Endari treatment ameliorates sickle cell-related disruption in intestinal barrier functions and is associated with prolonged survival in sickle cell mice.

BACKGROUND: Endari (L-glutamine) is a conditional amino acid that reduces the frequency of vaso-occlusive crisis (VOC) in sickle cell disease (SCD). AIM: To investigate whether Endari could ameliorate intestinal barrier function and improve survival outcomes in SCD. METHODS: We treated female Townes SCD mice with Endari and evaluated their intestinal barrier functions by measuring the recovery of orally administered fluorescein isothiocyanate (FITC)-conjugated dextran 4 kDa in serum, and serum intestinal fatty acid binding proteins (iFABP) and lipopolysaccharide (LPS) concentrations by ELISA. We also explored the impact the Endari has on the survival of the SCD mice that underwent repeated experimentally-induced VOC. RESULTS: Compared to SCD mice treated with water only, Endari-treated mice showed improved intestinal barrier functions, with decrease in the barrier permeability and reduction in the translocation of lipopolysaccharides from the intestinal lumen into the circulation. These changes occurred after only 4 weeks of Endari treatment. Improved intestinal barrier function was also associated with prolonged survival in Endari-treated SCD mice after repeated experimentally-induced VOC. CONCLUSION: Our findings provide the evidence supporting the beneficial effects of Enadri in improving intestinal barrier function and associated survival outcomes in SCD.

Research Note: Modified serum fluorescein isothiocyanate dextran (FITC-d) assay procedure to determine intestinal permeability in poultry fed diets high in natural or synthetic pigments.

Oral administration of fluorescein isothiocyanate dextran (FITC-d) has been used as an indicator for intestinal permeability in poultry research for several years. Under healthy conditions, tight junctions in the intestinal wall will not allow the 4-6kDa FITC-d to enter the bloodstream. Detection of FITC-d in serum (1-hour post-oral administration of FITC-d) has proven to be a reliable indicator of leaky gut syndrome (increased intestinal inflammation and disruption of tight junctions). Administration of supplementary phytobiotics in feed, particularly products with high beta-carotene levels or other pigments, has resulted in strong serum background fluorescence, which can render this assay unreliable. To account for this increase in background autofluorescence, the FITC-d assay procedure has been modified to accommodate these particular serum samples by including pre-administration serum collection from each treatment group to remove background fluorescence. The modified FITC-d procedure detailed will allow for analysis of intestinal permeability in pigmented serum.

Synergistic effect of the herbal mixture C5E on gemcitabine treatment in PANC­1 cells.

The present study aimed to determine the anticancer effect of the herbal mixture extract C5E in the pancreatic cancer cell line, PANC­1, in the absence or presence of gemcitabine treatment, a chemotherapeutic drug used for the treatment of pancreatic cancer. The anticancer effects of C5E, gemcitabine and C5E plus gemcitabine in PANC­1 cells following 72 h of treatment were investigated. The effect of each treatment on cell cycle arrest, apoptosis and the proportion of side population (SP) cells was determined using flow cytometric analysis following propidium iodide (PI), Annexin V­FITC/PI double staining and Hoechst 33342 staining, respectively. SP cells share similar characteristics to cancer stem­like cells, and a reduction in the SP is considered to be indicative of an anticancer effect. The percentage of SP cells and the cell viability of general PANC­1 cells were significantly decreased in response to all treatments. The percentage of SP cells was reduced from 8.2% (control) to 3.9, 7.2 and 5.1% following the treatment with C5E, gemcitabine and the co­treatment, respectively. All three treatments were discovered to inhibit cell viability by arresting the cell cycle at the S phase and promoted cell death by inducing early apoptosis, with the levels of apoptosis being increased from 1.9% (control) to 7.3, 2.5 and 12.0% following the treatment with C5E, gemcitabine and the co­treatment, respectively. The mRNA expression levels of sonic hedgehog, which is implicated in the development of certain types of cancer, were downregulated to a greater extent following the co­treatment with C5E and gemcitabine compared with the treatment with either C5E or gemcitabine alone. As the co­treatment with gemcitabine and C5E was more effective than each individual treatment, the present study suggested that the combined treatment may exhibit synergistic effects in PANC­1 cells.

