基于Nrf2/HO-1通路探讨藏红花素对后循环缺血性眩晕大鼠的影响及机制

Crocin Treats Posterior Circulation Ischemic Vertigo in Rats via Nrf2/HO-1 Pathway

目的研究藏红花素对后循环缺血性眩晕(Posterior circulation ischemic vertigo,PCIV)大鼠的影响,并基于核因子E2相关因子2/血红素加氧酶1(Nrf2/HO-1)通路探索其作用机制。方法将60只清洁级雄性SD大鼠按随机数字表法分为5组:假手术组、模型组、藏红花素(60 mg/kg)组、ML385(Nrf2抑制剂,30 mg/kg)组和藏红花素(60 mg/kg)+ML385(30 mg/kg)组。除假手术组外,其余4组通过结扎右侧颈总动脉和右侧锁骨下动脉构建PCIV动物模型。各组分别1次/d连续治疗1周后,通过电刺激逃避实验考察大鼠眩晕症状程度,检测前庭神经核血流量、血液流变学指标[全血黏度(低切、中切、高切)、血浆黏度]和红细胞参数[聚集指数(Erythrocyte aggregation index,EAI)、红细胞刚性指数(Index of rigidity of erythrocyte,IR)、红细胞压积(Hematocrit,HCT)、血沉(Erythrocyte sedimentation rate,ESR)],HE染色观察脑组织病理变化,检测脑组织乳酸(Lactic acid,Lac)、乳酸脱氢酶(Lactate dehydrogenase,LDH)、白细胞介素1β(Interleukin-1β,IL-1β)、白细胞介素-6(Interleukin-6,IL-6)、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、丙二醛(Malondialdehyde,MDA)、超氧化物歧化酶(Superoxide dismutase,SOD)、过氧化氢酶(Catalase,CAT)活性,Western blot法检测脑组织核因子E2相关因子2(Nuclear factor E2 related factor 2,Nrf2)、血红素加氧酶1(Heme oxygenase 1,HO-1)、胞浆NF-κB p65、胞核NF-κB p65蛋白表达水平。结果与模型组比较,藏红花素组电刺激逃避潜伏期缩短,前庭神经核血流量升高,全血黏度、血浆黏度、EAI、IR、HCT、ESR降低;脑组织结构疏松,神经元数量减少,空泡变性、胞核偏移、核膜边界不清等病理变化明显改善;脑组织Lac、LDH、IL-1β、IL-6、TNF-α、MDA含量降低,SOD、CAT活性升高,Nrf2、HO-1表达量升高,胞核NF-κB p65表达量和胞核NF-κB p65/胞浆NF-κB p65比值降低,差异有统计学意义(P<0.05)。ML385作用与藏红花素相反,ML385组眩晕症状及其他检测指标变化较模型组更加严重。ML385可明显逆转藏红花素对PCIV大鼠眩晕症状、前庭神经核血流量的改善作用及Nrf2、HO-1、胞核NF-κB p65蛋白表达等其他指标的调控作用。结论藏红花素可改善PCIV大鼠眩晕症状和前庭神经核血流量,抑制炎症反应和氧化应激损伤,减轻脑组织损伤,其机制可能与激活Nrf2/HO-1通路、抑制NF-κB核转位有关。

Objective To study the effect of crocin on posterior circulation ischemic vertigo(PCIV)in rats and to decipher the underlying mechanism based on the nuclear factor E2-related factor 2(Nrf2)/heme oxygenase 1(HO-1)pathway.Methods According to the random number table method,60 SPF male SD rats were assigned into 5 groups:sham,model,crocin(60 mg/kg),ML385(Nrf2 inhibitor,30 mg/kg),and crocin(60 mg/kg)+ML385(30 mg/kg).The rats in other 4 groups except the sham group were modeled for PCIV by ligating the right common carotid artery and the right subclavian artery.After continuous treatment for 1 week in each group once a day,the degree of vertigo was investigated by electrical stimulation escape test.The blood flow of vestibular nucleus,hemorheological indexes[whole blood viscosity(low,middle,and high shear)and plasma viscosity],and erythrocyte parameters[erythrocyte aggregation index(EAI),index of rigidity(IR),hematocrit(HCT),and erythrocyte sedimentation rate(ESR)]were measured.Hematoxylin-eosin staining was employed to observe the pathological changes of the brain tissue.The levels of lactic acid(Lac),lactate dehydrogenase(LDH),interleukin(IL)-1β,IL-6,tumor necrosis factor-α(TNF-α),and malondialdehyde(MDA)and the activities of superoxide dismutase(SOD)and catalase(CAT)in the brain tissue were measured.Western blotting was employed to determine the protein levels of Nrf2 and HO-1 in the brain tissue,nuclear factorκB p65(NF-κB p65)in the cytoplasm,and NF-κB p65 in the nucleus.Results Compared with the model group,crocin shortened the electrical stimulation escape latency,increased the blood flow of vestibular nucleus,decreased the whole blood viscosity,plasma viscosity,EAI,IR,HCT,and ESR,and alleviated the pathological changes such as loose brain tissue structure,reduced neurons,vacuolar degeneration,nuclear deviation,and unclear nuclear membrane boundary.Moreover,crocin lowered the levels of Lac,LDH,IL-1β,IL-6,TNF-α,and MDA in the brain tissue,increased the activities of SOD and CAT,up-regulated the expression of Nrf2 and HO-1,down-regulated the expression of NF-κB p65 in the nucleus,and decreased the ratio of nuclear NF-κB p65 to cytoplasmic NF-κB p65(all P<0.05).The effect of ML385 was opposite to that of crocin,and the changes of vertigo symptoms and other indicators in the ML385 group were more serious than those in the model group.ML385 obviously reversed the alleviating effects of crocin on vertigo symptoms,blood flow of vestibular nucleus,and expression of Nrf2,HO-1,and nuclear NF-κB p65 in the rat model of PCIV.Conclusion Crocin can alleviate vertigo symptoms,recover the blood flow of vestibular nucleus,inhibit inflammatory response and oxidative stress,and reduce brain tissue damage in the rat model of PCIV by activating the Nrf2/HO-1 pathway and inhibiting the NF-κB nuclear translocation.