鸡血藤总黄酮对急性缺血性脑卒中大鼠肠道屏障功能、氧化应激及ERK基因表达与DNA甲基化的影响

Effect of Total Flavonoids in Spatholobi Caulis on Intestinal Barrier Function,Oxidative Stress,Gene Expression and DNA Methylation of ERK in Acute Ischemic Stroke Rats

【目的】探讨鸡血藤总黄酮对急性缺血性脑卒中大鼠的治疗作用及机制。【方法】将60只大鼠随机分为正常组,模型组,中药低、中、高剂量组和阿司匹林组,每组10只。除正常组,其余各组大鼠构建大脑中动脉闭塞(MCAO)模型。造模成功后,中药低、中、高剂量组分别给予鸡血藤总黄酮100、200、400 mg·kg^(-1)·d^(-1)灌胃,阿司匹林组给予阿司匹林100 mg·kg^(-1)·d^(-1)灌胃,正常组与模型组给予等体积生理盐水灌胃,连续给药4周。给药结束后,进行悬尾实验(TST)、强迫游泳实验(FST)、旷野实验等行为学观察,苏木素-伊红(HE)染色法观察海马组织病理学变化,酶联免疫吸附分析(ELISA)检测血清二胺氧化酶(DAO)、D-乳酸水平,黄嘌呤氧化酶法检测海马组织超氧化物歧化酶(SOD)水平,硫代巴比妥酸法检测海马组织丙二醛(MDA)水平,实时定量聚合酶链反应(PCR)法检测海马组织细胞外信号调节激酶(ERK)mRNA表达水平,采用Sequenom Mass Array飞行时间质谱法检测海马组织ERK DNA甲基化水平。【结果】与正常组比较,模型组大鼠TST不动时间缩短,FST不动时间和圈内保留时间延长,血清中DAO、D-乳酸含量升高,海马组织中SOD水平降低、MDA水平升高,海马组织中ERK mRNA表达水平降低、ERK DNA甲基化水平升高(均P<0.05),海马组织可见明显病理损伤;与模型组比较,中药中、高剂量组大鼠TST不动时间延长、FST不动时间和圈内保留时间缩短,血清中DAO、D-乳酸含量降低,海马组织中SOD水平升高、MDA水平降低,海马组织中ERK mRNA表达水平升高、ERK DNA甲基化水平降低(均P<0.05),海马病理学变化得到改善;中药中、高剂量组上述各指标的改善效果优于阿司匹林组,或与阿司匹林组相当。【结论】鸡血藤总黄酮可有效治疗急性缺血性脑卒中大鼠,其机制可能与调节肠道屏障功能、减轻海马组织氧化应激及促进ERK mRNA表达、抑制ERK DNA甲基化水平有关。

Objective To investigate the therapeutic effects and mechanisms of total flavonoids in Spatholobi Caulis on acute ischemic stroke rats. Methods Sixty rats were randomly divided into normal group,model group,low-,medium-and high-dose of Chinese medicine groups and Aspirin group,with 10 rats in each group. Except for the normal group,the middle cerebral artery occlusion(MCAO)model was constructed in all groups. After successful modeling,the rats were given 100,200 and 400 mg·kg^(-1)·d^(-1)of total flavonoids in Spatholobi Caulis by gavage in the low-, medium-and high-dose of Chinese medicine groups, respectively, Aspirin group was given100 mg·kg^(-1)·d^(-1)of Aspirin by gavage,while the normal group and the model group were given equal volume of normal saline by gavage. The treatment in all groups lasted for 4 weeks. After the administration, behavioral observations were performed by tail suspension test(TST), forced swimming test(FST)and open field test,hippocampal histopathological changes were observed by hematoxylin-eosin(HE)staining,serum diamine oxidase(DAO)and D-lactate levels were detected by enzyme-linked immunosorbent assay(ELISA),superoxide dismutase(SOD)level in hippocampal tissue was determined by xanthine oxidase method, the level of malondialdehyde(MDA)in hippocampal tissue was determined by thiobarbituric acid method,mRNA expression of extracellular signal-regulated kinase(ERK)in hippocampal tissue was detected by real-time quantitative polymerase chain reaction(PCR),and DNA methylation of ERK in hippocampal tissue was measured by sequenom mass array timeof-flight mass spectrometry. Results Compared with the normal group,rats in the model group had shorter TST immobility time,longer FST immobility time and intra-circular retention time,higher serum levels of DAO and D-lactic acid,lower SOD and higher MDA levels in hippocampal tissue,lower mRNA expression level of ERK and higher DNA methylation level of ERK in hippocampal tissue(all P<0.05), and significant pathological damage in hippocampal tissue was seen. Compared with the model group,the TST immobility time was prolonged,FST immobility time and intracircular retention time were shortened,serum levels of DAO and D-lactic acid were reduced,SOD level in hippocampal tissue was increased,MDA level was reduced,mRNA expression level of ERK in hippocampal tissue was increased, DNA methylation level of ERK was reduced(all P<0.05), and hippocampal pathological changes were improved. The improvement of the above indexes in the medium-and high-dose of Chinese medicine groups was superior to or comparable to that in the Aspirin group. Conclusion Total flavonoid in Spatholobi Caulis is effective in treating acute ischemic stroke in rats. The mechanism may be related to the regulation of intestinal barrier function, reduction of oxidative stress in hippocampal tissue and promotion of m RNA expression of ERK and inhibit DNA methylation level of ERK in hippocampal tissue.