针刺调控脑缺血再灌注损伤大鼠海马区核心基因
circHDAC2 表达抗神经损伤的研究Acupuncture modulates expression of
circHDAC2 in hippocampus of cerebral ischemia reperfusion injury rats against neurological damage- 上海中医药杂志 2023年57卷第11期 页码：57-66
- 作者机构：
1.湖南中医药大学针灸推拿与康复学院（湖南 长沙 410208）
2.安徽中医药大学第二附属医院康复中心（安徽 合肥 230061）
3.成都中医药大学针灸推拿学院（四川 成都 610075）
4.湖南省妇幼保健院中医科（湖南 长沙 410008）
- 作者简介：
谢灿明，女，硕士研究生，主要从事针灸效应机制的研究工作
田浩梅，教授，硕士研究生导师； E-mail： 451358104@qq.com
- 基金信息：国家自然科学基金项目(82274662);湖南省自然科学基金项目(2021JJ30490);长沙市科技局自然科学基金项目(kq2014094);湖南省研究生科研创新项目(2022CX103)
DOI：10.16305/j.1007-1334.2023.2306014
中图分类号：
扫 描 看 全 文
谢灿明,江姗姗,王瑶,等.针刺调控脑缺血再灌注损伤大鼠海马区核心基因circHDAC2表达抗神经损伤的研究[J].上海中医药杂志,2023,57(11):57-66.
XIE Canming,JIANG Shanshan,WANG Yao,et al.Acupuncture modulates expression of circHDAC2 in hippocampus of cerebral ischemia reperfusion injury rats against neurological damage[J].Shanghai Journal of Traditional Chinese Medicine,2023,57(11):57-66.
谢灿明,江姗姗,王瑶,等.针刺调控脑缺血再灌注损伤大鼠海马区核心基因circHDAC2表达抗神经损伤的研究[J].上海中医药杂志,2023,57(11):57-66. DOI： 10.16305/j.1007-1334.2023.2306014.
XIE Canming,JIANG Shanshan,WANG Yao,et al.Acupuncture modulates expression of circHDAC2 in hippocampus of cerebral ischemia reperfusion injury rats against neurological damage[J].Shanghai Journal of Traditional Chinese Medicine,2023,57(11):57-66. DOI： 10.16305/j.1007-1334.2023.2306014.
摘要
目的,2,探讨针刺对脑缺血再灌注损伤（CIRI）大鼠脑保护作用与海马组织差异表达核心基因、转录网中信使RNAs（mRNAs）功能之间的联系，为针刺调控环状RNAs（circRNAs）抗CIRI后神经损伤研究提供参考。,方法,2,将54只SD大鼠随机分为假手术组、模型组和针刺组，每组18只，后两组采用改良Longa线栓法制备大脑中动脉阻塞再灌注模型，假手术组仅剥离血管，不插线栓。干预期内针刺组捆绑于鼠板并针刺“大椎”“水沟”“百会”穴，均匀捻转刺激1 min，频率为90次/min，15 min后行针1次，共留针30 min，每12 h干预1次，共7次。假手术组、模型组捆绑于鼠板不针刺。改良加西亚（Garcia） 评分法检测大鼠神经功能状况，2,3,5-氯化三苯基四氮唑（TTC）染色法观察脑梗死面积；通过基因芯片技术筛选缺血侧海马组织差异表达基因，整理出模型组/假手术组、针刺组/模型组共同差异表达的环状RNAs（co-DE circRNAs），根据,P,值、差异倍数（FC）、基因本体（GO）数据库条目富集数量筛选出高表达的核心co-DE circRNAs，分析核心co-DE circRNAs来源基因的GO功能；统计缺血侧海马CA1区尼氏染色阳性细胞数；通过实时荧光定量逆转录聚合酶链式反应（RT-qPCR）法验证核心co-DE circRNAs表达量，选取表达趋势与芯片结果一致的circRNAs进行竞争性内源RNA（ceRNA）网络预测，对预测网络中的mRNAs进行GO功能富集分析。,结果,2,干预前，与假手术组比较，各造模组改良Garcia 神经功能评分显著降低（,P,<,0.05）；干预后，与假手术组比较，模型组脑梗死面积比显著升高，改良 Garcia 神经功能评分和CA1区尼氏染色阳性细胞数显著降低（,P,<,0.05）；与模型组比较，针刺组脑梗死面积比显著降低，改良Garcia 神经功能评分和CA1区尼氏染色阳性细胞数显著升高（,P,<,0.05）；组内干预前后比较，针刺组改良Garcia 神经功能评分显著升高（,P,<,0.05）。芯片结果显示，模型组下调/针刺组上调、模型组上调/针刺组下调的co-DE circRNAs为23个，根据FC,>,1.25和GO数据库条目富集数量筛选出核心基因为环状RNA组蛋白去乙酰化酶2基因（,circHDAC2,）、环状RNA神经营养受体酪氨酸激酶2基因（,circNTRK2,）；GO数据库分析显示，二者富集于神经元的产生、神经系统发育、神经元分化等条目。验证其表达量发现，与假手术组比较，模型组大鼠缺血侧海马组织,circHDAC2,、,circNTRK2, 表达量均显著下调（,P,<,0.05）；与模型组比较，针刺组大鼠缺血侧海马组织,circHDAC2,、,circNTRK2,表达量均显著上调（,P,<,0.05）。,circHDAC2,表达趋势与芯片结果一致，由此对,circHDAC2, 构建相关 ceRNA调控网络，调控网络中mRNAs的GO分析主要富集在神经元的产生、神经元分化的调控、轴突引导、神经系统发育、神经元死亡的调节等条目。,结论,2,针刺可改善CIRI大鼠改良 Garcia神经功能评分与脑梗死面积比，降低缺血侧海马组织的神经元损伤，其作用可能与调控缺血侧海马组织,circHDAC2,表达以及激发调控网络中mRNA的神经元产生、神经元分化、神经元细胞体功能等有关。
Abstract
Objective,2,To explore the connection between the cerebral protective effect of acupuncture on cerebral ischemia reperfusion injury （CIRI） rats and the differentially expressed core genes in hippocampus and the functions of messenger RNAs （mRNAs） in the transcriptional network， so as to provide relevant reference information for the regulation of circular RNAs by acupuncture to resist nerve injury after CIRI.,Methods,2,Fifty-four male SD rats were randomly divided into sham group， MCAO/R