Supplementation with organic yeast-derived selenium provides immune protection against experimental necrotic enteritis in broiler chickens.

Necrotic enteritis (NE) is a potentially fatal poultry disease that causes enormous economic losses in the poultry industry worldwide. The study aimed to evaluate the effects of dietary organic yeast-derived selenium (Se) on immune protection against experimental necrotic enteritis (NE) in commercial broilers. Chickens were fed basal diets supplemented with different Se levels (0.25, 0.50, and 1.00 Se mg/kg). To induce NE, Clostridium perfringens (C. perfringens) was orally administered at 14 days of age post hatch. The results showed that birds fed 0.25 Se mg/kg exhibited significantly increased body weight gain compared with the non-supplemented/infected birds. There were no significant differences in gut lesions between the Se-supplemented groups and the non-supplemented group. The antibody levels against α-toxin and NetB toxin increased with the increase between 0.25 Se mg/kg and 0.50 Se mg/kg. In the jejunal scrapings and spleen, the Se-supplementation groups up-regulated the transcripts for pro-inflammatory cytokines IL-1ß, IL-6, IL-8, iNOS, and LITAF and avian ß-defensin 6, 8, and 13 (AvBD6, 8 and 13). In conclusion, supplementation with organic yeast-derived Se alleviates the negative consequences and provides beneficial protection against experimental NE.

Regulatory effects of Trichinella spiralis serpin-type serine protease inhibitor on endoplasmic reticulum stress and oxidative stress in host intestinal epithelial cells.

Endoplasmic reticulum stress (ERS) and oxidative stress (OS) are adaptive responses of the body to stressor stimulation. Although it has been verified that Trichinella spiralis (T. spiralis) can induce ERS and OS in the host, their association is still unclear. Therefore, this study explored whether T. spiralis-secreted serpin-type serine protease inhibitor (TsAdSPI) is involved in regulating the relationship between ERS and OS in the host intestine. In this study, mice jejunum and porcine small intestinal epithelial cells (IECs) were detected using qPCR, western blotting, immunohistochemistry (IHC), immunofluorescence (IF), and detection kits. The results showed that ERS- and OS-related indexes changed significantly after TsAdSPI stimulation, and Bip was located in IECs, indicating that TsAdSPI could induce ERS and OS in IECs. After the use of an ERS inhibitor, OS-related indexes were inhibited, suggesting that TsAdSPI-induced OS depends on ERS. When the three ERS signalling pathways, ATF6, IRE1, and PERK, were sequentially suppressed, OS was only regulated by the PERK pathway, and the PERK-eif2α-CHOP-ERO1α axis played a key role. Similarly, the expression of ERS-related indexes and the level of intracellular Ca2+ were inhibited after adding the OS inhibitor, and the expression of ERS-related indexes decreased significantly after inhibiting calcium transfer. This finding indicated that TsAdSPI-induced OS could affect ERS by promoting Ca2+ efflux from the endoplasmic reticulum. The detection of the ERS and OS sequences revealed that OS occurred before ERS. Finally, changes in apoptosis-related indexes were detected, and the results indicated that TsAdSPI-induced ERS and OS could regulate IEC apoptosis. In conclusion, TsAdSPI induced OS after entering IECs, OS promoted ERS by enhancing Ca2+ efflux, and ERS subsequently strengthened OS by activating the PERK-eif2α-CHOP-ERO1α axis. ERS and OS induced by TsAdSPI synergistically promoted IEC apoptosis. This study provides a foundation for exploring the invasion mechanism of T. spiralis and the pathogenesis of host intestinal dysfunction after invasion.

Accumulation of Alpha-Synuclein and Increase in the Inflammatory Response in the substantia nigra, Jejunum, and Colon in a Model of O3 Pollution in Rats.