DNA-crosslinked alginate and layered microspheres to modulate the release of encapsulated FITC-dextran.

Alginate can be gently crosslinked by calcium into hydrogels and microspheres for the encapsulation and release of proteins and drugs. However, the release is often over short periods unless alginate is also covalently modified or crosslinked. This research aims to sustain the release of encapsulated model drug FITC-dextran by covalently crosslinking alginate with short oligomers DNA because evidence suggests that DNA may also interact with alginate to further increase effective crosslinking. Furthermore, modulating the release of drugs from alginate in response to specific proteins could tailor release profiles to improve patient treatment. This research develops a DNA-crosslinked alginate hydrogel and layered alginate microspheres to encapsulate and then sustain the release FITC-dextran (model drug). An aptamer sequence to hen egg-white lysozyme is included in one DNA strand to allow for the disruption of the crosslinks by interactions with human lysozyme. Alginate was covalently modified with complementary strands of DNA to crosslink the alginate into hydrogels, which had increased crosslinking density when re-swollen (in comparison to controls crosslinked with PEG) and could sustained the release of encapsulated FITC-dextran. When an aptamer sequence for hen lysozyme was included in the DNA crosslinks, the hydrogels decrosslinked when incubated in human lysozyme for 60 days. In addition, calcium alginate microspheres were coated with 3 alternating layers of poly-Lysine, DNA-crosslinked alginate, and poly-L-lysine. FITC-dextran loaded into the microspheres released in a sustained manner past 30 days (into PBS at 37 °C) and would likely continue to release for far longer had the studies continued. When incubated with 3 µM of human lysozyme, a burst release of FITC-dextran occurred from both the hydrogels and microspheres, with no changes in the controls. The increased release was in bursts followed by similar sustained release rates suggesting that the human lysozyme temporarily disrupted the DNA crosslinks which were then re-established or were influenced by interactions between DNA and alginate. Importantly, covalently bound complementary strands of DNA could crosslink the alginate and additional interactions appeared to further sustain the release of encapsulated therapeutics.

Microfluidic Fabrication of Structure-Controlled Chitosan Microcapsules via Interfacial Cross-Linking of Droplet Templates.

In this work, a simple and flexible method for the fabrication of chitosan microcapsules with controllable structures and functions via the interfacial cross-linking reaction of the water-in-oil (W/O) emulsion templates is developed. The interfacial cross-linking reactions of chitosan and terephthalaldehyde (TPA) in W/O emulsion templates are comprehensively studied. The interfacial cross-linking reactions of the droplet templates in both batchwise and continuous conditions are studied. A poly(dimethylsiloxane) (PDMS) droplet-capture microfluidic chip is fabricated to investigate the interfacial reaction in continuous conditions online. In this study, the size and shell thickness of the microcapsules are affected by the preparation condition, such as the template size, emulsifier concentration, TPA concentration, and cross-linking time. Moreover, the size and shell thickness changes of chitosan microcapsules prepared in continuous conditions are much faster than those prepared in batchwise conditions. By regulating the preparation parameters, the microcapsules with controllable structures are fabricated in both batchwise and continuous conditions. The drug release behaviors of the microcapsules with controllable structures are studied. Furthermore, by adding magnetic nanoparticles to the aqueous solution, magnetic-responsive microcapsules are fabricated easily. This work provides valuable guidance for the controllable fabrication of chitosan microcapsules with designed structures and functions via single emulsion templates.

A 90 day oral toxicity study of blueberry polyphenols in ovariectomized sprague-dawley rats.

