Effects Of Intestinal Alkaline Phosphatase Research Articles

Intestinal alkaline phosphatase (IAP, IAP Enhancer) attenuates intestinal inflammation and alleviates insulin resistance.

In this study, we investigated the effects of intestinal alkaline phosphatase (IAP) in controlled intestinal inflammation and alleviated associated insulin resistance (IR). We also explored the possible underlying molecular mechanisms, showed the preventive effect of IAP on IR in vivo, and verified the dephosphorylation of IAP for the inhibition of intestinal inflammation in vitro. Furthermore, we examined the preventive role of IAP in IR induced by a high-fat diet in mice. We found that an IAP + IAP enhancer significantly ameliorated blood glucose, insulin, low-density lipoprotein, gut barrier function, inflammatory markers, and lipopolysaccharide (LPS) in serum. IAP could dephosphorylate LPS and nucleoside triphosphate in a pH-dependent manner in vitro. Firstly, LPS is inactivated by IAP and IAP reduces LPS-induced inflammation. Secondly, adenosine, a dephosphorylated product of adenosine triphosphate, elicited anti-inflammatory effects by binding to the A2A receptor, which inhibits NF-κB, TNF, and PI3K-Akt signalling pathways. Hence, IAP can be used as a natural anti-inflammatory agent to reduce intestinal inflammation-induced IR.

Phosphate Groups in the Lipid A Moiety Determine the Effects of LPS on Hepatic Stellate Cells: A Role for LPS-Dephosphorylating Activity in Liver Fibrosis.

Alkaline phosphatase (AP) activity is highly upregulated in plasma during liver diseases. Previously, we demonstrated that AP is able to detoxify lipopolysaccharide (LPS) by dephosphorylating its lipid A moiety. Because a role of gut-derived LPS in liver fibrogenesis has become evident, we now examined the relevance of phosphate groups in the lipid A moiety in this process. The effects of mono-phosphoryl and di-phosphoryl lipid A (MPLA and DPLA, respectively) were studied in vitro and LPS-dephosphorylating activity was studied in normal and fibrotic mouse and human livers. The effects of intestinal AP were studied in mice with CCL4-induced liver fibrosis. DPLA strongly stimulated fibrogenic and inflammatory activities in primary rat hepatic stellate cells (rHSCs) and RAW264.7 macrophages with similar potency as full length LPS. However, MPLA did not affect any of the parameters. LPS-dephosphorylating activity was found in mouse and human livers and was strongly increased during fibrogenesis. Treatment of fibrotic mice with intravenous intestinal-AP significantly attenuated intrahepatic desmin+− and αSMA+ −HSC and CD68+− macrophage accumulation. In conclusion, the lack of biological activity of MPLA, contrasting with the profound activities of DPLA, shows the relevance of LPS-dephosphorylating activity. The upregulation of LPS-dephosphorylating activity in fibrotic livers and the protective effects of exogenous AP during fibrogenesis indicate an important physiological role of intestinal-derived AP during liver fibrosis.

Effects of intestinal alkaline phosphatase on intestinal barrier function in a cecal ligation and puncture (CLP)-induced mouse model for sepsis.

Sepsis is a severe pathological condition associated with systemic inflammation, intestinal inflammation, and gastrointestinal barrier dysfunction. Intestinal alkaline phosphatase (IAP) has been demonstrated to detoxify lipopolysaccharide, an important mediator in the pathophysiology of sepsis. We investigated the effect of treatment with IAP on intestinal permeability, intestinal inflammation, and bacterial translocation. OF-1 mice were divided into 4 groups (n=12/group), undergoing either a sham or cecal ligation and puncture (CLP) procedure to induce sepsis. Mice received IAP or a vehicle intraperitoneally 5minutes prior to the onset of the CLP or sham procedure, which was repeated every 12hours for two consecutive days. After two days, in vivo intestinal permeability, intestinal inflammation, and bacterial translocation were determined. CLP-induced sepsis resulted in significantly more weight loss, worse clinical disease scores, bacterial translocation, and elevated inflammatory cytokines. Intestinal permeability was increased up to 5-fold (P<.001). IAP activity was significantly increased in septic animals. Treatment with IAP had no effect on clinical outcomes but reduced the increased permeability of the small intestine by 50% (P=.005). This reduction in permeability was accompanied by a modified gene expression of claudin-1 (P=.025), claudin-14 (P=.035), and interleukin 12 (P=.015). A discriminant analysis showed that treatment with IAP is linked to modified mRNA levels of several tight junction proteins and cytokines. Treatment with IAP diminished CLP-induced intestinal barrier disruption, associated with modified expression of several cytokines and claudins. Nevertheless, this effect did not translate into better clinical outcomes in our experimental setup.

