Hou Qiuke, Huang Yongquan, Zhang Changrong, Zhu Shuilian, Li Peiwu, Chen Xinlin, Hou Zhengkun, Liu Fengbin
Department of Gastroenterology, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, China.
Department of Orthopedics, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, China.
J Neurogastroenterol Motil. 2018 Oct 1;24(4):656-668. doi: 10.5056/jnm18037.
BACKGROUND/AIMS: MicroRNAs (miRNAs) were reported to be responsible for intestinal permeability in diarrhea-predominant irritable bowel syndrome (IBS-D) rats in our previous study. However, whether and how miRNAs regulate visceral hypersensitivity in IBS-D remains largely unknown.
We established the IBS-D rat model and evaluated it using the nociceptive visceral hypersensitivity test, myeloperoxidase activity assay, restraint stress-induced defecation, and electromyographic (EMG) activity. The distal colon was subjected to miRNA microarray analysis followed by isolation and culture of colonic epithelial cells (CECs). Bioinformatic analysis and further experiments, including dual luciferase assays, quantitative real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay, were used to detect the expression of miRNAs and how it regulates visceral hypersensitivity in IBS-D rats.
The IBS-D rat model was successfully established. A total of 24 miRNAs were differentially expressed in the distal colon of IBS-D rats; 9 were upregulated and 15 were downregulated. Among them, the most significant upregulation was miR-200a, accompanied by downregulation of cannabinoid receptor 1 (CNR1) and serotonin transporter (SERT). MiR-200a mimic markedly inhibited the expression of CNR1/SERT. Bioinformatic analysis and luciferase assay confirmed that CNR1/SERT are direct targets of miR-200a. Rescue experiments that overexpressed CNR1/SERT significantly abolished the inhibitory effect of miR-200a on the IBS-D rats CECs.
This study suggests that miR-200a could induce visceral hyperalgesia by targeting the downregulation of CNR1 and SERT, aggravating or leading to the development and progression of IBS-D. MiR-200a may be a regulator of visceral hypersensitivity, which provides potential targets for the treatment of IBS-D.
背景/目的：在我们之前的研究中，据报道微小RNA（miRNA）与腹泻型肠易激综合征（IBS-D）大鼠的肠道通透性有关。然而，miRNA是否以及如何调节IBS-D中的内脏超敏反应在很大程度上仍然未知。
我们建立了IBS-D大鼠模型，并使用伤害性内脏超敏反应试验、髓过氧化物酶活性测定、束缚应激诱导排便和肌电图（EMG）活动对其进行评估。对远端结肠进行miRNA微阵列分析，随后分离并培养结肠上皮细胞（CEC）。采用生物信息学分析和进一步的实验，包括双荧光素酶测定、定量实时聚合酶链反应、蛋白质印迹和酶联免疫吸附测定，来检测miRNA的表达及其如何调节IBS-D大鼠的内脏超敏反应。
成功建立了IBS-D大鼠模型。共有24种miRNA在IBS-D大鼠的远端结肠中差异表达；9种上调，15种下调。其中，上调最显著的是miR-200a，同时伴随着大麻素受体1（CNR1）和5-羟色胺转运体（SERT）的下调。miR-200a模拟物显著抑制CNR1/SERT的表达。生物信息学分析和荧光素酶测定证实CNR1/SERT是miR-200a 的直接靶点。过表达CNR1/SERT的挽救实验显著消除了miR-200a对IBS-D大鼠CEC的抑制作用。
本研究表明，miR-200a可通过靶向下调CNR1和SERT诱导内脏痛觉过敏，加重或导致IBS-D的发生和发展。miR-200a可能是内脏超敏反应的调节因子，为IBS-D的治疗提供了潜在靶点。