Regular consumption of polyphenol-rich fruits and vegetables is associated with beneficial health outcomes. To increase polyphenol intakes, consumers are increasingly using herbal and botanical dietary supplements containing concentrated polyphenol extracts. However, the safety of this consumption modality has not been vetted. To address this, ovariectomized Sprague-Dawley (OVX-SD) rats were orally gavaged with purified blueberry polyphenols at 0-1000 mg total polyphenols/kg bw/d for 90d. No differences in behavior, body weight, or food consumption were observed. No tumors or macroscopic changes were observed, and histopathological analyses showed no differences among groups. Although several statistically significant differences between treatment and control groups were observed in urine (color and pH) and blood (monocyte count, total cholesterol, and chloride ion concentration) analyses, these parameters were within normal ranges and not considered biologically significant. Intestinal permeability assessed via FITC-dextran showed increased intestinal permeability in the highest dose, though no morphological differences were found throughout the gastrointestinal tract. Given the lack of other systemic changes, this finding is likely of minimal physiological importance. These results indicate a NOAEL for blueberry polyphenols in OVX-SD rats is ≥ 1000 mg total polyphenols/kg bw/d, which translates to a 70 kg human consuming ~10 g polyphenols. Keywords: Blueberry, Polyphenol, Sub-chronic toxicity.

Barbaloin Attenuates Mucosal Damage in Experimental Models of Rat Colitis by Regulating Inflammation and the AMPK Signaling Pathway.

BACKGROUND Barbaloin is one of the main medicinal ingredients of aloe vera, which displays various anti-inflammatory and anti-apoptosis properties in several inflammatory and fibrotic diseases. Our study evaluated its efficacy against dextran sulfate sodium (DSS)-induced colitis in rats. MATERIAL AND METHODS Ulcerative colitis (UC) rat models were established in vivo, and after barbaloin treatment, body weight and inflammation index were measured. Additionally, the signaling mechanism by which barbaloin protects against UC was investigated using LPS-infected Caco-2 cells. RESULTS Barbaloin could significantly reverse UC-induced weight loss and colon injury. Further, it could effectively increase the mRNA expression of IL-4 and IL-10 in colon tissues, while decreasing the expression of IFN-γ, IL-6, IL-1ß, and TNF-alpha. Furthermore, it significantly enhanced UC-inhibited atresia band 1 (ZO-1), occludin, and E-cadherin, and was also found to activate the AMPK signaling pathway. Additionally, si-RAN-induced knockdown, and overexpression assay showed that barbaloin could inhibit the UC-enhanced MLCK signaling pathway by activating the AMPK signaling pathway. CONCLUSIONS Barbaloin can effectively inhibit inflammation and reverse epithelial barrier function to protect against UC, possibly via activation of the AMPK signaling pathway.

Photobiomodulation Mitigates Cerebrovascular Leakage Induced by the Parkinsonian Neurotoxin MPTP.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is commonly used to model Parkinson's disease (PD) as it specifically damages the nigrostriatal dopaminergic pathway. Recent studies in mice have, however, provided evidence that MPTP also compromises the integrity of the brain's vasculature. Photobiomodulation (PBM), the irradiation of tissue with low-intensity red light, mitigates MPTP-induced loss of dopaminergic neurons in the midbrain, but whether PBM also mitigates MPTP-induced damage to the cerebrovasculature has not been investigated. This study aimed to characterize the time course of cerebrovascular disruption following MPTP exposure and to determine whether PBM can mitigate this disruption. Young adult male C57BL/6 mice were injected with 80 mg/kg MPTP or isotonic saline and perfused with fluorescein isothiocyanate FITC-labelled albumin at various time points post-injection. By 7 days post-injection, there was substantial and significant leakage of FITC-labelled albumin into both the substantia nigra pars compacta (SNc; p < 0.0001) and the caudate-putamen complex (CPu; p ≤ 0.0003); this leakage partly subsided by 14 days post-injection. Mice that were injected with MPTP and treated with daily transcranial PBM (670 nm, 50 mW/cm2, 3 min/day), commencing 24 hours after MPTP injection, showed significantly less leakage of FITC-labelled albumin in both the SNc (p < 0.0001) and CPu (p = 0.0003) than sham-treated MPTP mice, with levels of leakage that were not significantly different from saline-injected controls. In summary, this study confirms that MPTP damages the brain's vasculature, delineates the time course of leakage induced by MPTP out to 14 days post-injection, and provides the first direct evidence that PBM can mitigate this leakage. These findings provide new understanding of the use of the MPTP mouse model as an experimental tool and highlight the potential of PBM as a therapeutic tool for reducing vascular dysfunction in neurological conditions.

Green tea polyphenols enhance gingival keratinocyte integrity and protect against invasion by Porphyromonas gingivalis.