This work aimed to study the effect of repeated exposure to low doses of ozone on alpha-synuclein and the inflammatory response in the substantia nigra, jejunum, and colon. Seventy-two male Wistar rats were divided into six groups. Each group received one of the following treatments: The control group was exposed to air. The ozone groups were exposed for 7, 15, 30, 60, and 90 days for 0.25 ppm for four hours daily. Afterward, they were anesthetized, and their tissues were extracted and processed using Western blotting, immunohistochemistry, and qPCR. The results indicated a significant increase in alpha-synuclein in the substantia nigra and jejunum from 7 to 60 days of exposure and an increase in NFκB from 7 to 90 days in the substantia nigra, while in the jejunum, a significant increase was observed at 7 and 15 days and a decrease at 60 and 90 days for the colon. Interleukin IL-17 showed an increase at 90 days in the substantia nigra in the jejunum and increases at 30 days and in the colon at 15 and 90 days. Exposure to ozone increases the presence of alpha-synuclein and induces the loss of regulation of the inflammatory response, which contributes significantly to degenerative processes.

Lemon Flavonoid Extract Eriomin Improves Pro/Antioxidant Status and Interferes with Cholesterol Metabolism without Affecting Serum Cholesterol Levels in Aged Rats.

This study aimed to assess the antioxidant capacity of lemon flavonoid extract Eriomin® (LE) and its impact on cholesterol metabolism in the context of healthy aging. We orally treated 24-month-old male Wistar rats with an LE (40 mg/kg) suspended in 0.3 mL of sunflower oil. At the same time, control groups received an equal volume of sunflower oil (CON) or remained untreated (ICON) daily for 4 weeks. We examined LE's effects on superoxide dismutase and catalase- and glutathione-related enzyme activities, the concentration of lipid peroxides and protein carbonyls, total oxidant status (TOS) and antioxidant status (TAS), and oxidative stress index (OSI) in the liver, jejunum, and ileum. We also measured total cholesterol, its biosynthetic precursors (lanosterol, lathosterol, desmosterol), its degradation products (bile acid precursors) in the serum, liver, jejunum, and ileum, and serum phytosterols (intestinal absorption markers). LE reduced TOS, TAS, and OSI (p < 0.05) compared with control values, indicating its consistent antioxidant action in all examined organs. LE lowered hepatic desmosterol (p < 0.05) while also reducing 7α- and 24-hydroxycholesterol levels in the liver and ileum (p < 0.01). Serum cholesterol, hepatic gene expression, and the immunostaining intensity of CYP7A1 were unchanged. In conclusion, LE exerted non-enzymatic antioxidant effects and reduced cholesterol degradation, reducing its biosynthesis products, thereby maintaining serum cholesterol levels.

Iron uptake mediated by the plant-derived chelator nicotianamine in the small intestine.

Iron is an essential metal for all living organisms that is absorbed in the intestinal cells as a heme-chelated or free form. It is unclear how important plant-derived chelators, such as nicotianamine (NA), an organic small molecule that is ubiquitous in crops, vegetables, and various other foods, contribute to iron bioavailability in mammals. We performed electrophysiological assays with Xenopus laevis oocytes and radioactive tracer experiments with Caco-2 cells. The findings revealed that the proton-coupled amino acid transporter SLC36A1 (PAT1) transports iron in the form of NA-Fe (II) complex in vitro. Decreased expression of hPAT1 by RNA interference in Caco-2 cells reduced the uptake of NA-59Fe (II) complex. The uptake of inorganic 59Fe (II) was relatively unaffected. These results imply that PAT1 transports iron as a NA-Fe (II) complex. The rate of 59Fe absorption in the spleen, liver, and kidney was higher when mice were orally administered NA-59Fe (II) compared with free 59Fe (II). The profile of site-specific PAT1 expression in the mouse intestine coincided with those of NA and iron contents, which were the highest in the proximal jejunum. Orally administered NA-59Fe (II) complex in mice was detected in the proximal jejunum by thin layer chromatography. In contrast, much less 59Fe (or NA) was detected in the duodenum, where the divalent metal transporter SLC11A2 (DMT1) absorbs free Fe (II). The collective results revealed the role of PAT1 in NA-Fe (II) absorption in the intestine and potential implication of NA in iron uptake in mammals.

