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  • br Experimental Procedures br Acknowledgments br Introductio

    2019-08-15


    Experimental Procedures
    Acknowledgments
    Introduction The ability of the affiliative and nurture hormone oxytocin (OT) to attenuate stress has been observed across psychological, neurological, and physiological levels [1], [2]. We and others have demonstrated that, in the gut, OT can lessen the inflammatory stress that accompanies disorders such as irritable bowel disease [3], [4]. The stress-attenuating effect of OT may serve an important role during early postnatal gut development, as implicated by the dynamic regulation of OT receptor (OTR) expression in rat enterocytes [5]. Furthermore, mice lacking the OTR have accelerated gastrointestinal transit time, increased macromolecular permeability, more severe experimentally induced colitis, reduced villus height and shorter crypts, as well as reduced proliferation [6]. OTR expression peaks during the milk suckling period [5] when maternal gut-originating microbiota colonize the newborn gut [7], [8] and while it is concomitantly exposed to a high OT concentration in milk [9], [10], [11]. Moreover, our prior in vitro studies with enterocytic Caco2BB cells show that OT downregulates the Akt/mTORC1 (mammalian target of rapamycin complex 1) anabolic signaling pathway [12] and activates some endoplasmic reticulum (ER) stress sensors that elicit the unfolded protein response (UPR) [13]. The precise physiological role of OT in newborn enterocytes remains to be elucidated. Enterocytes sample bacterial products, such as lipopolysaccharide (LPS), with Toll-like receptors (TLRs). These receptors activate both the canonical pro-inflammatory transcriptional program mediated by nuclear factor kappa-light-chain-enhancer of activated Mifepristone (NF-κB) [14], [15] and the UPR [16]. Inflammation stress can result in chaperone failure and subsequent accumulation of misfolded protein cargo in the ER, which is often a prelude to apoptosis [17]. ER stress under these conditions may initiate the UPR transcriptional program, which limits mRNA translation except for selected proteins (e.g., chaperones) to preserve the ER by clearing misfolded proteins [18]. At least three proteins are anchored in the ER membrane that sense stress and activate the UPR. These include (1) ATF6 (activating transcription factor 6), (2) PERK (protein kinase RNA-like endoplasmic reticulum kinase) and (3) IRE1a (inositol requiring enzyme 1a) [19]. The ultimate outcome of the UPR, whether it is cell preservation or the initiation of apoptosis to limit organ damage, is dependent upon the severity of the initiating pathological process. For example, the activation of PERK by severe, prolonged stress, may inactivate its substrate, the eukaryotic initiation translation factor 2a (eIF2a), but also activate the pro-apoptotic C/EBP homologous protein (CHOP) transcription factor [19]. On the other hand, a splice variant of the UPR, mediated by IRE1a and spliced X-box binding protein 1 (XBP1s), may constitutively maintain the quality of protein folding [20]. In this study we assessed the impact of OT-treatment upon UPR signaling molecules during or after exposure to LPS, which mimics bacterial endotoxin ingestion with breast milk, on enterocytic Caco2BB cells. Protein extracts of cells stimulated in vitro were assayed using automated immunocapillary electrophoresis. We show that OT activated select UPR mediators and inhibited and partially reversed LPS-induced inflammation. UPR induction by OT-rich milk may precondition enterocytes in the developing mammal to resist cellular stress associated with microbial colonization and may also impact enterocyte differentiation through effects on cellular metabolism [21]. OT may also protect other cell types, such as neurons, from stress-related complications during postnatal development.
    Materials and methods
    Results Our experimental Mifepristone conditions were optimized to coordinate the temporal parameters of NF-κB activation and the UPR to the time course of relevant intracellular signaling in response to OT that we have previously published [12], [13], [24]. This included physiological, and not necessarily pathological, LPS stimulation (e.g., lower dose and shorter duration of exposure vs. pathological conditions of 6–72h with a higher dose [27], [28], [29], [30]) so that we could assess signaling after 30min of exposure to OT and/or LPS.