Intestinal alkaline phosphatase and sodium butyrate may be beneficial in attenuating LPS-induced intestinal inflammation.

In this study, we evaluated the effect of intestinal alkaline phosphatase (IAP) and sodium butyrate (NaBu) on lipopolysaccharide (LPS)-induced intestinal inflammation. Intestinal alkaline phosphatase and RelA/p65 (NF-κB) gene expressions in porcine jejunum explants were evaluated following exposure to sodium butyrate (NaBu) and essential oil from Brazilian red pepper (EO), alone or in combination with NaBu, as well as exogenous IAP with or without LPS challenge. Five piglets weighing approximately 20 kg each were sacrificed, and their jejunum were extracted. The tissues were segmented into 10 parts, which were exposed to 10 treatments. Gene expressions of IAP and RelA/p65 (NF-κB) in jejunal explants were evaluated via RT-PCR. We found that EO, NaBu, and exogenous IAP were able to up-regulate endogenous IAP and enhance RelA/p65 (NF-κB) gene expression. However, only NaBu and exogenous IAP down-regulated LPS-induced inflammatory response via RelA/p65 (NF-κB). In conclusion, we demonstrated that exogenous IAP and NaBu may be beneficial in attenuating LPS-induced intestinal inflammation.

Effect of intestinal alkaline phosphatase on expression of Muc2, Stat4 and P-Stat4 in colitis in mice

目的：探讨肠道碱性磷酸酶（intestine alkaline phosphatase,IAP）对2,4,6-三硝基苯磺酸（2,4,6-trinitro-benzenesulfonic acid,TNBS）诱导的小鼠结肠炎中Mux2、Stat4和磷酸化Stat4（P-Stat4）表达的影响.方法：以对TNBS敏感的45只Balb/C小鼠为研究对象,随机分为3组.分别给予对照组（control）、模型组（TNBS）和治疗组（TNBS/IAP）生理盐水灌肠、TNBS灌肠加生理盐水灌胃和TNBS灌肠加IAP灌胃.于1 wk后评估小鼠肠道炎症情况,用HE染色检测肠道病理改变,免疫组织化学染色检测小鼠肠道上皮中Mux2、Stat4和P-Stat4的表达情况.用不同浓度的IAP预处理DC2.4细胞系,用蛋白免疫印迹试验检测LPS诱导后细胞中Stat4和P-Stat4的表达.结果：与对照组比较,模型组小鼠结肠炎症明显,而治疗组较模型组改善.在小鼠结肠上皮中,Mux2染色阳性率在3组间有差异（χ^2=19.62,P〈0.05）;模型组低于对照组（13.33%vs 93.33%）（χ^2=19.29,P〈0.05）;治疗组高于模型组（60.00%vs 13.33%）（χ^2=7.033,P〈0.05）.Stat4染色阳性率在3组间有差异（χ^2=7.22,P〈0.05）;模型组高于对照组（66.67%vs20.00%）（χ^2=6.652,P〈0.05）;治疗组和模型组（50.00%vs 66.67%）之间的差异无统计学意义（χ^2=3.333,P〉0.05）.P-Stat4染色阳性率在3组间有差异（χ^2=12.95,P〈0.05）;模型组高于对照组（60.00%vs 6.67%）（χ^2=9.6,P〈0.05）;治疗组低于模型组（13.33%vs 60.00%）（χ^2=7.033,P〈0.05）.20、100 I U/m L的I A P处理DC2.4后,Stat4和P-Stat4的表达下调（P〈0.05）.结论：IAP促进结肠炎小鼠肠道Muc2的表达,下调Stat4的磷酸化,可缓解TNBS诱导的小鼠结肠炎.