Selenium exerts protective effects against heat stress-induced barrier disruption and inflammation response in jejunum of growing pigs.

BACKGROUND: Heat stress (HS) has a negative impact on the intestinal barrier and immune function of pigs. Selenium (Se) may improve intestinal health through affecting selenoproteins. Thus we investigate the protective effect of new organic Se (2-hydroxy-4-methylselenobutanoic acid, HMSeBA) on jejunal damage in growing pigs upon HS and integrate potential roles of corresponding selenoproteins. RESULTS: HS decreased the villus height and increased (P < 0.05) the protein abundance of HSP70, and downregulated (P < 0.05) protein levels of tight junction-related proteins (CLDN-1 and OCLD). HS-induced jejunal damage was associated with the upregulation of four inflammation-related genes and ten selenoprotein-encoding genes, downregulation (P < 0.05) of four selenoprotein-encoding genes and decreased (P < 0.05) the protein abundance of GPX4 and SELENOS. Compared with the HS group, HMSeBA supplementation not only elevated the villus height and the ratio of V/C (P < 0:05), but also reduced (P < 0.05) the protein abundance of HSP70 and MDA content, and increased (P < 0.05) the protein abundance of OCLD. HMSeBA supplementation downregulated the expression of seven inflammation-related genes, changed the expression of 12 selenoprotein-encoding genes in jejunum mucosa affected by HS, and increased the protein abundance of GPX4, TXNRD1 and SELENOS. CONCLUSION: Organic Se supplementation beyond nutritional requirement alleviates the negative effect of HS on the jejunum of growing pigs, and its protective effect is related to the response of corresponding selenoproteins. © 2021 Society of Chemical Industry.

Duodenal and proximal jejunal motility inhibition associated with bisacodyl-induced colonic high-amplitude propagating contractions.

Bisacodyl is a stimulant laxative often used in manometric studies of pediatric constipation to determine if it can initiate propulsive high-amplitude propagating contractions (HAPCs). Whereas the effects of bisacodyl infusion on colonic motility are well described, the effects of the drug on other regions of the gut after colonic infusion are not known. The aim of the present study was to characterize the effects of bisacodyl on both colonic and small bowel motility. Twenty-seven children (9.3 ± 1.2 yr) undergoing simultaneous high-resolution antroduodenal and colonic manometry were included. Small bowel and colonic motor patterns were assessed before and after colonic infusion of bisacodyl. Patients were divided into two groups: responders and nonresponders based on the presence of high-amplitude propagating contractions (HAPCs) after bisacodyl infusion. Nineteen patients were responders. A total of 188 postbisacodyl HAPCs was identified with a mean count of 10.4 ± 5.5 (range, 3-22), at a frequency of 0.6 ± 0.2/min and mean amplitude of 119.8 ± 23.6 mmHg. No motor patterns were induced in the small bowel. However, in the 19 responders the onset of HAPCs was associated with a significant decrease in small bowel contractile activity. In the nonresponders, there was no detectable change in small bowel motility after bisacodyl infusion. Bisacodyl-induced HAPCs are associated with a significant reduction in small bowel motility probably mediated by extrinsic sympathetic reflex pathways. This inhibition is potentially related to rectal distension, caused by the HAPC anal propulsion of colonic content.NEW & NOTEWORTHY The present study has shown, for the first time, that the presence of high-amplitude propagating contractions induced by bisacodyl is associated with a significant reduction in small bowel motility. These findings support of possible existence of a reflex pathway that causes inhibition of small bowel motility in response to rectal distension.

Requirement for the intestinal epithelial insulin-like growth factor-1 receptor in the intestinal responses to glucagon-like peptide-2 and dietary fat.

