Mechanism of electroacupuncture on rats with primary dysmenorrhea based on microRNA expression spectrum and PI3K/Akt/mTOR signaling pathway

To investigate the effect of electroacupuncture (EA) on microRNA (miRNA) expression spectrum and PI3K/Akt/mTOR signaling pathway in uterine tissue of rats with primary dysmenorrhea (PDM), and to explore the potential mechanism of EA in the treatment of PDM. Thirty female SD rats, weighted (200 ± 20) g were randomly divided into control group, model group and EA group, 10 rats in each group. By using subcutaneous injection of estradiol diphenhydrate combined with intraperitoneal injection of oxytocin, PDM models were established. Rats in the EA group received EA at “Sanyinjiao”(SP6) and “Guanyuan”(CV4) at dense waves and a frequency of 50 Hz, once a day, 20 min each time, for 10 consecutive days. After the 10-day intervention, samples were collected and transmission electron microscopy was used to observe the ultrastructural changes of the cells in uterine tissue in each group. With RNA-seq method, the changes of miRNA expression spectrum in rat uterine tissue were detected. Bioinformatics analysis such as GO functional annotation and KEGG pathway was performed according to differentially expressed miRNAs. Differentially expressed miRNAs were verified by qRT-PCR. Endometrial stromal cells were selected as the target cells and transfected; and they were divided into control group, NC mimics group, mimic miR-144-3p group, NC inhibitor group and inhibitor miR-144-3p group. The apoptosis was determined by using flow cytometrydetect apoptosis, the miRNA and protein expression of PI3K/Akt/mTOR signaling pathway were detected by qRT-PCR and Western blot in each group separately. 1. Transmission electron microscope. (1) Control group: no obvious morphological changes in the uterine tissue. (2) Model group: fibroblasts in uterine tissue were irregular, the edema was presented in cellular cytoplasm, the nuclei were irregular and mitochondria swollen seriously; the rough endoplasmic reticulum was expanded moderately. (3) EA group: fibroblasts were spindle-shaped and pyknotic, the cytoplasm increased in electron density, the nuclei were slightly irregular and pyknotic, mitochondria were oval in shape, with little swelling and vacuolation; the rough endoplasmic reticulum was expanded slightly and retained, with a small amount of degranulation. 2. Compared with the control group, there were 26 differentially expressed miRNAs in the uterine tissue of rats with PDM. After EA intervention, the expression of miR-144-3p was significantly up-regulated. GO functional analysis of differentially expressed miRNAs in PDM rats after EA showed that the biological functions involved calcium transmembrane transporter activity, mitogen-activated protein kinase binding, epithelial cell migration, tissue migration, etc. 3. KEGG pathway analysis showed that PI3K/Akt signaling pathway, MAPK signaling pathway and calcium signaling pathway were enriched. Overexpression of miR-144-3p increased the apoptosis of endometrial stromal cells ( P < 0.01), and decreased the mRNA and protein expression of PI3K, Akt and mTOR ( P < 0.01). EA can optimize the cell morphology in the uterine tissue of rats with PDM and affect the miRNA expression spectrum, which may be associated with the effect of EA for up-regulating miR-144-3p expression in endometrial stromal cells, suppressing PI3K/Akt/mTOR signaling pathway and causing apoptosis. 目的:探究电针对原发性痛经 (primary dysmenorrhea, PDM) 大鼠子宫组织形态,microRNA(miRNA)表达谱及PI3K/Akt/mTOR信号通路的影响, 探究电针治疗PDM的潜在机制.方法:将30只体质量 (200±20) g的雌性SD大鼠随机分为空白组,模型组和电针组, 每组10只.采用皮下注射苯甲酸雌二醇联合腹腔注射缩宫素法制备PDM模型大鼠.电针组大鼠取“三阴交”“关元”, 加以电针, 施以密波,50 Hz, 20 min/次, 1次/天, 连续10天后取材.透射电镜观察各组大鼠子宫组织细胞超微结构的改变, RNA-seq法检测各组大鼠子宫组织miRNA表达谱的变化, 根据差异表达的miRNAs进行GO功能注释和KEGG通路等生物信息学分析, qRT-PCR验证差异表达miRNAs.选择子宫内膜基质细胞作为目的细胞并转染, 分对照组,mimics negative control (NC) 组,miR-144-3p mimics组,inhibitor NC组和miR-144-3p inhibitor组, 运用流式细胞仪检测细胞凋亡情况, qRT-PCR,Western blot法检测各组细胞中PI3K/Akt/mTOR信号通路miRNA和蛋白的表达水平.结果:1透射电镜示: (1) 空白组大鼠子宫组织未见明显形态学改变. (2) 模型组大鼠子宫组织成纤维细胞呈不规则形, 细胞胞质明显水肿, 细胞核严重不规则形, 线粒体严重肿胀, 粗面内质网中度扩张. (3) 电针组大鼠子宫组织成纤维细胞大致呈梭形, 固缩, 细胞质电子密度增高, 细胞核呈轻度不规则, 轻微固缩, 线粒体呈卵圆形, 少量肿胀,空泡化, 粗面内质网轻微扩张,潴留, 少量脱颗粒.2与空白组相比, 模型组大鼠子宫组织中差异表达的miRNA有26种, 电针干预后, 其中的miR-144-3p表达显著上调.电针调控PDM中差异表达miRNAs GO功能分析显示所涉及的生物功能包括钙离子跨膜转运蛋白活性,丝裂原活化蛋白激酶结合和上皮细胞迁移.3 KEGG pathway分析发现富集较多的通路有PI3K/Akt信号通路,MAPK信号通路,钙信号通路等.过表达miR-144-3p可使子宫内膜基质细胞出现凋亡升高 ( P <0.01) , 并使PI3K,Akt,mTOR的mRNA和蛋白表达水平下降 ( P <0.01) . 结论: 电针能改善PDM大鼠子宫组织细胞形态, 影响PDM大鼠子宫组织miRNA表达谱, 其机制可能与电针上调子宫内膜基质细胞中miR-144-3p的表达,抑制 PI3K/Akt/mTOR信号通路,促进凋亡有关.