Acupuncture modulates expression of <italic style="font-style: italic">circHDAC2</italic> in hippocampus of cerebral ischemia reperfusion injury rats against neurological damage

XIE Canming; JIANG Shanshan; WANG Yao; XIANG Jing; LIU Xiaoyue; TANG Hong; LI Zhanfu; WANG Hongjuan; CHEN Chutao; TIAN Haomei

doi:10.16305/j.1007-1334.2023.2306014

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Acupuncture modulates expression of circHDAC2 in hippocampus of cerebral ischemia reperfusion injury rats against neurological damage

更新时间：2023-11-24

- Acupuncture modulates expression of circHDAC2 in hippocampus of cerebral ischemia reperfusion injury rats against neurological damage
- Shanghai Journal of Traditional Chinese Medicine Vol. 57, Issue 11, Pages: 57-66(2023)
- 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：
- 基金信息：
- DOI：10.16305/j.1007-1334.2023.2306014
  CLC：
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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

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Acupuncture modulates expression of circHDAC2 in hippocampus of cerebral ischemia reperfusion injury rats against neurological damage

DOI：10.16305/j.1007-1334.2023.2306014

摘要

Abstract

关键词

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