The intestinal hormone, glucagon-like peptide-2 (GLP-2), enhances the enterocyte chylomicron production. However, GLP-2 is known to require the intestinal-epithelial insulin-like growth factor-1 receptor (IE-IGF-1R) for its other actions to increase intestinal growth and barrier function. The role of the IE-IGF-1R in enterocyte lipid handling was thus tested in the GLP-2 signaling pathway, as well as in response to a Western diet (WD). IE-IGF-1R knockout (KO) and control mice were treated for 11 days with h(GLY2 )GLP-2 or fed a WD for 18 weeks followed by a duodenal fat tolerance test with C14 -labeled triolein. Human Caco-2BBE cells were treated with an IGF-1R antagonist or signaling inhibitors to determine triglyceride-associated protein expression. The IE-IGF-1R was required for GLP-2-induced increases in CD36 and FATP-4 in chow-fed mice, and for expression in vitro; FATP-4 also required PI3K/Akt. Although WD-fed IE-IGF-1R KO mice demonstrated normal CD36 expression, the protein was incorrectly localized 2h post-duodenal fat administration. IE-IGF-1R KO also prevented the WD-induced increase in MTP and decrease in APOC3, increased jejunal mucosal C14 -fat accumulation, and elevated plasma triglyceride and C14 -fat levels. Collectively, these studies elucidate new roles for the IE-IGF-1R in enterocyte lipid handling, under basal conditions and in response to GLP-2 and WD-feeding.

COL1A1 as a potential new biomarker and therapeutic target for type 2 diabetes.

Type 2 diabetes (T2D) is a public health problem with a rising incidence worldwide. In this study, a potential new biomarker for T2D and mechanisms underlying the hypoglycemic effects of Enteromorpha prolifera oligosaccharide were investigated. Tandem mass tag labeling with LC-MS/MS was used to identify the differentially expressed proteins (DEPs) between the jejunum of diabetic rats and control rats. Correlations between glycometabolic parameters and DEPs were revealed by a network analysis. The expression levels of target genes in key metabolic pathways were further evaluated to identify candidate biomarkers. Among 6810 total proteins, approximately 88 % were quantified, of which 148 DEPs with a fold change of <0.83 or>1.2 and a corrected p-value of <0.05 were identified. A KEGG enrichment analysis indicated that the hypoglycaemic effects of E. prolifera oligosaccharide involved the PI3K/AKT and extracellular matrix receptor interaction signaling pathways. More importantly, Col1a1 was the most significant gene in the extracellular matrix receptor interaction pathway and was linked to hypoglycaemic activity for the first time. Thus, Col1a1 is a novel potential therapeutic target for alleviating T2D.

Enterococcus faecium PNC01 isolated from the intestinal mucosa of chicken as an alternative for antibiotics to reduce feed conversion rate in broiler chickens.

BACKGROUND: The development and utilization of probiotics had many environmental benefits for replacing antibiotics in animal production. Bacteria in the intestinal mucosa have better adhesion to the host intestinal epithelial cells compared to bacteria in the intestinal contents. In this study, lactic acid bacteria were isolated from the intestinal mucosa of broiler chickens and investigated as the substitution to antibiotic in broiler production. RESULTS: In addition to acid resistance, high temperature resistance, antimicrobial sensitivity tests, and intestinal epithelial cell adhesion, Enterococcus faecium PNC01 (E. faecium PNC01) was showed to be non-cytotoxic to epithelial cells. Draft genome sequence of E. faecium PNC01 predicted that it synthesized bacteriocin to perform probiotic functions and bacteriocin activity assay showed it inhibited Salmonella typhimurium from invading intestinal epithelial cells. Diet supplemented with E. faecium PNC01 increased the ileal villus height and crypt depth in broiler chickens, reduced the relative length of the cecum at day 21, and reduced the relative length of jejunum and ileum at day 42. Diet supplemented with E. faecium PNC01 increased the relative abundance of Firmicutes and Lactobacillus, decreased the relative abundance of Bacteroides in the cecal microbiota. CONCLUSION: E. faecium PNC01 replaced antibiotics to reduce the feed conversion rate. Furthermore, E. faecium PNC01 improved intestinal morphology and altered the composition of microbiota in the cecum to reduce feed conversion rate. Thus, it can be used as an alternative for antibiotics in broiler production to avoid the adverse impact of antibiotics by altering the gut microbiota.