group and acupuncture group， with 18 rats in each group. Rats in the latter two groups were used to form middle cerebral artery occlusion and reperfusion model by the modified Longa thread embolism method， and rats in the sham group were stripped of the blood vessels only without inserting the thread embolism. During the intervention period， rats in the acupuncture group were bound and fixed， and received acupuncture treatment at GV14， GV20 and GV26. The needles were twisted 1 min （frequency 90 times/min） and repeated 15 min later， needles retained for 30 min in total. Acupuncture was performed once every 12 h for a total of 7 sessions. Rats in the sham group and MCAO/R group were only bound to the board and didn’t get needled. The modified Garcia score was used to detect the neurological function status of rats， and TTC staining was used to observe the area of cerebral infarction. Differentially expressed genes in the hippocampal tissue on the ischemic side were screened by gene microarray technology， and the differentially expressed cyclic RNAs （co-DE circRNAs） in the MCAO/R and sham group， and the acupuncture group and MCAO/R group were sorted out. The core co-DE circRNAs with high expression were screened according to ,P,-value， Fold Change （FC） and enrichment number of gene ontology （GO） entries， and the GO function of the core co-DE circRNAs derived genes was analyzed. The number of Nissl's staining positive cells in the ischemic hippocampal CA1 region was counted， and the core co-DE circRNAs expression was verified by real-time fluorescence quantitative reverse transcriptase polymerase chain reaction （RT-qPCR）. The circRNA with the same expression trend as that of the chip was selected for competitive endogenous RNA （ceRNA） network prediction， and the mRNAs in the prediction network was analyzed for GO functional enrichment.,Results,2,Before intervention， the modified Garcia neurological function score was significantly decreased in each modeling group compared with the sham group （,P,<,0.05）； after intervention， the cerebral infarction area ratio was significantly increased， and the modified Garcia neurological function score and the number of Nissl's staining positive cells in the CA1 area were significantly decreased in the MCAO/R group compared with the sham group （,P,<,0.05）； the cerebral infarct area ratio was significantly decreased， and the modified Garcia neurological function score and the number of Nissl's staining positive cells in CA1 area were significantly increased in the acupuncture group compared with the MCAO/R group （,P,<,0.05）； comparing before and after intervention within the group， the modified Garcia neurological function score in the acupuncture group was significantly increased （,P,<,0.05）. The microarray results showed there were 23 co-DE circRNAs that were down-regulated in the MCAO/R group/up-regulated in the acupuncture group and up-regulated in the MCAO/R group/down-regulated in the acupuncture group， and the core genes： circular RNA ,HDAC2, （,circHDAC2,） and circular RNA ,NTRK2, （,circNTRK2,） were screened out according to the Fold Change （FC,>,1.25） and the enrichment number of GO entries. GO analysis showed that both were enriched to neuron generation， nervous