The effect of intestinal alkaline phosphatase on intestinal epithelial cells, macrophages and chronic colitis in mice

AimsIntestinal alkaline phosphatase (IAP) is an intestinal brush border enzyme that is shown to function as a gut mucosal defense factor, but its defensive mechanism remains unclear. The aims of this study were to evaluate the effect of IAP on intestinal epithelial cells and macrophages, and on chronic colitis in interleukin-10-deficient (IL-10−/−) mice. Main methodsHuman intestinal epithelial cells COLO 205 and peritoneal macrophages from IL-10−/− mice were pretreated with IAP and then stimulated with lipopolysaccharide (LPS). IL-8 secretion from COLO205 cells and TNF-α, IL-6, IL-12 from peritoneal macrophages were measured by ELISA. Electrophoretic mobility shift assay was used to assess the DNA binding activity of NF-κB and IκBα phosphorylation/degradation was evaluated by immunoblot assay in COLO 205. For the in vivo study, colitis was induced in IL-10−/− mice with piroxicam, the mice were then treated with 100 or 300units of IAP by oral gavage for 2weeks. Colitis was quantified by histopathologic scoring, and the phosphorylation of IκBα in the colonic mucosa was assessed using immunohistochemistry. Key findingsIAP significantly inhibited LPS-induced inflammatory cytokine production in both IECs and peritoneal macrophages. IAP also attenuated LPS-induced NF-κB binding activity and IκBα phosphorylation/degradation in IECs. Oral administration of IAP significantly reduced the severity of colitis and down-regulated colitis-induced IκBα phosphorylation in IL-10−/− mice. SignificanceIAP may inhibit the activation of intestinal epithelial cells and peritoneal macrophages, and may attenuate chronic murine colitis. This finding suggests that IAP supplementation is a potential therapeutic option for inflammatory bowel disease.

P017 The effect of intestinal alkaline phosphatase on intestinal epithelial cells, macrophages and chronic colitis in interleukin-10-deficient mice

abate metastasis dissemination by maintaining lymphatic vessel junctions’ integrity. Methods: The azoxymethane (AOM)/dextran sulfate sodium (DSS) CAC model was used. In addition, colitic mice were orthotopically injected with CT26 colon cancer cells. Modulation of lymphangiogenesis was obtained by systemic inhibition of VEGFR3 or adenoviral overexpression of VEGFC. Tumor density and size were measured at the end of the AOM/DSS protocol by visual, endoscopic and histological inspection. In the xenotransplantation model, liver, lung and draining lymphnodes were processed for metastasis quantification by FACS analysis. Whole mounts of colons were stained to analyse area density and dimension of lymphatic vessels (LVs) within peritumoral and tumoral regions. Moreover, VE-cadherin distribution and expression were studied in vivo on LVs and in vitro on Human intestinal lymphatic endothelial cells (HILEC) stimulated with VEGFC or with a VEGFR3 inhibitor. Results: In both the AOM/DSS and the xenotransplantation models, systemic inhibition of VEGFR3 inhibited lymphangiogenesis in peritumoral and tumoral regions, reducing both area density and LVs dimension. In addition, it reduced tumor density and size, together with an abatement of metastatic dissemination, when compared to untreated animals. In contrast, tumor density and growth, including metastasis formation was enhanced by VEGFC, which in turn increased lymphangiogenesis within the same regions. Whole mount staining showed that VEGFC altered VE-cadherin expression both in vivo and in vitro, whereas VEGFR3 inhibitor kept endothelial junction integrity, thus linking the VEGFC/VEGFR3 pathway to VE-cadherin-dependent metastasis dissemination. Conclusions: Our findings demonstrate that VEGFC/VEGFR3dependent lymphangiogenesis plays a key role both in CAC growth and metastasis dissemination. Our study reveals a novel mechanism of control of lymphatic junction integrity which may be a promising target for the treatment of CAC and the associated metastatic process.