Portal Venous Flow Is Increased by Jejunal but Not Colonic Hydrogen Sulfide in a Nitric Oxide-Dependent Fashion in Rats.

Hydrogen sulfide (H2S) is a recently discerned endogenous signaling molecule that modulates the vascular system. Endogenous hydrogen sulfide has been shown to dilate both the mesenteric and portal vasculature. Gut microbiome, via sulfur reducing bacteria, is another source of H2S production within the gut lumen; this source of H2S is primarily produced and detoxified in the colon under physiologic conditions. Nitric oxide (NO), a major endogenous vasodilator in the portal circulation, participates in H2S-induced vasodilation in some vascular beds. We hypothesize that jejunal but not colonic H2S increases portal vein flow in a NO-dependent fashion. To evaluate the effects of luminal H2S, venous blood flow, portal venous pressure, and systemic venous pressure were measured in rats after administration of either vehicle or an H2S donor (NaHS) into the jejunum or the colon. We found that portal venous pressure and systemic pressure did not change and were similar between the three study groups. However, portal venous blood flow significantly increased following jejunal administration of NaHS but not in response to colonic NaHS or vehicle administration. To test the contribution of NO production to this response, another group of animals was treated with either an NO synthase inhibitor (N-Ω-nitro-L-arginine, L-NNA) or saline prior to jejunal NaHS infusion. After L-NNA pretreatment, NaHS caused a significant fall rather than increase in portal venous flow compared to saline pretreatment. These data demonstrate that H2S within the small intestine significantly increases portal venous blood flow in a NO-dependent fashion.

Mechanisms of deoxynivalenol-induced endocytosis and degradation of tight junction proteins in jejunal IPEC-J2 cells involve selective activation of the MAPK pathways.

Mycotoxin contamination in foods is a major risk factor for human and animal health due to its prevalence in cereals and their by-products. Deoxynivalenol (DON), mainly produced by Fusarium genera, is the most common mycotoxin detected in cereal products. Deoxynivalenol disrupts intestinal barrier function and decreases protein levels of tight junction proteins (TJP). However, the overall mechanism by which DON regulates specific TJP turnover and epithelial cell integrity remains unclear. Herein, we show that DON (2 µM) decreases the protein stability and accelerates the degradation of TJP in the lysosome. Interestingly, pretreatment of cells with dynasore (a dynamin-dependent endocytosis inhibitor) protected against DON-induced degradation of claudin-3 and 4. Immunofluorescence analysis also shows that the decreased membrane presence of claudin-4 and ZO-1 induced by DON is reversible with dynamin inhibition, whereas the pretreatment with cytochalasin D (an actin-dependent endocytosis inhibitor) reverses the degradation of claudin-1 and 4 induced by DON. We also show that the endocytosis and degradation of claudin-1 is regulated by p38 mitogen-activated protein kinase (MAPK), whereas the endocytosis of claudin-4 and ZO-1 is mediated by c-Jun-N-terminal kinase (JNK). Resveratrol, with JNK inhibitory activity, also prevents the endocytosis and degradation of claudin-4 and ZO-1 and protects against DON-induced decrease in transepithelial electrical resistance (TEER) and increase in FITC-dextran permeability. Collectively, this study, for the first time, shows that DON accelerates the endocytosis and degradation of TJP and this is regulated by the activation of p38 MAPK and JNK signaling pathways. Therefore, natural bioactive compounds with p38 MAPK and JNK inhibitory activities may be effective in preventing the DON-induced TJP disruption and preserve gut barrier function in vivo.

The Protect Effects of Chitosan Oligosaccharides on Intestinal Integrity by Regulating Oxidative Status and Inflammation under Oxidative Stress.

