1、中国病理生理杂志 Chinese Journal of Pathophysiology 2023，39（2）：220-232杂志网址：http:/针刺调控CIRI大鼠缺血侧海马组织差异表达circRNAs的功能研究*江姗姗1，唐红1，汪红娟1，吕倩忆2，谢灿明1，王瑶1，陈楚淘1，田浩梅1（1湖南中医药大学针灸推拿与康复学院，湖南 长沙 410208；2成都市第二人民医院，四川 成都 610021）摘要 目的：探讨针刺对脑缺血再灌注损伤（CIRI）大鼠脑组织的保护作用，观察针刺对CIRI大鼠缺血侧海马组织环状RNAs（circRNAs）差异表达的影响，并对其进行基因本体（GO）分析。方法：68
2、周龄SD大鼠54只，运用随机数字法随机分为造模组和假手术组（sham组），造模成功后再随机分为模型组（model组）和针刺组（AC组），每组18只。采用改良Longa线栓法制备大脑中动脉闭塞再灌注（MCAO/R）模型，激光散斑成像仪监测造模前、MCAO手术后及再灌注后脑血流量，假手术组只剥离血管，不插入线栓；干预期间模型组和假手术组只捆绑不针刺，针刺组捆绑+针刺。采用改良加西亚（Garcia）评分法对神经功能进行评定，TTC染色法检测脑梗死面积，Western blot法检测神经元核抗原（NeuN）的表达，尼氏染色法观察缺血侧海马组织神经元损伤程度，基因芯片微阵列分析筛选出缺血侧海马组织差异表
3、达的circRNAs，并对模型组/假手术组、针刺组/模型组共同差异表达circRNAs的来源基因进行GO分析。结果：与假手术组比较，模型组大鼠脑梗死面积比显著升高（P0.01），Garcia神经功能评分、NeuN表达量和海马CA1区尼氏染色阳性细胞数显著降低（P0.05或P0.01）；与模型组比较，针刺组脑梗死面积比显著降低（P0.01），神经功能评分、NeuN表达量和尼氏染色阳性细胞数显著升高（P0.05或P1.25，P0.05）；其中模型组/假手术组、针刺组/模型组共同差异表达的circRNAs个数为23个；GO分析显示共同差异表达circRNAs的来源基因功能涉及神经系统发育，神经元的产
4、生、发育、分化及投射，头部、大脑及海马的发育，突触的形成、发育、延伸及运输等。结论：CIRI大鼠缺血侧海马组织circRNAs在造模后及针刺干预后均存在差异表达。针刺能显著改善CIRI大鼠的神经功能和脑梗死面积，减轻海马组织神经元损伤，其机制可能与针刺调控缺血侧海马组织多种circRNAs的差异表达及激发其促进神经元发育分化、抗神经损伤等功能有关。关键词 针刺；脑缺血再灌注损伤；神经元损伤；环状RNA；差异表达中图分类号 R743；R363.2 文献标志码 A doi：10.3969/j.issn.1000-4718.2023.02.004Research on function of acu
5、puncture regulating differential expression of circRNAs in hippocampus of CIRI ratsJIANG Shanshan1，TANG Hong1，WANG Hongjuan1，L Qianyi2，XIE Canming1，WANG Yao1，CHEN Chutao1，TIAN Haomei1（1School of Acupuncture，Massage and Rehabilitation，Hunan University of Chinese Medicine，Changsha 410208，China；2Chengd
6、u Second Peoples Hospital，Chengdu 610021，China.E-mail：）ABSTRACT AIM：To explore the protective effect of acupuncture on brain tissue of rats with cerebral ischemia reperfusion injury（CIRI），observe the effect of acupuncture on the differential expression of circular RNA（circRNA）in the hippocampus of t
7、he ischemic side of CIRI rats，and carry out gene ontology（GO）analysis on it.METHODS：Fifty four 68 week old SD rats were randomly divided into modeling group and sham operation group（sham group）.CIRI rats 文章编号 1000-4718（2023）02-0220-13 收稿日期 2022-08-22 修回日期 2022-12-23*基金项目 国家自然科学基金资助项目（No.81874508；No.
8、82274662）；湖南省自然科学基金资助项目（No.2020JJ4065；No.2021JJ30490）；长沙市科技局自然科学基金项目（No.kq2014094）；湖南省研究生科研创新项目（No.CX20220799）；湖南中医药大学研究生创新课题（No.2021CX39；No.2022CX97）通讯作者 Tel：13548574270；E-mail：220were randomly divided into model group（model group）and acupuncture group（AC group）with 18 rats in each group.Establishm
9、ent of middle cerebral artery occlusion reperfusion（MCAO/R）model by using Longa monofilament method.The cerebral blood flow was monitored by laser speckle imager.In the sham operation group，only the blood vessels were stripped without inserting thread plugs.The acupuncture group was bound+acupunctur
10、e every 12 h，30 minutes for 7 times，during which twirling manipulation was performed.The nerve function was evaluated by the modified Garcia scoring method.Cerebral infarction area was measured by TTC staining.Detection of the expression of neuronal nuclear antigen（NeuN）by Western blot.Observation o
11、f neuronal damage in ischemic hippocampus by Nissl staining.Gene chip microarray analysis was used to screen out the circRNA differentially expressed in the hippocampus of ischemic side，and GO analysis was performed on the source genes of common differentially expressed circRNA in model group vs sha
12、m group and AC group vs model group.RESULTS：Before intervention，compared with the sham group，the Garcia neural function score in the modeling group was significantly lower（P0.01）.After intervention，compared with the sham group，the score of the Model group decreased significantly（P0.01）.Compared with
13、 the model group，the score of AC group increased significantly（P0.01）.Compared with before intervention，the score of AC group increased significantly after intervention（P0.01）.Compared with the sham group，the area ratio of cerebral infarction in the model group was significantly increased（P0.01），the
14、 expression of NeuN was significantly decreased（P0.01），and the number of Nielsen staining positive cells in hippocampal CA1 neurons in the ischemic side was significantly decreased（P0.05）.Compared with the model group，the area ratio of cerebral infarction in the AC group was significantly reduced（P0
15、.01），the expression of NeuN was significantly increased（P0.05），and the number of neurons with positive Nissl staining was significantly increased（P1.25，P1.25、P0.05的筛选标准，寻找假手术组、模型组、针刺组间差异表达的circRNAs，并对差异表达circRNAs的来源基因进行GO功能富集分析。Figure 1.Establishment of middle cerebral artery occlusion/reperfusion（M
16、CAO/R）rat model.The scale bar=0.5 mm.A：laser speckle imager showed the cerebral blood flow in the right cerebral hemisphere is full before modeling；B：after MCAO，the cerebral blood flow decreased significantly；C：after reperfusion，the cerebral blood flow recovered.图1大脑中动脉闭塞再灌注大鼠模型的建立2234统计学处理本实验采用完全随机设计，所有数据为计量资料，用SPSS 25.0软件进行统计学分析，组内干预前后比较：差值符合正态分布使用配对t检验；不符合则使用配对秩和检验。各组间比较：所有数据进行正态性检验，满足正态性分布使用单因素方差分析，方差齐者用 LSD 检验，方差不齐者用 Tamhanes T2检验，数据用均数标准差（meanSD）表示；不符合正态性分布则使用非参数检验，以中位数与四分位数间距 median（Q）表示。