system development， neuronal differentiation， etc. Verification of their expression found that compared with the sham group， the expressions of ,circHDAC2, and ,circNTRK2, in the hippocampal tissue of the ischemic side of rats in the MCAO/R group were significantly down-regulated （,P,<,0.05）； compared with the MCAO/R group， the expressions of ,circHDAC2, and ,circNTRK2, in the hippocampal tissue of the ischemic side of rats in the acupuncture group were significantly up-regulated （,P,<,0.05）. The ,circHDAC2, expression trend was consistent with the microarray results， so the GO analysis of the mRNAs in the regulatory network of ,circHDAC2, was mainly enriched to the regulation of neuron generation， neuron differentiation， axon guidance， nervous system development， neuron death， and so on.,Conclusion,2,Acupuncture increases the modified Garcia neurological function score， decreases cerebral infarct area ratio in CIRI rats and reduces neuronal damage in the hippocampal tissue of the ischemic side in CIRI rats， which may be related to the regulation of ,circHDAC2, expression in the hippocampal tissue of the ischemic side by acupuncture and the stimulation of neuronal production， neuronal differentiation， and neuronal cell body of mRNAs in the regulatory network.
关键词
脑卒中针刺脑缺血再灌注损伤大鼠环状RNA信使RNA作用机制神经损伤
Keywords
cerebral apoplexyacupuncturecerebral ischemia-reperfusion injuryratcircular RNAmessenger RNAmechanism of actionneurological injury
references
PAUL S， CANDELARIO-JALIL E. Emerging neuroprotective strategies for the treatment of ischemic stroke： An overview of clinical and preclinical studies［J］. Exp Neurol， 2021， 335： 113518.
GBD 2019 STROKE COLLABORATORS. Global， regional， and national burden of stroke and its risk factors， 1990-2019： a systematic analysis for the Global Burden of Disease Study 2019［J］. Lancet Neurol， 2021， 20（10）： 795-820.
SALMENA L， POLISENO L， TAY Y， et al. A ceRNA hypothesis： the Rosetta Stone of a hidden RNA language？［J］. Cell， 2011， 146（3）： 353-358.
NAJAFI S， AGHAEI ZARCH S M， MAJIDPOOR J， et al. Recent insights into the roles of circular RNAs in human brain development and neurologic diseases［J］. Int J Biol Macromol， 2023， 225： 1038-1048.
WANG S， LI N， MENG X. Silencing of the circ-Arhgap5 RNA protects neuronal PC12 cells against injury and depends on the miR-29a-3p/Rock1 axis［J］. Metab Brain Dis， 2023， 38（4）： 1285-1296.
何香花，张为，杨泽，等. CircRNAs与阿尔茨海默病相关性的研究进展与未来方向［J］. 中国老年保健医学，2023， 21（3）： 112-116.
LI F， LI C， LI X， et al. Altered circular RNA expression profiles in the non-ischemic thalamus in focal cortical infarction mice［J］. Aging （Albany NY）， 2020，12（13）： 13206-13219.
安小琼，谢鹏，朱晓西，等. 小鼠脑缺血再灌注损伤模型中circRNA的m6A修饰及表达谱分析［J］. 中国生物化学与分子生物学报，2022， 38（10）： 1403-1417.
陈博威，唐荣梅，徐雅倩，等. 大脑中动脉栓塞模型大鼠海马组织circRNA-miRNA-mRNA三元转录网络分析［J］. 中国病理生理杂志，2022， 38（3）： 479-486.
肖姮，阳仁达，田浩梅，等. 针刺联合亚低温对脑缺血/再灌注损伤大鼠Bcl-2、Bax、Caspase-3蛋白表达的影响［J］. 湖南中医药大学学报，2016， 36（2）： 58-61.
刘琴，林亚平，陈文，等. 针刺联合亚低温对脑缺血再灌注损伤大鼠脑组织p-Raf1、p-ERK1/2的影响［J］. 湖南中医药大学学报，2016， 36（1）： 58-61.
SHEN J， CHEN L， CHENG J， et al. Circular RNA sequencing reveals the molecular mechanism of the effects of acupuncture and moxibustion on endometrial receptivity in patients undergoing infertility treatment［J］. Mol Med Rep， 2019， 20（2）： 1959-1965.