The aim of this study was to evaluate the effects of the dietary supplementation of chitosan oligosaccharides (COS) on intestinal integrity, oxidative status, and the inflammation response with hydrogen peroxide (H2O2) challenge. In total, 30 rats were randomly assigned to three groups with 10 replications: CON group, basal diet; AS group, basal diet + 0.1% H2O2 in drinking water; ASC group, basal diet + 200 mg/kg COS + 0.1% H2O2 in drinking water. The results indicated that COS upregulated (p < 0.05) villus height (VH) of the small intestine, duodenum, and ileum; mucosal glutathione peroxidase activity; jejunum and ileum mucosal total antioxidant capacity; duodenum and ileum mucosal interleukin (IL)-6 level; jejunum mucosal tumor necrosis factor (TNF)-α level; duodenum and ileum mucosal IL-10 level; the mRNA expression level of zonula occludens (ZO)-1 in the jejunum and ileum, claudin in the duodenum, nuclear factor-erythroid 2-like 2 in the jejunum, and heme oxygenase-1 in the duodenum and ileum; and the protein expression of ZO-1 and claudin in jejunum; however, it downregulated (p < 0.05) serum diamine oxidase activity and D-lactate level; small intestine mucosal malondialdehyde content; duodenum and ileum mucosal IL-6 level; jejunum mucosal TNF-α level; and the mRNA expression of IL-6 in the duodenum and jejunum, and TNF-α in the jejunum and ileum. These results suggested COS could maintain intestinal integrity under oxidative stress by modulating the intestinal oxidative status and release of inflammatory cytokines.

Ammonia exposure causes the disruption of the solute carrier family gene network in pigs.

Ammonia is the main harmful gas in livestock houses. However, the toxic mechanism of ammonia is still unclear. Therefore, we examined the effects of ammonia exposure on different tissues of fattening pigs by histological analysis and transcriptome techniques in this study. The results showed that there were varying degrees of pathological changes in liver, kidney, hypothalamus, jejunum, lungs, spleen, heart and trachea of fattening pigs under ammonia exposure. Notably, the extent of damage in liver, kidney, jejunum, lungs, hypothalamus and trachea was more severe than that in heart and spleen. Transcriptome results showed that ammonia exposure caused changes in 349, 335, 340, 229, 120, 578, 407 and 115 differentially expressed genes in liver, kidney, spleen, lung, trachea, hypothalamus, jejunum and heart, respectively. Interestingly, the changes in solute vector (SLC) family genes were found in all 8 tissues, and the verified gene results (SLC11A1, SLC17A7, SLC17A6, SLC6A4, SLC22A7, SLC25A3, SLC28A3, SLC7A2, SLC6A6, SLC38A5, SLC22A12, SLC34A1, SLC26A1, SLC26A6, SLC27A5, SLC22A8 and SLC44A4) were consistent with qRT-PCR results. In conclusion, ammonia exposure can cause pathological changes in many tissues and organs of fattening pigs and changes in the SCL family gene network. Importantly, the SCL family is involved in the toxic mechanism of ammonia. Our findings will provide a new insight for better assessing the mechanism of ammonia toxicity.

Melatonin-Activated Receptor Signaling Pathways Mediate Protective Effects on Surfactant-Induced Increase in Jejunal Mucosal Permeability in Rats.

A well-functional intestinal mucosal barrier can be compromised as a result of various diseases, chemotherapy, radiation, and chemical exposures including surfactants. Currently, there are no approved drugs targeting a dysfunctional intestinal barrier, which emphasizes a significant medical need. One candidate drug reported to regulate intestinal mucosal permeability is melatonin. However, it is still unclear if its effect is primarily receptor mediated or antioxidative, and if it is associated with enteric neural pathways. The aim of this rat intestinal perfusion study was to investigate the mechanisms of melatonin and nicotinic acetylcholine receptors on the increase in intestinal mucosal clearance of 51Cr-labeled ethylenediaminetetraacetate induced by 15 min luminal exposure to the anionic surfactant, sodium dodecyl sulfate. Our results show that melatonin abolished the surfactant-induced increase in intestinal permeability and that this effect was inhibited by luzindole, a melatonin receptor antagonist. In addition, mecamylamine, an antagonist of nicotinic acetylcholine receptors, reduced the surfactant-induced increase in mucosal permeability, using a signaling pathway not influenced by melatonin receptor activation. In conclusion, our results support melatonin as a potentially potent candidate for the oral treatment of a compromised intestinal mucosal barrier, and that its protective effect is primarily receptor-mediated.

