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电针预处理对大脑中动脉栓塞小鼠脑微血管内皮细胞功能的影响

The effect of electroacupuncture pretreatment on the functioning of microvascular endothelial cells in the cerebrum after middle cerebral artery obstruction
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摘要 目的观察电针预处理对大脑中动脉栓塞(MCAO)小鼠脑微血管内皮细胞功能的影响,并进一步探讨miRNA-155靶向阴阳因子1(YY1)/p53通路在电针预处理调节MCAO小鼠脑微血管内皮细胞功能中的作用。方法按随机数字表法将42只小鼠随机分为假手术组、模型组、电针预处理组、miR-155抑制剂组(电针预处理联合miR-155抑制剂处理)、miR-155激动剂组(电针预处理联合miR-155激动剂处理)、miR-155抑制剂阴性对照组(电针预处理联合miR-155抑制剂阴性对照处理)和miR-155激动剂阴性对照组(电针预处理联合miR-155激动剂阴性对照处理),每组6只小鼠。电针预处理组、miR-155抑制剂组、miR-155激动剂组、miR-155抑制剂阴性对照组和miR-155激动剂阴性对照组均接受电针预处理,假手术组和模型组不接受任何预处理。miR-155抑制剂组、miR-155激动剂组、miR-155抑制剂阴性对照组和miR-155激动剂阴性对照组于电针预处理结束后,且造模前2 h进行对应的侧脑室注射。7组小鼠均于电针预处理最后1次治疗结束48 h后进行MCAO模型制作。于造模成功24 h和72 h后,采用改良神经功能缺损评分(mNSS)评估模型组、假手术组和电针预处理组小鼠的神经功能缺损程度。mNSS评分结束后对7组小鼠取材。采用苏木精伊红(HE)染色法观察缺血区脑组织的损伤情况;采用原位末端转移酶标记技术(TUNEL)染色法观察内皮细胞的凋亡情况;采用酶联免疫吸附法(ELISA)检测模型组、假手术组和电针预处理组血清中细胞间粘附分子-1(ICAM-1)的水平;采用免疫荧光检测脑微血内皮细胞凋亡数及ICAM-1、Bcl-2、Bax阳性细胞数;采用实时荧光定量聚合酶链式反应(qRT-PCR)检测法检测I/R区miRNA-155、YY1、p53表达水平。结果造模成功24 h和72 h后,电针预处理组的mNSS评分分别为(6.17±1.17)分和(4.00±0.63)分,均显著低于模型组同时间点,差异均有统计学意义(P<0.05)。HE染色结果显示,电针预处理组造模成功24 h和72 h后的细胞形态较模型组更规则。TUNEL染色结果显示,电针预处理组造模成功24 h和72 h后的凋亡细胞数量显著低于模型组同时间点(P<0.05)。ELISA检测结果显示,电针预处理组造模成功24 h和72 h后的ICAM-1表达显著低于模型组同时间点(P<0.05)。免疫荧光检测结果显示,电针预处理组造模成功24 h和72 h后ICAM-1的相对表达量显著低于模型组同时间点(P<0.05);电针预处理组造模成功24 h和72 h后的Bax/Bcl-2比值显著低于模型组。qRT-PCR检测结果显示,电针预处理组造模成功24 h和72 h后的miRNA-155、YY1、p53的相对表达量显著低于模型组同时间点。miR-155抑制剂组造模成功24 h和72 h后miRNA-155的表达量显著低于miR-155抑制剂对照组同时间点(P<0.05);miR-155抑制剂组造模成功24 h和72 h后p53的表达量显著低于miR-155抑制剂对照组同时间点(P<0.05)。免疫荧光检测结果显示,miR-155抑制剂组造模成功24 h和72 h后ICAM-1表达量显著低于miR-155抑制剂对照组同时间点(P<0.05);miR-155抑制剂组造模成功24 h和72 h后的Bax与Bcl-2比值显著低于miR-155抑制剂对照组同时间点(P<0.05)。结论电针预处理可以改善脑缺血再灌注小鼠的脑微血管内皮细胞功能,并可能通过下调miRNA-155介导的YY1/p53通路来减轻脑缺血再灌注损伤小鼠脑微血管内皮细胞的损伤。 Objective To observe any effect of electroacupuncture pretreatment(EP)on the function of cerebral microvascular endothelial cells in mice with middle cerebral artery obstruction(MCAO),and to explore the role of miRNA-155 RNA and the p53 pathway in determining it.Methods Forty-two mice were randomly divided into a sham operation group,a model group,an EP group,an miR-155 inhibitor(MI)group,an miR-155 agonist(MA)group,an miR-155 inhibitor negative control(MIN)group and an miR-155 agonist negative control(MAN)group.Each group had 6 rats.All except the sham-operated and model groups received EP,followed by injecting into the corresponding lateral ventricles inhibitor and agonist in groups MI and MA,and placebos in group MIN and MAN.All 7 groups were subjected to MCAO modeling 48 hours after the EP.Twenty-four and 72 hours after the modeling,neurological deficits were quantified using modified neurological severity scoring(mNSS).All of the mice were then sacrificed and any brain tissue damage was observed using hematoxylin-eosin(HE)staining.Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling(TUNEL)was used to observe any apoptosis of endothelial cells,and enzyme-linked immunosorbent assays(ELISA)were employed to detect the level of intercellular cell adhesion molecule-1(ICAM-1)in the serum.Apoptotic endothelial cells in the cerebral microbleed and ICAM-1,Bcl-2 and Bax positive cells were detected using immunofluorescence.Quantitative real-time polymerase chain reactions(qRT-PCR)were carried to observe the expressions of miRNA-155 RNA and YY1 and p53 proteins in the ischemia/reperfusion(I/R)region.Results Twenty-four and 72 hours after the MCAO the average mNSS score of the EP group was significantly lower than the model group′s average.HE staining showed that the cell morphology of the EP group was more regular.The number of apoptotic cells,the expression of ICAM-1,the relative expression of ICAM-1,the Bax/Bcl-2 ratio,as well as the relative expression of miRNA-155 RNA and YY1 and p53 proteins were all then significantly lower in the EP group than in the model group,on average.The expression of miRNA-155 RNA,p53 protein and ICAM-1,as well as the Bax to Bcl-2 ratio in the MI group was then significantly lower than in the MIN group,on average.Conclusion Electroacupuncture pretreatment improves microvascular endothelial cell functioning in the cerebrum after ischemia and reperfusion(at least in mice)and may attenuate cerebral cell injury by down-regulating the miRNA-155-mediated YY1/p53 pathway.
作者 王颖 许将 王辉煌 王辉 周翔 李雪静 Wang Ying;Xu Jiang;Wang Huihuang;Wang Hui;Zhou Xiang;Li Xuejing(The Affiliated Huai'an Hospital of Xuzhou Medical University,Huai'an 223022,China)
出处 《中华物理医学与康复杂志》 CAS CSCD 北大核心 2024年第6期492-499,共8页 Chinese Journal of Physical Medicine and Rehabilitation
基金 国家自然科学基金(82105004) 淮安市科技项目(HAB202027)。
关键词 电针 脑缺血再灌注 阴阳因子1 P53 Electroacupuncture Cerebral ischemia and reperfusion YinYang-1 transcription factor p53 pathway
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