Abstract
Exogenous intestinal alkaline phosphatase (IAP), an enzyme produced endogenously at the brush edge of the intestinal mucosa, may mitigate the increase in aberrant intestinal permeability increased during sepsis. The aim of this study was to test the efficacy of the inhibitory effect of IAP on acute intestinal inflammation and to study the molecular mechanisms underlying IAP in ameliorating intestinal permeability. We used an in vivo imaging method to evaluate disease status and the curative effect of IAP. Two Escherichia coli (E.coli) B21 strains, carrying EGFP labeled enhanced green fluorescent protein (EGFP) and RFP labeled red fluorescent protein (RFP), were constructed as tracer bacteria and were administered orally to C57/B6N mice to generate an injection peritonitis (IP) model. The IP model was established by injecting inflammatory lavage fluid. C57/B6N mice bearing the tracer bacteria were subsequently treated with (IP+IAP group), or without IAP (IP group). IAP was administered to the mice via tail vein injections. The amount of tracer bacteria in the blood, liver, and lungs at 24 h post-injection was analyzed via flow cytometry (FCM), in vivo imaging, and Western blotting. Intestinal barrier function was measured using a flux assay with the macro-molecule fluorescein isothiocyanate dextran, molecular weight 40kD, (FD40). To elucidate the molecular mechanism underlying the effects of IAP, we examined the levels of ERK phosphorylation, and the expression levels of proteins in the ERK-SP1-VEGF and ERK-Cdx-2-Claudin-2 pathways. We observed that IAP inhibited the expression of Claudin-2, a type of cation channel-forming protein, and VEGF, a cytokine that may increase intestinal permeability by reducing the levels of dephosphorylated ERK. In conclusion, exogenous IAP shows a therapeutic effect in an injection peritonitis model. This including inhibition of bacterial translocation. Moreover, we have established an imaging methodology for live-animals can effectively evaluate intestinal permeability and aberrant bacterial translocation in IP models.
Highlights
The gastrointestinal tract harbors a massive pool of microbes [1,2,3,4,5,6,7]
When the host is attacked by acute pathogens, the bacteria may move across the intestinal wall, which is known as gut-origin bacteria translocation (BT) [8,9]
The number of clones formed on an LB plate containing ampicillin, or kanamycin, was recorded
Summary
The gastrointestinal tract harbors a massive pool of microbes [1,2,3,4,5,6,7]. When the host is attacked by acute pathogens, the bacteria may move across the intestinal wall, which is known as gut-origin bacteria translocation (BT) [8,9]. Peritonitis is a commonly observed condition that may cause systemic inflammatory response syndrome, sepsis, and multiple organ failure [10,11]. Does the peritoneal cavity act as the source of systemic infection, but the impaired intestinal barrier facilitates the transmission of gut microbes into the blood stream. These effects are due primarily to changes in the expression of inflammatory factors, such as VEGF, that alter intestinal permeability [12] and intestinal tight junction proteins, such as those of the Claudin family [13,14]. Finding mechanisms that regulate these pathways and developing agents to protect intestinal barrier function may help improve the prognosis of these patients [15,16]
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