚合酶链式反应(RT-qPCR)测定海马组织Nrf2、血红素氧合酶-1(HO-1)、Janus激酶2(JAK2)、信号转导和转录激活因子3(STAT3)m RNA表达水平,蛋白免疫印迹法(Western Blot)测定海马组织Nrf2、HO-1、JAK2、STAT3蛋白表达水平。[结果]与正常对照组比较,脑损伤组大鼠靶象限停留时间及穿越平台次数显著减少(P<0.05)。血管组织SOD表达水平,海马组织Nrf2、HO-1 m RNA及蛋白表达水平显著降低(P<0.05)。血清sEPCR、sTM、ET-1含量、血管组织MDA及TNF-α表达水平、脑组织TNF-α、IL-1β及IL-6水平、海马组织JAK2、STAT3 mRNA及蛋白水平显著升高(P<0.05)。与脑损伤组比较,通窍活血汤低、中、高剂量组及神经节苷脂组大鼠脑损伤组大鼠靶象限停留时间及穿越平台次数显著增加(P<0.05)。血管组织SOD表达水平、海马组织Nrf2、HO-1 mRNA及蛋白表达水平显著升高(P<0.05)。血清s EPCR、sTM、ET-1含量、血管组织MDA及TNF-α表达水平、脑组织TNF-α、IL-1β及IL-6表达水平、海马组织JAK2、STAT3 mRNA及蛋白表达水平显著降低(P<0.05),且通窍活血汤不同剂量组间存在显著差异(P<0.05)。[结论]通窍活血汤能够显著抑制缺血缺氧性脑损伤大鼠神经损伤及脑组织炎症反应,抑制血管氧化应激损伤,发挥脑组织及血管的保护作用,其机制可能与调节Nrf2通路有关。

[Objective]To study the vascular protection and neurological improvement effects of Tongqiao Huoxue Decoction on ischemichypoxic brain injury in newborn rats and the regulation of Nrf2 pathway.[Methods]Neonatal rats were randomly divided into normal control group(12 rats)and model group(60 rats).The model of ischemic hypoxic brain injury rats was established by carotid artery ligation method,and the rats were randomly divided into brain injury group,Tongqiao Huoxue Decoction low-dose group,medium-dose group,and high-dose group and ganglioside group,12 rats per group.Tongqiao Huoxue Decoction low-dose,medium-dose and high-dose groups were given Tongqiao Huoxue Decoction by intragastric administration at the dose of 4,8 and 16 g/kg,respectively,ganglioside group was given intraperitoneal injection of ganglioside at the dose of 10 mg/kg,once a day for 14 days.Morris water maze determination of rats in the 90 s target quadrant residence time and the number of cross platform.Enzyme-linked immunosorbent(ELISA)determination of serum sEPCR,sTM,ET-1 level,vascular tissue SOD,MDA and TNF-α level,the brain tissue TNF-αand IL-6,IL-1β level,Hematoxylin-eosin(HE)staining was used to determine histopathological changes in the hippocampus,and real-time quantitative polymerase chain reaction(RT-qPCR)was used to determine mRNA levels of Nrf2,HO-1,JAK2 and STAT3 in the hippocampus.The protein levels of Nrf2,HO-1,JAK2 and STAT3 in the hippocampus were determined by Western Blot.[Results]Compared with normal control group,the target quadrant residence time and the times of crossing the platform of rats in brain injury group were significantly reduced(P<0.05).SOD level in vascular tissue,mRNA and protein levels of Nrf2 and HO-1 in hippocampus were significantly decreased(P<0.05).Serum sEPCR,sTM,ET-1 level,vascular tissue MDA and the level of TNF-α,brain tissue TNF-α and IL-6,IL-1β level,hippocampus JAK2 and STAT3 mRNA and protein levels significantly increased(P<0.05);compared with brain injury group,the target quadrant residence time and the number of crossing the platform of rats in brain injury group were significantly increased in Tongqiao Huoxue Decoction low-dose,medium-dose and high-dose groups and ganglioside group(P<0.05).And SOD level in vascular tissue,mRNA and protein levels of Nrf2 and HO-1 in hippocampal tissue were significantly increased(P<0.05);serum sEPCR,sTM,ET-1 level,vascular tissue MDA and the level of TNF-α,brain tissue TNF-α beta and IL-6,IL-1βlevel,hippocampus JAK2 and STAT3 mRNA and protein levels significantly reduced(P<0.05),and there was significant difference between different dose groups of Tongqi Huoxue Decoction(P<0.05).[Conclusion]Tongqiao Huoxue Decoction can significantly inhibit nerve injury and brain tissue inflammation in rats with ischemic hypoxic brain injury,inhibit vascular oxidative stress injury,and play a protective role in brain tissue and blood vessels.The mechanism may be related to the regulation of Nrf2 pathway.