The gingival epithelium, a stratified squamous tissue that acts as an interface between the external environment and the underlying connective tissue, plays an active role in maintaining periodontal health. The aim of the present study was to investigate the ability of green tea catechins to enhance gingival epithelial barrier function and protect against the disruption of epithelial integrity induced by Porphyromonas gingivalis. Both the green tea extract and epigallocatechin-3-gallate (EGCG) dose- and time-dependently increased the transepithelial electrical resistance (TER) of a gingival keratinocyte model and decreased the permeability of the cell monolayer to fluorescein isothyocyanate-conjugated 4.4-kDa dextran. This was associated with the increased expression of zonula occludens-1 (ZO-1) and occludin, two tight junction proteins. Treating the gingival keratinocyte monolayer with P. gingivalis caused a reduction in TER and affected the distribution of ZO-1 and occludin, allowing P. gingivalis to translocate through the cell monolayer. These deleterious effects mediated by P. gingivalis were abolished by the green tea extract and EGCG. This protection may be in part related to the ability of tea catechins to inhibit the protease activities of P. gingivalis. Given the above properties, green tea catechins may represent promising preventive and therapeutic molecules against periodontal disease.

The influence of pH and enzyme cross-linking on protein delivery properties of WPI-beet pectin complexes.

The incorporation of bioactive proteins and peptides into food is associated with the loss of bioactivity due to deactivation in complex food matrices and in digestion systems. In this study, two different types of protein carriers, i.e. biopolymer complexation and complex coacervation were fabricated using whey protein isolation (WPI, 6wt%) and beet pectin (BP, 1.25 and 1.00wt%) at pH5.5 and 3.5, respectively. The release of the encapsulated FITC-BSA, a model bioactive protein, in both carriers in the absence and presence of laccase was investigated at both pH7.0 and 4.0. Release of FITC-BSA from both lyophilized WPI-beet pectin biopolymer complexation and complex coacervation were biphasic with an initial burst release followed by a slower release phase. The addition of laccase in biopolymer complexation increased the loading efficiency from 44.95% to 52.15% and slowed down the burst release of FITC-BSA but did change the biphasic release pattern. Laccase-cross linked WPI (6wt%)-BP (1wt%) complex coacervation had highest FITC-BSA loading efficiency (96.90%). The release of the embedded FITC-BSA in this carrier at both pH4 and 7 was in a gradual manner and the profile can be fit to zero order kinetics over the 72h study period suggesting enzymatically reinforced complex coacervation between the protein and the negatively charged beet pectin can restrain the burst release of FITC-BSA. These results indicate that laccase cross-linked WPI-beet pectin complex coacervation can be a good carrier system for delivering hydrophilic bioactive proteins or peptides successfully with enhanced loading parameters and sustained release profiles.

Reduced fasting periods increase intestinal permeability in chickens.

Fasting of up to 24 hr has been shown to increase intestinal permeability (IP) in chickens. The aim of this study was to determine whether fasting duration of 4.5 and 9 hr increased IP and whether l-glutamine (a non-essential amino acid) supplementation before fasting provided some protection of barrier function as shown in other species. Ross 308 male broilers (n = 96) were fed either a control diet or the same diet supplemented with 1% glutamine from d0 to d38 post-hatch. On d37, the birds were assigned to single-bird metabolism cages and were fasted for either 0, 4.5, 9 or 19.5 hr. This study design was 2 × 4 factorial with two levels of glutamine and four levels of fasting. Birds in the 0-hr fasting group had free access to feed. All birds had ad libitum access to water. To measure IP on day 38, following their respective fasting periods, birds were administered two separate oral gavages of fluorescein isothiocyanate dextran (FITC-d) followed by lactulose, mannitol and rhamnose (LMR) sugars, 60 min apart. Whole blood was collected from the jugular vein 90 min post-LMR sugar gavage. FITC-d and L/M/R ratios were measured by spectrophotometry and high-performance ionic chromatography respectively. Lipopolysaccharide (LPS) endotoxins in plasma of the birds fed the control diet were also measured using chicken-specific LPS antibody ELISA. Serum FITC-d and plasma L/M and L/R ratios for 4.5, 9 and 19.5 hr were significantly (p < .05) higher compared to the non-fasting group. However, IP was not different in the glutamine-supplemented group (p > .05) compared to the control group. LPS concentrations measured by the ELISA were below the detectable range. We conclude that fasting periods of 4.5 and 9 hr increased IP compared to non-fasted birds and dietary glutamine supplementation did not ameliorate changes in IP.

