栀子甙对慢性脑缺血认知障碍大鼠神经炎症反应的调节

Effects of Geniposide on the Neuroinflammation in Chronic Cerebral Hypoperfusion Rat Model

目的观察栀子甙对慢性脑缺血认知障碍大鼠的保护作用,并探讨其作用机制。方法采用双侧颈总动脉永久性结扎建立慢性脑缺血大鼠模型,假手术组作为正常对照。在手术8周后,采用Morris水迷宫实验筛选符合痴呆标准的大鼠,随机分为生理盐水组、栀子甙低剂量〔50 mg/(kg·d)〕治疗组及栀子甙高剂量〔100 mg/(kg·d)〕治疗组。按其分组连续灌胃干预4周后,再行Morris水迷宫实验评估学习记忆能力,HE染色观察皮层海马病理改变;Western blot检测额叶和海马胶质纤维酸性蛋白(glial fibrillary acidic portein,GFAP)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和核转录因子kapa B(nuclear factor-kapa B,NF-κB)蛋白的表达;ELISA检测额叶和海马炎性因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素(interleukin,IL)-6的水平。结果连续干预4周后,与假手术组相比,生理盐水组大鼠在水迷宫测试中,逃避潜伏期延长以及目标象限活动减少(P<0.05),且行为学测试成绩差于干预前(P<0.05);与假手术组相比,栀子甙低剂量治疗组和栀子甙高剂量治疗组逃避潜伏期延长以及目标象限活动也减少(P<0.05),但是相应成绩优于生理盐水组(P<0.05),且与干预前成绩相比无明显变化(P>0.05)。HE染色结果显示,生理盐水组大鼠海马和皮层神经元退化明显,细胞数目明显减少,排列松散,层次减少紊乱,深染细胞多见;与生理盐水组相比,栀子甙低剂量治疗组和栀子甙高剂量治疗组相应病理损害减轻。与假手术组相比,生理盐水组、栀子甙低剂量治疗组及栀子甙高剂量治疗组大鼠海马和额叶GFAP、iNOS、NF-κB的表达及炎性因子TNF-α和IL-6的水平均增加(P<0.05),但与生理盐水组相比,栀子甙低剂量治疗组及栀子甙高剂量治疗组相应值均减少(P<0.05)。结论栀子甙可能通过抑制星形胶质细胞活化,减少iNOS、NF-κB的表达及炎性因子TNF-α和IL-6的释放介导其对慢性脑缺血认知障碍大鼠的保护作用。

Objective To investigate the effects and the mechanism of geniposide on the neuroinflammation occured in the neurodegeneration course of a chronic cerebral hypoperfusion rat model.Methods Permanent bilateral common carotid arteries occlusions was performed to induce gradient cognitive deficit in rats.The sham group was used as control group.Then 18 rats that met the Screening Criteria were randomly selected 8 weeks post surgery,and were randomly divided into three groups,the 2-VO rats with saline solution group(2-VO+saline group),2-VO rats with50 mg/kg per day geniposide group(2-VO+G50)and 2-VO rats with 100 mg/kg per day geniposide group(2-VO+G100).All intervention groups were daily administered with geniposide or saline for 4 weeks.The sham-operated rats were administrated with saline.Then the rats were tested for Morris water maze to evaluate the memory and learning ability.Rats were sacrificed to obtain cortex and hippocampus tissues for HE staining and to detect expression level of glial fibrillary acidic protein(GFAP),inducible nitric oxide synthase(iNOS)and nuclear factor-kappa B(NF-κB),and the level of inflammatory factors tumor necrosis factor-α(TNF-α)and interleukin(IL)-6.Results The 2-VO+saline group rats showed significant longer escape latency and less percent time in target quadrant,compared with sham-operation group(P<0.05).The escape latency of 2-VO+G50 and 2-VO+G100 groups were shorter than the 2-VO+saline group(P<0.05),but still longer than the sham group(P<0.05),the percent time in target quadrant of which were more than the 2-VO+saline group and less than the sham group.However,there was no significant difference between these two groups.HE staining of sham group showed that neurons in the cortex and hippocampus lined up in order,cellar nucleus were big and globular.HE staining results showed that there were obviously neuoral cells loss,severe cytomorphosis,structural disappearance and nuclear fragmentation in the 2-VO+saline group.The 2-VO+G50 and 2-VO+G100 groups showed less neurodamage than the 2-VO+saline group with less neuoral cells loss,cytomorphosis and ambiguous nucleus.GFAP,iNOS,NF-κB were all highly expressed in the process of cognitive dysfunction in rats after chronic cerebral ischemia,however geniposide intervention(50 and 100 mg/kg per day)significantly decreased the expression of the above proteins.In addition,much more TNF-αand IL-6 were released in brain induced by chronic cerebral ischemia,and the levels were decreased after chronic geniposide oral treatment.No significant differences were detected between 2-VO+G50 and 2-VO+G100 groups.Conclusion These findings demonstrated that geniposide significantly prevented cognition deterioration induced by chronic cerebral hypoperfusion in rats.Geniposide inhibited neuroinflammation occurred in the process of chronic cerebral ischemia probably via reducing iNOS and NF-κB expression and suppressing the release of inflammatory factor TNF-αand IL-6.