Department of Obstetrics and Gynecology, Xiangya Hospital, Central South University, Changsha, Hunan, China.
Department of Obstetrics and Gynecology, Xiangya Hospital, Central South University, Changsha, Hunan, China
J Investig Med. 2020 Aug;68(6):1171-1178. doi: 10.1136/jim-2020-001412. Epub 2020 Jul 21.
The precise mechanisms that lead to parturition remain unclear. In our initial complementary DNA (cDNA) microarray experiment, we found that the neuromedin B receptor (NMBR) was differentially expressed in the human myometrium during spontaneous or oxytocin-induced labor. We have previously shown that neuromedin B (NMB) could induce interleukin 6 (IL-6) and type 2 cyclo-oxygenase enzyme (COX-2) expression in the primary human myometrial cells via nuclear factor kappa B (NF-κB) transcription factor p65 (p65) and Jun proto-oncogene, activator protein 1 (AP-1) transcription factor subunit (c-Jun). This study is aimed to investigate whether NMBR is required for NMB-induced effect. Primary myometrial cell culture was established to provide a suitable model to investigate the mechanism of NMB in labor initiation. Immunochemical staining was conducted to validate the NMBR expression in primary myometrial cells. The mRNA and protein expression of NMBR, p65, c-Jun, COX-2 and IL-6 were assessed by Quantitative Real Time PCR (RT-qPCR) and western blotting. Lentiviruses with shRNAs targeting NMBR or containing cDNA sequence of NMBR were transfected to primary myometrial cells to knockdown or overexpress NMBR. Cell death was determined by annexin V and propidium iodide staining and analyzed by flow cytometry. The upregulation of COX-2 and IL-6 and phosphorylation of p65 and c-Jun were significantly attenuated by knockdown of NMBR and enhanced by overexpressed NMBR following NMB treatment, with no significant change in total p65 and c-Jun. In summary, this study showed that NMBR-mediated NMB-induced NF-κB and AP-1 activation, which in turn, induce expression of IL-6 and COX-2 in primary myometrial cells.
导致分娩的确切机制仍不清楚。在我们最初的 cDNA（互补 DNA）微阵列实验中，我们发现神经调节素 B 受体（NMBR）在自发性或催产素诱导的分娩过程中在人子宫肌中差异表达。我们之前已经表明，神经调节素 B（NMB）可以通过核因子 kappa B（NF-κB）转录因子 p65（p65）和 Jun 原癌基因，激活蛋白 1（AP-1）转录因子亚基（c-Jun）诱导原代人子宫肌细胞中白细胞介素 6（IL-6）和 2 型环氧化酶酶（COX-2）的表达。本研究旨在探讨 NMBR 是否是 NMB 诱导作用所必需的。建立原代子宫肌细胞培养物，为研究 NMB 在分娩启动中的作用机制提供合适的模型。进行免疫化学染色以验证原代子宫肌细胞中 NMBR 的表达。通过定量实时 PCR（RT-qPCR）和 Western blot 评估 NMBR、p65、c-Jun、COX-2 和 IL-6 的 mRNA 和蛋白表达。用靶向 NMBR 的 shRNA 或包含 NMBR cDNA 序列的慢病毒转染原代子宫肌细胞，以敲低或过表达 NMBR。通过 Annexin V 和碘化丙啶染色测定细胞死亡，并通过流式细胞术进行分析。NMB 处理后，NMBR 敲低显著减弱了 COX-2 和 IL-6 的上调以及 p65 和 c-Jun 的磷酸化，而过表达 NMBR 则增强了 COX-2 和 IL-6 的上调以及 p65 和 c-Jun 的磷酸化，而总 p65 和 c-Jun 无明显变化。总之，这项研究表明，NMBR 介导的 NMB 诱导的 NF-κB 和 AP-1 激活，进而诱导原代子宫肌细胞中 IL-6 和 COX-2 的表达。