Puerarin may protect against Schwann cell damage induced by glucose fluctuation.

Puerarin is one of the major active ingredients in Gegen, a traditional Chinese herb that has been reported to have a wide variety of beneficial pharmacology functions. Previous studies have implicated that the damaging effects of hyperglycemia resulting from oxidative stress and glucose fluctuation may be more dangerous than constant high glucose in the development of diabetes-related complications. The present study focuses on the effects of puerarin on glucose fluctuation-induced oxidative stress-induced Schwann cell (SC) apoptosis in vitro. Primarily cultured SCs were exposed to different conditions and the effect of puerarin on cell viability was determined by MTT assays. Intracellular reactive oxygen species (ROS) generation and mitochondrial transmembrane potential were detected by flow cytometry analysis. Apoptosis was confirmed by the Annexin V-FITC/PI and TUNEL method. Quantitative real-time reverse transcriptase polymerase chain reaction was performed to analyze the expression levels of bax and bcl-2. Western blot was performed to analyze the expression levels of some important transcription factors and proteins. The results showed that incubating SCs with intermittent high glucose for 48 h decreased cell viability and increased the number of apoptotic cells whereas treating with puerarin protected SCs against glucose fluctuation-induced cell damage. Further study demonstrated that puerarin suppressed activation of apoptosis-related proteins including PARP and caspase-3, downregulation of bcl-2, and upregulation of intracellular distribution of bax from cytosol to mitochondria, which was induced by glucose fluctuation. Moreover, puerarin inhibited the elevation of intracellular ROS and mitochondrial depolarization induced by glucose fluctuation. These results suggest that puerarin may protect SCs against glucose fluctuation-induced cell injury through inhibiting apoptosis as well as oxidative stress.

Plumbagin, a plant-derived naphthoquinone metabolite induces mitochondria mediated apoptosis-like cell death in Leishmania donovani: an ultrastructural and physiological study.

Naphthoquinones are known to exhibit a broad range of biological activities against microbes, cancer and parasitic diseases and have been widely used in Indian traditional medicine. Plumbagin is a plant-derived naphthoquinone metabolite (5-hydroxy-2-methyl-1,4-naphthoquinone) reported to inhibit trypanothione reductase, the principal enzyme and a validated drug target involved in detoxification of oxidative stress in Leishmania. Here, we report the mechanistic aspects of cell death induced by plumbagin including physiological effects in the promastigote form and ultrastructural alterations in both promastigote and amastigote forms of Leishmania donovani which till now remained largely unknown. Our observations show that oxidative stress induced by plumbagin resulted in depolarization of the mitochondrial membrane, depletion in ATP levels, elevation of cytosolic calcium, increase in caspase 3/7-like protease activity and lipid peroxidation in promastigotes. Apoptosis-like cell death induction post plumbagin treatment was confirmed by biochemical assays like Annexin V/FITC staining, TUNEL as well as morphological and ultrastructural studies. These findings collectively highlight the mode of action and importance of oxidative stress inducing agents in effectively killing both forms of the Leishmania parasite and opens up the possibility of exploring plumbagin and its derivatives as promising candidates in the chemotherapy of Leishmaniasis.

Chitosan wound dressing with hexagonal silver nanoparticles for hyperthermia and enhanced delivery of small molecules.

Chitosan films were synthesized with hexagonal silver nanoparticles (Ag NP). The unique shape and size of the Ag NP shift the optical absorption into the infrared. Stimulation of the nanoparticles with infrared light was used to generate heat and facilitate intracellular delivery of fluorescently-labeled dextran molecules. Chitosan films prepared with hexagonal or spherical Ag NP were characterized by optical and thermal analyses, and X-ray diffraction. There were found to be slight differences between how the chitosan molecular chains interface with the Ag NP depending upon shape of the nanoparticle. Viability of cells associated with dermal wound healing was evaluated on chitosan films prepared with hexagonal or spherical Ag NP, with both keratinocytes and fibroblasts having normal or moderately enhanced growth on films containing hexagonally-shaped nanoparticles.