MicroRNA Expression Profiling in Porcine Liver, Jejunum and Serum upon Dietary DON Exposure Reveals Candidate Toxicity Biomarkers.

Deoxynivalenol (DON), a frequent mycotoxin worldwide, impairs human and animal health. The response of microRNAs, small non-coding RNAs, to DON has been scarcely investigated, but holds remarkable potential for biomarker applications. Hence, we aimed to investigate DON-induced changes in the microRNA expression in porcine liver, jejunum and serum by combining targeted and untargeted analyses. Piglets received uncontaminated feed or feed containing 900 µg/kg and 2500 µg/kg DON for four weeks, followed by a wash-out period. In tissue, only slight changes in microRNA expression were detected, with ssc-miR-10b being downregulated in liver of DON-exposed piglets. In serum, several microRNAs were differentially expressed upon DON exposure, four of which were validated by qPCR (ssc-miR-16, ssc-miR-128, ssc-miR-451, ssc-miR-205). The serum microRNA response to DON increased over time and declined after removal of contaminated diets. Receiver operating curve analyses for individual microRNAs were significant, and a combination of the four microRNAs increased the predictive capacity for DON exposure. Predicted microRNA target genes showed enrichment of several pathways including PIK3-AKT, Wnt/ß-catenin, and adherens junctions. This study gives, for the first time, a comprehensive view of the porcine microRNA response to DON, providing a basis for future research on microRNAs as biomarkers for mycotoxins.

Microcirculatory Function during Endotoxemia-A Functional Citrulline-Arginine-NO Pathway and NOS3 Complex Is Essential to Maintain the Microcirculation.

Competition for the amino acid arginine by endothelial nitric-oxide synthase (NOS3) and (pro-)inflammatory NO-synthase (NOS2) during endotoxemia appears essential in the derangement of the microcirculatory flow. This study investigated the role of NOS2 and NOS3 combined with/without citrulline supplementation on the NO-production and microcirculation during endotoxemia. Wildtype (C57BL6/N background; control; n = 36), Nos2-deficient, (n = 40), Nos3-deficient (n = 39) and Nos2/Nos3-deficient mice (n = 42) received a continuous intravenous LPS infusion alone (200 µg total, 18 h) or combined with L-citrulline (37.5 mg, last 6 h). The intestinal microcirculatory flow was measured by side-stream dark field (SDF)-imaging. The jejunal intracellular NO production was quantified by in vivo NO-spin trapping combined with electron spin-resonance (ESR) spectrometry. Amino-acid concentrations were measured by high-performance liquid chromatography (HPLC). LPS infusion decreased plasma arginine concentration in control and Nos3-/- compared to Nos2-/- mice. Jejunal NO production and the microcirculation were significantly decreased in control and Nos2-/- mice after LPS infusion. No beneficial effects of L-citrulline supplementation on microcirculatory flow were found in Nos3-/- or Nos2-/-/Nos3-/- mice. This study confirms that L-citrulline supplementation enhances de novo arginine synthesis and NO production in mice during endotoxemia with a functional NOS3-enzyme (control and Nos2-/- mice), as this beneficial effect was absent in Nos3-/- or Nos2-/-/Nos3-/- mice.

Nanoparticulate tablet dosage form of lisofylline-linoleic acid conjugate for type 1 diabetes: in situ single-pass intestinal perfusion (SPIP) studies and pharmacokinetics in rat.