WANG Y， HU W， HAN J， et al. Electroacupuncture alleviates perioperative hypothalamus-pituitary-adrenal axis dysfunction via circRNA-miRNA-mRNA networks［J］. Front Mol Neurosci， 2023， 16： 1115569.
LONGA E Z， WEINSTEIN P R， CARLSON S， et al. Reversible middle cerebral artery occlusion without craniectomy in rats［J］. Stroke， 1989， 20（1）： 84-91.
李忠仁. 实验针灸学［M］. 北京：中国中医药出版社，2007.
华兴邦，周浩良. 大鼠穴位图谱的研制［J］. 实验动物与动物实验，1991（1）： 1-5.
GARCIA J H， WAGNER S， LIU K F， et al. Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats. Statistical validation［J］. Stroke， 1995， 26（4）： 627-634.
SWANSON R A， MORTON M T， TSAO-WU G， et al. A semiautomated method for measuring brain infarct volume［J］. J Cereb Blood Flow Metab， 1990， 10（2）： 290-293.
AI L Y， DU M Z， CHEN Y R， et al. Integrated analysis of lncRNA and mRNA expression profiles indicates age-related changes in meniscus［J］. Front Cell Dev Biol， 2022， 10： 844555.
何兴伟. 中风病从督脉论治探讨［J］. 中国中医基础医学杂志，2006， 12（8）： 561，563.
SHAO T Y， DING M R， YE Z X， et al. Governor vessel acupuncture for acute ischemic stroke： a systematic review and meta-analysis［J］. Ann Palliat Med， 2021， 10（7）： 7236-7246.
鲍春蓉，钟颖茜，林万庆，等. 基于数据挖掘的针刺治疗中风后平衡功能障碍选穴规律分析［J］. 上海中医药杂志，2021， 55（9）： 21-25.
郑慧娥，何灏龙，陈芯仪，等. 针刺对CIRI大鼠缺血侧海马区miRNA表达及miR-20a-5p和miR-22-5p的影响［J］. 湖南中医药大学学报，2020， 40（10）： 1226-1231.
唐红，郑慧娥，汪红娟，等. 针刺对脑缺血再灌注损伤模型大鼠海马细胞凋亡的影响［J］. 神经解剖学杂志，2023， 39（3）： 326-332.
XU S， ZHOU L， PONNUSAMY M， et al. A comprehensive review of circRNA： from purification and identification to disease marker potential［J］. PeerJ， 2018， 6： e5503.
袁阳，陈怀龙，张高峰，等. 环状RNA在脑缺血性损伤中作用机制的研究进展［J］. 国际麻醉学与复苏杂志，2021， 42（1）： 103-107.
张亚敏，徐虹，孙华，等. 针刺百会、足三里穴对脑缺血再灌注损伤大鼠脑组织及血清miRNA-29、miRNA-320表达的影响［J］. 针灸临床杂志，2015， 31（7）： 76-80.
郑慧娥，田浩梅，何灏龙，等. 针刺结合亚低温对脑缺血再灌注损伤大鼠脑缺血侧海马区差异miRNA功用及miR-106b-5p表达的影响［J］. 中国中医基础医学杂志，2021， 27（11）： 1759-1764.
NEWRZELLA D， PAHLAVAN P S， KRÜGER C， et al. The functional genome of CA1 and CA3 neurons under native conditions and in response to ischemia［J］. BMC Genomics， 2007， 8： 370.
XU Y， WANG Q， CHEN J， et al. Updating a strategy for histone deacetylases and its inhibitors in the potential treatment of cerebral ischemic stroke［J］. Dis Markers， 2020， 2020： 8820803.
吴芳芳. 环状RNA TLK1调控缺血性脑卒中神经损伤及其机制研究［D］. 南京：东南大学，2021.
0
浏览量
0
下载量
0
CSCD
0
CNKI被引量
- 网络PDF下载
- Meta-XML下载
- BibTeX下载
- Endnote下载
- RefMan 下载
- NoteExpress下载
- NoteFirst下载
关联资源
相关文章
相关作者
相关机构
- 技术支持由北京北大方正电子有限公司提供 沪ICP备13044091号
沪公网安备 31011502015228号 - 本系统建议在Chrome、 IE9+ 以上版本浏览器阅读本站内容，360浏览器请切换至极速模式
- Cookies帮助我们提供服务并提供个性化体验。使用本网站，即表示您同意我们使用Cookies
- 总访问量：72685 今日访问量：76