Filpa Viviana, Carpanese Elisa, Marchet Silvia, Pirrone Cristina, Conti Andrea, Rainero Alessia, Moro Elisabetta, Chiaravalli Anna Maria, Zucchi Ileana, Moriondo Andrea, Negrini Daniela, Crema Francesca, Frigo Gianmario, Giaroni Cristina, Porta Giovanni
Department of Medicine and Surgery, University of Insubria, Varese, Italy.
Department of Internal Medicine and Therapeutics, Section of Pharmacology, University of Pavia, Pavia, Italy.
Am J Physiol Gastrointest Liver Physiol. 2017 Apr 1;312(4):G374-G389. doi: 10.1152/ajpgi.00386.2016. Epub 2017 Feb 2.
Neuronal and inducible nitric oxide synthase (nNOS and iNOS) play a protective and damaging role, respectively, on the intestinal neuromuscular function after ischemia-reperfusion (I/R) injury. To uncover the molecular pathways underlying this dichotomy we investigated their possible correlation with the orthodenticle homeobox proteins OTX1 and OTX2 in the rat small intestine myenteric plexus after in vivo I/R. Homeobox genes are fundamental for the regulation of the gut wall homeostasis both during development and in pathological conditions (inflammation, cancer). I/R injury was induced by temporary clamping the superior mesenteric artery under anesthesia, followed by 24 and 48 h of reperfusion. At 48 h after I/R intestinal transit decreased and was further reduced by -propyl-l-arginine hydrochloride (NPLA), a nNOS-selective inhibitor. By contrast this parameter was restored to control values by 1400W, an iNOS-selective inhibitor. In longitudinal muscle myenteric plexus (LMMP) preparations, iNOS, OTX1, and OTX2 mRNA and protein levels increased at 24 and 48 h after I/R. At both time periods, the number of iNOS- and OTX-immunopositive myenteric neurons increased. nNOS mRNA, protein levels, and neurons were unchanged. In LMMPs, OTX1 and OTX2 mRNA and protein upregulation was reduced by 1400W and NPLA, respectively. In myenteric ganglia, OTX1 and OTX2 staining was superimposed with that of iNOS and nNOS, respectively. Thus in myenteric ganglia iNOS- and nNOS-derived NO may promote OTX1 and OTX2 upregulation, respectively. We hypothesize that the neurodamaging and neuroprotective roles of iNOS and nNOS during I/R injury in the gut may involve corresponding activation of molecular pathways downstream of OTX1 and OTX2. Intestinal ischemia-reperfusion (I/R) injury induces relevant alterations in myenteric neurons leading to dismotility. Nitrergic neurons seem to be selectively involved. In the present study the inference that both neuronal and inducible nitric oxide synthase (nNOS and iNOS) expressing myenteric neurons may undergo important changes sustaining derangements of motor function is reinforced. In addition, we provide data to suggest that NO produced by iNOS and nNOS regulates the expression of the vital transcription factors orthodenticle homeobox protein 1 and 2 during an I/R damage.
神经元型和诱导型一氧化氮合酶（nNOS和iNOS）在缺血再灌注（I/R）损伤后分别对肠道神经肌肉功能发挥保护和损伤作用。为了揭示这种二分法背后的分子途径，我们研究了它们与大鼠小肠肌间神经丛中orthodenticle同源框蛋白OTX1和OTX2在体内I/R后的可能相关性。同源框基因对于在发育过程以及病理状况（炎症、癌症）下肠道壁稳态的调节至关重要。I/R损伤通过在麻醉下临时夹闭肠系膜上动脉诱导，随后进行24小时和48小时的再灌注。I/R后48小时，肠道转运下降，并且被nNOS选择性抑制剂盐酸-N-丙基-L-精氨酸（NPLA）进一步降低。相比之下，该参数通过iNOS选择性抑制剂1400W恢复到对照值。在纵行肌肌间神经丛（LMMP）制备物中，I/R后24小时和48小时，iNOS、OTX1和OTX2的mRNA和蛋白水平升高。在这两个时间段，iNOS和OTX免疫阳性的肌间神经元数量增加。nNOS的mRNA、蛋白水平和神经元数量未改变。在LMMP中，OTX1和OTX2的mRNA和蛋白上调分别被1400W和NPLA降低。在肌间神经节中，OTX1和OTX2染色分别与iNOS和nNOS的染色重叠。因此，在肌间神经节中，iNOS和nNOS衍生的NO可能分别促进OTX1和OTX2的上调。我们推测，在肠道I/R损伤期间，iNOS和nNOS的神经损伤和神经保护作用可能涉及OTX1和OTX2下游分子途径的相应激活。肠道缺血再灌注（I/R）损伤会导致肌间神经元发生相关改变，进而导致运动障碍。含氮能神经元似乎被选择性地累及。在本研究中，表达神经元型和诱导型一氧化氮合酶（nNOS和iNOS）的肌间神经元可能发生重要变化，维持运动功能紊乱这一推断得到了加强。此外，我们提供的数据表明，在I/R损伤期间，iNOS和nNOS产生的NO调节重要转录因子orthodenticle同源框蛋白1和2的表达。