Lisofylline (LSF) is an anti-inflammatory molecule with high aqueous solubility and rapid metabolic interconversion to its parent drug, pentoxifylline (PTX) resulting in very poor pharmacokinetic (PK) parameters, necessitating high dose and dosing frequency. In the present study, we resolved the physicochemical and pharmacokinetic limitations associated with LSF and designed its oral dosage form as a tablet for effective treatment in type 1 diabetes (T1D). Self-assembling polymeric micelles of LSF (lisofylline-linoleic acid polymeric micelles (LSF-LA PLM)) were optimized for scale-up (6 g batch size) and lyophilized followed by compression into tablets. Powder blend and tablets were evaluated as per USP. LSF-LA PLM tablet so formed was evaluated for in vitro release in simulated biological fluids (with enzymes) and for cell viability in MIN-6 cells. LSF-LA PLM in tablet formulation was further evaluated for intestinal permeability (in situ) along with LSF and LSF-LA self-assembled micelles (SM) as controls in a rat model using single-pass intestinal perfusion (SPIP) study. SPIP studies revealed 1.8-fold higher oral absorption of LSF-LA from LSF-LA PLM as compared to LSF-LA SM and ~5.9-fold higher than LSF (alone) solution. Pharmacokinetic studies of LSF-LA PLM tablet showed greater Cmax than LSF, LSF-LA, and LSF-LA PLM. Designed facile LSF-LA PLM tablet dosage form has potential for an immediate decrease in the postprandial glucose levels in patients of T1D.

Antispasmodic activity and mechanism of action of polyherbal formulation DCD-684 on rabbit jejunum.

Digas colic drops (DCD-684) is a polyherbal formulation containing decoctions of five medicinal plants namely Carum carvi L., Foeniculum vulgare Mill, Mentha arvensis L., Mentha piperita L. and Zingiber officinale Roscoe. These plants have been extensively used in traditional medicine for the treatment of various gastrointestinal diseases including abdominal colic. This study was conducted to determine the spasmolytic effect of DCD-684 (100% v/v) and its individual plant components on isolated rabbit jejunum (in vitro) and their possible mechanism of action. The effects were evaluated on spontaneous and pre-contracted tissues using KCl (80mM) and other contractile agonists including acetylcholine (0.3µM), carbamylcholine (0.3µM), serotonin (10 µM) and histamine (100µM) in the presence and absence of DCD-684. The various concentrations of DCD-684 (0.1-3% v/v) demonstrated spasmolytic effects on both spontaneous (IC50=0.75%) and KCl-induced contractions (IC50=1.6%), respectively. It also inhibited the contractions induced by acetylcholine (IC50=0.45%), carbamylcholine (IC50=0.95%), serotonin (IC50=0.95%) and histamine (IC50=0.87%). The DCD-684 exhibited synergistic effect due to its five plant components suggesting that spasmolytic cascade is probably governed by muscarinic and/or nicotinic receptors, serotonergic histaminergic, as well as calcium channel blocking mechanisms. Thereby, providing the pharmacological basis of its therapeutic use in the gastrointestinal motility disorders and related inflammatory ailments.

GC/MS analysis, anti-leishmanial and relaxant activity of essential oil of Chenopodium ambrosioides (L.) from Malakand region.

Chenopodium ambrosioides is abundantly available in Malakand region. As constituents and concentrations of essential oils vary based on its geographical location, we carried our current study to extract and evaluate its possible relaxant activity in rabbits' jejunum and anti-leishmanial activity against promastigotes of Leishmania tropica. The essential oil was obtained from aerial fresh parts through steam distillation followed by GC/MS analysis. Antispasmodic activity was performed on spontaneous and KCl induced contractions. Curves for calcium concentration response (CCRCs) were prepared with and without different concentrations of essential oils and verapamil - a standard calcium channel blocker as per our reported procedures. GC/MS analysis indicated that the essential oil contains 4-carene (56.59%) and o-cymene (41.46%), the two most abundant compounds previously reported from this species. The LD50 value for acute toxicity is 279.66±2.2mg/kg. The essential oil have significant antileishmanial activity with LC50 of Log10 (1.83±0.0026) ×10-6mg/ml, potent relaxant activity on rabbits' jejunal preparations with respective EC50 = 1.46±0.15mg/ml for spontaneous activity. For KCl (80mM) induced contractions, EC50=0.26±0.02mg/ml. In CCRCs, the oil produced a right shift as exhibited by verapamil. More, its relaxant activity, which is mediated through calcium channel blocking mechanism, proves a rationale for its traditional use in gut spasm.