Bistoletti Michela, Micheloni Giovanni, Baranzini Nicolò, Bosi Annalisa, Conti Andrea, Filpa Viviana, Pirrone Cristina, Millefanti Giorgia, Moro Elisabetta, Grimaldi Annalisa, Valli Roberto, Baj Andreina, Crema Francesca, Giaroni Cristina, Porta Giovanni
Department of Medicine and Surgery, University of Insubria, Varese, Italy.
Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy.
PeerJ. 2020 Feb 13;8:e8442. doi: 10.7717/peerj.8442. eCollection 2020.
Inflammatory bowel diseases are associated with remodeling of neuronal circuitries within the enteric nervous system, occurring also at sites distant from the acute site of inflammation and underlying disturbed intestinal functions. Homeoproteins orthodenticle OTX1 and OTX2 are neuronal transcription factors participating to adaptation during inflammation and underlying tumor growth both in the central nervous system and in the periphery. In this study, we evaluated OTX1 and OTX2 expression in the rat small intestine and distal colon myenteric plexus after intrarectal dinitro-benzene sulfonic (DNBS) acid-induced colitis.
OTX1 and OTX2 distribution was immunohistochemically investigated in longitudinal muscle myenteric plexus (LMMP)-whole mount preparations. mRNAs and protein levels of both OTX1 and OTX2 were evaluated by qRT-PCR and Western blotting in LMMPs.
DNBS-treatment induced major gross morphology and histological alterations in the distal colon, while the number of myenteric neurons was significantly reduced both in the small intestine and colon. mRNA levels of the inflammatory markers, TNFα, pro-IL1β, IL6, HIF1α and VEGFα and myeloperoxidase activity raised in both regions. In both small intestine and colon, an anti-OTX1 antibody labeled a small percentage of myenteric neurons, and prevalently enteric glial cells, as evidenced by co-staining with the glial marker S100β. OTX2 immunoreactivity was present only in myenteric neurons and was highly co-localized with neuronal nitric oxide synthase. Both in the small intestine and distal colon, the number of OTX1- and OTX2-immunoreactive myenteric neurons significantly increased after DNBS treatment. In these conditions, OTX1 immunostaining was highly superimposable with inducible nitric oxide synthase in both regions. OTX1 and OTX2 mRNA and protein levels significantly enhanced in LMMP preparations of both regions after DNBS treatment.
These data suggest that colitis up-regulates OTX1 and OTX2 in myenteric plexus both on site and distantly from the injury, potentially participating to inflammatory-related myenteric ganglia remodeling processes involving nitrergic transmission.
炎症性肠病与肠神经系统内神经回路的重塑有关，这种重塑也发生在远离急性炎症部位且导致肠道功能紊乱的区域。同源异形蛋白正齿突蛋白OTX1和OTX2是神经元转录因子，在中枢神经系统和外周组织中参与炎症过程中的适应性反应以及肿瘤生长。在本研究中，我们评估了直肠内二硝基苯磺酸（DNBS）酸诱导的结肠炎后大鼠小肠和远端结肠肌间神经丛中OTX1和OTX2的表达。
在纵行肌肌间神经丛（LMMP）整装标本中通过免疫组织化学研究OTX1和OTX2的分布。通过qRT-PCR和蛋白质印迹法评估LMMP中OTX1和OTX2的mRNA和蛋白质水平。
DNBS处理导致远端结肠出现明显的大体形态和组织学改变，而小肠和结肠中肌间神经元的数量均显著减少。两个区域中炎症标志物TNFα、前IL1β、IL6、HIF1α和VEGFα的mRNA水平以及髓过氧化物酶活性均升高。在小肠和结肠中，抗OTX1抗体标记了一小部分肌间神经元，且主要是肠胶质细胞，与胶质细胞标志物S100β共染色证明了这一点。OTX2免疫反应性仅存在于肌间神经元中，并且与神经元型一氧化氮合酶高度共定位。在小肠和远端结肠中，DNBS处理后OTX1和OTX2免疫反应性肌间神经元的数量均显著增加。在这些情况下，两个区域中OTX1免疫染色与诱导型一氧化氮合酶高度重叠。DNBS处理后，两个区域的LMMP制剂中OTX1和OTX2的mRNA和蛋白质水平均显著增强。
这些数据表明，结肠炎可上调肌间神经丛中OTX1和OTX2的表达，无论在损伤部位还是远离损伤部位，可能参与涉及一氧化氮能传递的炎症相关肌间神经节重塑过程。
