银杏内酯治疗缺血性脑卒中的临床疗效及作用机制研究

Clinical efficacy and mechanism of action of ginkgolide in the treatment of ischaemic stroke

目的观察银杏内酯治疗缺血性脑卒中的临床疗效,并探究其改善大鼠脑缺血再灌注损伤预后的机制。方法临床研究中,收集2022年1月至2023年9月安徽医科大学第一附属医院北区住院部60例缺血性脑卒中患者,按照随机数字表法分为观察组和对照组,各30例。对照组常规使用抗血小板药物、调脂稳斑、降血压、神经功能保护药物,观察组在此基础上联用银杏内酯注射液,疗程为10天。对比两组患者治疗前后神经功能(NIHSS)评分、临床疗效、日常生活活动能力(Barthel)评分及外周血单核细胞(PBMC)中Kelch样ECH关联蛋白1(Keap1)和核因子E2相关因子2(Nrf2)基因及蛋白表达水平。在动物实验中,30只SPF级雄性SD大鼠采用Zea-Longa线栓法建立大鼠脑缺血模型,所有大鼠分为假手术组(NC组)、模型组、银杏内酯组,各10只。以Zea-Longa评分法评估各组大鼠神经功能损伤程度,HE染色检测脑组织病理学改变,TTC染色检测脑梗死体积百分率,ELISA法检测脑组织和血清中白细胞介素(IL)-2和IL-6及肿瘤坏死因子-α(TNF-α)水平,Real-time PCR和Western blot方法分别检测脑组织和PBMC中Keap1和Nrf2基因及蛋白表达水平。结果临床研究中,观察组总有效率(93.33%)高于对照组(70.00%)(P<0.05)。与对照组治疗后相比,观察组治疗后NIHSS评分显著降低(P<0.05),Barthel指数评分显著升高(P<0.01),Keap1蛋白表达水平明显下调(P<0.01),Nrf2mRNA及蛋白水平明显上调(P<0.01)。动物实验中,与模型组比较,银杏内酯组大鼠神经功能评分显著降低(P<0.01);大鼠脑组织病理状况显著改善;脑组织梗死体积缩小(P>0.05);脑组织匀浆及血清中炎性因子TNF-α、IL-2、IL-6表达均明显下调(P<0.01);脑组织及PBMC中Keap1mRNA及蛋白表达水平明显下调(P<0.01),Nrf2mRNA及蛋白表达水平明显上调(P<0.01)。结论银杏内酯可能通过Keap1/Nrf2信号通路,抑制氧化应激和炎症反应,改善脑卒中血脑屏障功能,降低脑损伤,提高神经功能。

Objective To observe the clinical efficacy of ginkgolide in the treatment of ischaemic stroke and to investigate its mecha⁃nism for improving the prognosis of cerebral ischaemia-reperfusion injury in rats.Methods In clinical studies,60 patients with ischemic stroke were collected from the Inpatient Department of the North District of the First Affiliated Hospital of Anhui Medical University from Janu⁃ary 2022 to September 2023.They were divided into an observation group and a control group according to the random number table method,with 30 cases in each group.The control group routinely used antiplatelet drugs,lipid regulation and stabilisation of plaque,blood pressure low⁃ering and neurological function protection drugs,and the observation group received combined infection with ginkgolide injection on this basis for a period of 10 days.Neurological function(NIHSS)scores,clinical efficacy,ability to perform activities of daily living(Barthel)scores,and gene and protein expression levels of Kelch-like echassociated protein 1(Keap1)and nuclear factor erythroid 2 related factor2(Nrf2)in periph⁃eral blood mononuclear cells(PBMCs)were compared between the two groups before and after treatment.In animal experiments,the Zea-Longa suture method was used to establish a rat cerebral ischemia model,in which there were 10 rats in each of the sham-operated group(NC group),the model group,and the ginkgolide group.Zea-Longa score was used to evaluate the degree of neurological injury in each group.The pathological changes of brain tissue were detected by HE staining,the percentage of cerebral infarction volume was detected by TTC staining,and the levels of interleukin(IL-2),IL-6 and tumor necrosis factor-α(TNF-α)in brain tissue and serum were detected by ELISA.The expres⁃sion levels of Keap1 and Nrf2 gene and protein in brain tissue and PBMC were detected by Real-time PCR and Western blot,respectively.Re⁃sults In the clinical study,the total effective rate of the observation group(93.33%)was higher than that of the control group(70.00%)(P<0.05).Compared with the control group after treatment,the NIHSS score of the observation group was significantly lower(P<0.05),the Barthel Index score was significantly higher(P<0.01),the Keap1 protein expression level was significantly down-regulated(P<0.01),and the Nrf2 mRNA and protein levels were significantly up-regulated after treatment(P<0.01).In the animal experiment,compared with the model group,the neural function score of ginkgolide group was significantly reduced(P<0.01).The pathological status of brain tissue in rats was improved significantly.The volume of cerebral infarction was significantly decreased(P>0.05).The expressions of inflammatory factors TNF-α,IL-2 and IL-6 in brain homogenate and serum were significantly down-regulated(P<0.01).Keap1 mRNA and protein expression levels in brain tis⁃sue and PBMC were significantly down-regulated(P<0.01),while Nrf2 mRNA and protein expression levels were significantly up-regulated(P<0.01).Conclusions Ginkgolide may inhibit oxidative stress and inflammatory response through the Keap1/Nrf2 signaling pathway,thereby improving stroke blood-brain barrier function,reducing brain injury,and enhancing neurological function.