College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, 730070, Gansu, China.
BMC Vet Res. 2022 Apr 19;18(1):143. doi: 10.1186/s12917-022-03243-2.
Neuromedin U (NMU) plays an important role in activating the group 2 innate lymphoid cells (ILC2s) and initiating the host's anti-parasitic immune responses. It is aimed to explore the distribution characteristics of NMU in the sheep small intestine and the influence of Moniezia benedeni infection on them. In the present study, the pET-28a-NMU recombinant plasmids were constructed, and Escherichia coli. BL21 (DE3) were induced to express the recombinant protein. And then, the rabbit anti-sheep NMU polyclonal antibody was prepared and immunofluorescence staining was performed with it. The expression levels of NMU in the intestine of normal and Moniezia benedeni-infected sheep were detected by ELISA.
The results showed that the molecular weight of the obtained NMU recombinant protein was consistent with the expected molecular (13 kDa) and it was expressed in the form of inclusion body. The titer and specificity of obtained rabbit anti-sheep NMU polyclonal antibody were good. The results of immunofluorescence analysis showed that the nerve fibers which specifically expressed NMU mainly extended from the ganglion in the submucosal to lamina propria (LP) in the sheep small intestine, and the expression level was relatively high; especially on the nerve fibers of LP around the intestinal glands. The expression levels were gradually increased from the duodenum to the ileum, and the levels in the jejunum and ileum were significantly higher than that in the duodenum (P < 0.05). In addition, scattered NMU positive cells were distributed in the epithelium of the jejunal crypts. Moniezia benedeni infection increased the expression of NMU in each intestinal segment, especially in the jejunum and ileum there were significant increase (P < 0.05).
It was suggested that Moniezia benedeni infection could be detected by the high expression of NMU in sheep enteric nervous, and which laid the foundation for further studies on whether NMU exerts anti-parasitic immunity by activating ILC2s. In addition, NMU was expressed in some intestinal gland epitheliums, which also provided a basis for studying its roles in regulation of the immune homeostasis. The present study laid the foundation for further revealing the molecular mechanism of sheep's neural-immune interaction network perceiving the colacobiosis of parasites.
神经调节素 U（NMU）在激活 2 型固有淋巴细胞（ILC2）并启动宿主抗寄生虫免疫反应方面发挥着重要作用。本研究旨在探讨 NMU 在绵羊小肠中的分布特征以及莫尼茨绦虫感染对其的影响。本研究构建了 pET-28a-NMU 重组质粒，诱导大肠杆菌 BL21（DE3）表达重组蛋白。然后，制备兔抗绵羊 NMU 多克隆抗体，并对其进行免疫荧光染色。通过 ELISA 检测正常和莫尼茨绦虫感染绵羊肠道中 NMU 的表达水平。
结果表明，获得的 NMU 重组蛋白的分子量与预期的分子质量（13 kDa）一致，且以包涵体的形式表达。获得的兔抗绵羊 NMU 多克隆抗体的效价和特异性均良好。免疫荧光分析结果显示，特异性表达 NMU 的神经纤维主要从黏膜下神经节向小肠固有层（LP）延伸，表达水平相对较高；特别是在肠腺周围的 LP 神经纤维上。表达水平从十二指肠到回肠逐渐增加，空肠和回肠的表达水平显著高于十二指肠（P<0.05）。此外，在空肠隐窝的上皮中分布有散在的 NMU 阳性细胞。莫尼茨绦虫感染增加了每个肠段 NMU 的表达，特别是在空肠和回肠中，表达水平显著增加（P<0.05）。
提示莫尼茨绦虫感染可通过绵羊肠内神经 NMU 的高表达来检测，为进一步研究 NMU 是否通过激活 ILC2 发挥抗寄生虫免疫作用奠定了基础。此外，NMU 在一些肠腺上皮中表达，这也为研究其在调节免疫稳态中的作用提供了依据。本研究为进一步揭示绵羊神经-免疫相互作用网络感知寄生虫共生的分子机制奠定了基础。
