Objectives: To explore the mechanisms of acupuncture against cerebral ischemia/reperfusion injury (CIRI) through observing the expression of circular RNA HDAC2 (circHDAC2) in the hippocampus of rats.
Methods: SD rats were randomly divided into sham-operation, model and acupuncture groups, with 13 rats in each group. The rat model of CIRI was established by middle cerebral artery occlusion. In the acupuncture group, acupuncture was delivvered at "Dazhui" (GV14), "Shuigou" (GV26) and "Baihui" (GV20), and the needles were retained for 30 min each time and acupuncture was conducted once every 12 h for a total of 7 sessions. Before and after intervention, using modified Garcia scale, the neurological function of the rats were evaluated, and TTC staining was employed to determine the cerebral infarct area. Gene chip technology was used to screen the circRNAs with differential expressions in the ischemic hippocampus, and the circRNAs with co-differential expression (co-DE circRNAs) in the model group/sham-operation group, and the acupuncture group/model group separately. Among those circRNAs, the core circRNAs were screened according to P value, fold change (FC) and gene ontology (GO) analysis；and their expressions in the ischemic hippocampus were determined using quantitative real-time PCR (qPCR). Based on the verification results, a competing endogenous RNA (ceRNA) prediction network was constructed. The expression levels of microRNA (miRNA) and mRNA with high node centrality in the prediction network were validated by qPCR.
Results: Before intervention, compared with the sham-operation group, the modified Garcia score of each modeling group decreased (P<0.01). After intervention, the modified Garcia score was reduced and the cerebral infarct area ratio increased (P<0.01) in the model group when compared with the sham-operation group. In the acupuncture group, the modified Garcia score was higher and the cerebral infarct area ratio lower (P<0.01) than those of the model group. The microarray results of gene chip showed that 16 co-DE circRNAs were down-regulated in the model group and up-regulated in the acupuncture group, and 7 co-DE circRNAs up-regulated in the model group and down-regulated in the acupuncture group. The core circHDAC2 and circNTRK2 were screened according to P value, FC and the enrichment number of GO entries. QPCR results showed that, compared with the sham-operation group, the expression of circHDAC2 and circNTRK2 of the ischemic hippocampal tissue was down-regulated in the model group (P<0.01)；and that of circHDAC2 and circNTRK2 up-regulated in the acupuncture group when compared with the model group (P<0.01). The relevant ceRNA regulatory network was constructed for circHDAC2 and the prediction results showed that the regulatory networks contained 12 miRNAs and 31 mRNAs. Results of verifying miRNA with high network node centrality and mRNA relevant with nerve regulation showed that, when compared with the sham-operation group, the expression levels of miR-29a, miR-29b and the solute carrier family 30 member 3 (SLC30A3) mRNA in the ischemic hippocampus were down-regulated (P<0.01)；and those of miR-3065 and mercaptopyruvate sulfurtransferase (MPST) up-regulated (P<0.01) in the model group. Compared with the model group, the expressions of miR-29a, miR-29b and SLC30A3 mRNA in the ischemic hippocampus were up-regulated (P<0.01, P<0.05), while that of miR-3065 down-regulated (P<0.05) in the acupuncture group.
Conclusions: Acupuncture significantly improves the neurological function and reduces the cerebral infarct area in CIRI rats, which may be related to the regulation of hippocampal circHDAC2/miR-3065/SLC30A3 axis.
目的: 通过对脑缺血再灌注损伤（CIRI）大鼠海马组织环状RNA HDAC2（circHDAC2）表达的研究，探讨针刺抗CIRI的作用机制。方法: SD大鼠随机分为假手术组、模型组、针刺组，每组13只。采用线栓法复制大脑中动脉阻塞再灌注模型。针刺组针刺“大椎”“水沟”“百会”，每次30 min，每隔12 h治疗1次，共7次。干预前后采用改良Garcia评分法评价大鼠神经功能，TTC染色法观察并计算脑梗死面积百分比，基因芯片技术筛选3组大鼠缺血侧海马组织差异表达的环状RNAs（circRNAs），并筛选模型组/假手术组、针刺组/模型组共同差异表达circRNAs（co-DE circRNAs），根据P值、差异倍数（FC）、基因本体（GO）条目富集数量筛选出其中的核心circRNAs，通过实时荧光定量PCR法验证其在缺血侧海马组织的表达水平，根据验证结果构建竞争性内源RNA（ceRNA）预测网络，利用实时荧光定量PCR法验证预测网络中节点中心度高的微小RNA（miRNA）和mRNA的表达水平。结果: 干预前，与假手术组比较，各造模组大鼠改良Garcia神经功能评分降低（P<0.01）。干预后，与假手术组比较，模型组改良Garcia神经功能评分降低、脑梗死面积百分比升高（P<0.01）；与模型组比较，针刺组改良Garcia神经功能评分升高，脑梗死面积百分比降低（P<0.01）。芯片结果显示：模型组下调和针刺组缺血侧海马组织中上调的co-DE circRNAs为16个，模型组上调和针刺组下调的co-DE circRNAs为7个；根据P值、FC和GO条目富集数量筛选出核心基因circHDAC2和circNTRK2。验证其表达量结果显示，与假手术组比较，模型组大鼠缺血侧海马组织circHDAC2、circNTRK2表达水平下调（P<0.01）；与模型组比较，针刺组大鼠circHDAC2、circNTRK2表达水平均上调（P<0.01）。对circHDAC2 构建相关 ceRNA调控网络，预测结果显示调控网络中包含miRNA 12个、mRNA 31个。对调控网络中节点中心度高的miRNA和与神经系统密切相关的mRNA的验证结果显示，与假手术组比较，模型组大鼠缺血侧海马组织 miR-29a、miR-29b、溶质载体家族30成员3（SLC30A3）mRNA表达水平下调（P<0.01），miR-3065、巯基丙酮酸硫转移酶（MPST）mRNA表达水平上调（P<0.01）；与模型组比较，针刺组大鼠miR-29a、miR-29b、SLC30A3 mRNA表达水平上调（P<0.01，P<0.05），miR-3065表达水平下调（P<0.05）。结论: 针刺可提高CIRI大鼠的神经功能、降低脑梗死面积，可能与针刺调控海马circHDAC2/miR-3065/SLC30A3轴有关。.
Keywords: Acupuncture; Cerebral ischemia/reperfusion injury; Circular RNA; MicroRNA.