龙生蛭胶囊保护脑缺血再灌注损伤大鼠的作用及机制研究  

Protective effect and mechanism of Longshengzhi capsules on cerebral ischemia-reperfusion injury in rats

作　　者：方欢乐[1] 李晓明[1] 周亚明 张鑫 刘小溪 陈衍斌 FANG Huanle;LI Xiaoming;ZHOU Yaming;ZHANG Xin;LIU Xiaoxi;CHEN Yanbin(Medical College of Xi’an Peihua University,Xi’an 710125,China;Scientific Research Department,Buchang Pharma Co.,Ltd.,Xi’an 710075,China;Xi’an Jiaotong University Health Science Center,Xi’an 710061,China)

机构地区：[1]西安培华学院医学院,西安710125 [2]陕西步长制药有限公司科研部,西安710075 [3]西安交通大学医学部,西安710061

出　　处：《中国药房》2024年第7期813-818,共6页China Pharmacy

基　　金：陕西省重点研发计划项目(No.2021SF-362);陕西省教育厅重点科学研究计划协同创新项目(No.21JY001);西安培华学院校级科研项目(No.PHKT2220)。

摘　　要：目的探讨龙生蛭胶囊对脑缺血再灌注损伤大鼠的脑保护作用及机制。方法通过改良线栓法建立大鼠大脑中动脉阻塞(MCAO)模型。实验分为假手术组(仅分离血管不插线栓,予同体积生理盐水)、模型组(造模,予同体积生理盐水)、尼莫地平组(阳性对照,造模,给药剂量为20 mg/kg)和龙生蛭胶囊低、中、高剂量组(造模,给药剂量依次为0.72、1.44、2.88 g/kg),每组10只。各组大鼠灌胃相应药液/生理盐水,每天1次,连续7 d。末次给药1 h后,采用Zea Longa评分法对各组大鼠的神经功能缺失情况进行评分;ABC-酶联免疫吸附试验法检测大鼠血清肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)水平;TTC染色观察大鼠脑梗死体积并计算脑梗死体积比;苏木精-伊红染色观察大鼠脑组织病理形态的变化;免疫组化法检测大鼠脑组织NLRP3蛋白阳性表达情况;实时荧光定量PCR法检测大鼠脑组织Toll样受体4(TLR4)、核因子κB(NF-κB)mRNA表达;Western blot法检测大鼠脑组织TLR4、NLRP3、磷酸化NF-κB(p-NF-κB)及细胞核内NF-κB蛋白表达情况。结果与假手术组比较,模型组大鼠神经功能缺失评分,血清TNF-α和IL-6水平,脑梗死体积比,脑组织NF-κB和TLR4 mRNA的相对表达水平,以及脑组织TLR4、NLRP3、p-NF-κB蛋白的相对表达水平及其细胞核内NF-κB蛋白的相对表达水平均显著升高(P<0.01);大鼠脑组织皮层神经细胞间隙增大、水肿明显,可见大量炎性细胞浸润;大鼠脑组织中NLRP3蛋白阳性表达明显增多。与模型组比较,龙生蛭胶囊中、高剂量组及尼莫地平组大鼠以上指标水平均有不同程度逆转,大部分差异有统计学意义(P<0.05或P<0.01);病理形态观察结果明显好转,大鼠脑组织中NLRP3蛋白阳性表达均明显减少。结论龙生蛭胶囊可能通过抑制TLR4/NF-κB/NLRP3信号通路,抑制神经炎症反应,从而达到对大鼠脑缺血再灌注损伤的保护作用。OBJECTIVE To explore the protective effect and mechanism of Longshengzhi capsules on cerebral ischemiareperfusion injury in rats.METHODS The model of middle cerebral artery occlusion(MCAO)was established by using the improved thread occlusion method.The experiment was divided into six groups:sham surgery group(only separating blood vessels without inserting thread plugs,given the same volume of normal saline),model group(modeling,given the same volume of normal saline),nimodipine group(positive control,modeling,dose of 20 mg/kg),and low-dose,medium-dose,and high-dose groups of Longshengzhi capsules(modeling,doses of 0.72,1.44 and 2.88 g/kg,respectively),with 10 mice in each group.Each group was given corresponding medication solution/normal saline by gavage,once a day,for 7 consecutive days.One hour after the last administration,the Zea Longa scoring method was used to score the neurological deficits in each group of rats,and the ABC enzyme-linked immunosorbent assay was used to detect the serum levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in rats;TTC staining was used to observe the volume of cerebral infarction in rats and calculate the cerebral infarction volume ratio.Hematoxylin eosin staining was used to observe the pathological changes in the brain tissue of rats.Immunohistochemical staining was used to detect the positive expression of NLRP3 protein in the brain tissue of rats.Real-time fluorescence quantitative PCR was used to detect mRNA relative expressions of Toll-like receptor 4(TLR4)and nuclear factor-κB(NF-κB)in the brain tissue of rats.Western blot assay was adopted to detect the relative expressions of TLR4,NLRP3 and phosphorylated NF-κB(p-NF-κB)protein in the brain tissue of rats and its intracellular NF-κB protein.RESULTS Compared with the sham surgery group,the neural dysfunction score,serum levels of TNF-αand IL-6,cerebral infarction volume ratio,relative expression levels of NF-κB and TLR4 mRNA,as well as protein relative expressions of TLR4,NLRP3 and p-NF-κB in th

关 键 词：龙生蛭胶囊 脑缺血再灌注损伤 炎症因子 TLR4/NF-κB/NLRP3信号通路 

分 类 号：R965[医药卫生—药理学]