Protective Effect of Intestinal Alkaline Phosphatase in Staphylococcus aureus Infection in Mice

The brush border enzyme intestinal alkaline phosphatase (IAP) is known to exert inhibitory effects against LPS and Gram-negative sepsis, but its impact on Gram-positive infections has not been previously examined. Based on our recent observation that IAP can inactivate lipoteichoic acid (LTA), a component of Gram-positive bacterial cell walls, we hypothesized that IAP may have beneficial effects against Staphylococcus aureus infection.

The effect of intestinal alkaline phosphatase on abscess fluid-induced inflammation

The effect of intestinal alkaline phosphatase on abscess fluid-induced inflammation

595 ALKALINE PHOSPHATASE IS A PROTECTIVE ENZYME DURING LIVER FIBROGENESIS

Background and Aim: Serum alkaline phosphatase (AP) levels serve as a marker for many liver diseases. Recent studies indicate that AP may act as a protective enzyme by dephosphorylation of LPS because dephosphorylation blocks toxicity of this product. Gut-derived LPS is known to aggravate liver damage and fibrosis and we hypothesized that higher levels of AP may represent a physiological response upon higher levels of this toxin during fibrosis. We therefore studied hepatic expression levels and effects of intestinal AP during fibrogenesis. Methods: Intestinal AP knock-out C57BL/6 mice (iAP KO) were examined at 6 weeks of age and compared to age-matched wildtype. Liver fibrogenesis was induced by CCl4 administration to Balb/c mice for 1 day (acute studies) or 8 weeks (chronic studies). The latter group received saline or calf-intestinal AP (5 units, i.v.), from week 6 to 8 (3x/week, n = 6/group). Results: iAP KO mice displayed higher intrahepatic expression levels of fibrogenic markers (PAR-1, Collagen I) paralleled by an increase in macrophages of the pro-fibrotic M2 phenotype relative to WT. So, lack of intestinal AP stimulates fibrogenesis within the liver. We subsequently examined intrahepatic AP expression in normal mice, after CCl4-induced acute liver damage and after chronic CCL4 administration, characterized by liver fibrosis. Results show a gradual increase in intrahepatic AP-activity from normal to fibrotic animals. Histochemical analysis revealed that this APactivity is able to dephosphorylate the lipid A moiety of LPS. We further explored the role of AP by injecting iAP to mice with CCL4-induced fibrosis. Data show a reduced hepatic AP activity, paralleled by significant lower expression levels of desmin and significant lower accumulation of M2 macrophages in iAP-treated mice compared to control. Conclusions: Based on the increased hepatic fibrogenic activity in intestinal AP-KO mice, and the attenuated fibrogenesis in fibrotic mice receiving intestinal AP, we conclude that iAP attenuates fibrosis. The enhanced expression of AP in fibrotic livers and the demonstration that this AP activity is able to dephosphorylate LPS, suggests that endogenous AP serves as a protective enzyme after liver damage. Enhanced AP activity during disease may therefore reflect a physiological response to LPS.

Aspirin Inhibition of In Vivo Effects of Intestinal Alkaline Phosphatase on Platelets

SummaryIntravenous injections of 10 mg/kg purified intestinal alkaline phosphatase (AlPh) into (I) normal (non-aspirinized) rabbits and (II) aspirinized rabbits, show that aspirin inhibits significantly the AlPh effects on platelet aggregation and adhesion. Since aspirin does not change the assayable injected AlPh activity or its electrophoretic distribution, and whereas aspirin inhibits the platelet ADP release, it is very possible that the effects of AlPh on platelets are due to platelet ADP release by AlPh.Aspirin also inhibits the hypercoagulable effect of the injected AlPh. If aspirin acetylates the platelet membrane by its acetyl group, this could compete with HF (Hageman factor) which is a sialoglycoprotein, and could thus prevent the activation of HF on the surface of platelets.