Combining Chemical Permeation Enhancers for Synergistic Effects.

Currently, macromolecular drugs such as proteins are mainly administered by means of injections due to their low intestinal epithelial permeability and poor stability in the gastrointestinal tract. This study investigated binary combinations of chemical drug absorption enhancers to determine if synergistic drug absorption enhancement effects exist. Aloe vera, Aloe ferox and Aloe marlothii leaf gel materials, as well as with N-trimethyl chitosan chloride (TMC), were combined in different ratios and their effects on the transepithelial electrical resistance (TEER), as well as the transport of FITC-dextran across Caco-2 cell monolayers, were measured. The isobole method was applied to determine the type of interaction that exists between the absorption enhancers combinations. The TEER results showed synergism existed for the combinations between A. vera and A. marlothii, A. marlothii and A. ferox as well as A. vera and TMC. Antagonism interactions also occurred and can probably be explained by chemical reactions between the chemical permeation enhancers, such as complex formation. In terms of FITC-dextran transport, synergism was found for combinations between A. vera and A. marlothii, A. marlothii and A. ferox, A. vera and TMC, A. ferox and TMC and A. marlothii and TMC, whereas antagonism was observed for A. vera and A. ferox. The combinations where synergism was obtained have the potential to be used as effective drug absorption enhancers at lower concentrations compared to the single components.

Effect of dexamethasone in feed on intestinal permeability, differential white blood cell counts, and immune organs in broiler chicks.

We have previously shown that intestinal barrier function can be adversely affected by poorly digested diets or feed restriction, resulting in increased intestinal inflammation-associated permeability. Three experiments were conducted in broilers to evaluate the effect of dexamethasone (DEX) treatment on systemic fluorescein isothiocyanate-dextran (FITC-D; 3-5 kDa) levels, indicative of increased gut epithelial leakage. Experiment 1 compared DEX injections of 1 mg/kg, once per day on d 3, 5, and 9, with feed administration at 0.57, 1.7, or 5.1 ppm d 4 to 10, with FITC-D serum concentrations 2.5 h after gavage with 4.16 mg/kg FITC-D. All DEX treatments resulted in marked (2 to 6X; P<0.05) increased serum FITC-D levels. Feed DEX administration resulted in greater (P<0.05) gut permeability than injection at any dose, with numerically optimal effects at the lowest dose tested. In experiments 2 and 3, chicks were randomly assigned to a starter ration containing either control (CON) or DEX treated feed (0.57 ppm/kg; d 3 to 10 experiment 2, d 4 to 10 experiment 3). At d 10, all chicks were treated by oral gavage with FITC-D and serum samples were obtained as described above. Samples of the liver were aseptically collected, homogenized, diluted 1:4 wt/vol in sterile saline, and serial dilutions were plated on tryptic soy agar to evaluate total numbers of aerobic bacteria in the liver as an index of bacterial translocation (BT). In both experiments, FITC-D absorption was significantly enhanced (P<0.05) in DEX-treated chicks, again indicating increased paracellular leakage across the gut epithelium associated with dissolution of tight junctions. Experiment 2 differential cell counts showed an increased heterophil/lymphocyte ratio, and immune organ (spleen and bursa of Fabricius) weights for experiments 2 and 3 were decreased (P<0.05) from controls. In experiments 2 and 3, dietary DEX administration resulted in numerically (experiment 2) or significantly (P<0.05) increased enteric BT to the liver, supporting the observation that dietary DEX causes a stress-like inflammatory GI response, which may contribute to subclinical or clinical disease, and may be a useful model for ongoing disease mitigation research related to stress-related diseases of GIT origin.

Sodium caprate-induced increases in intestinal permeability and epithelial damage are prevented by misoprostol.

Epithelial damage caused by intestinal permeation enhancers is a source of debate concerning safety. The medium chain fatty acid, sodium caprate (C10), causes reversible membrane perturbation at high dose levels required for efficacy in vivo, so the aim was to model it in vitro. Exposure of Caco-2 monolayers to 8.5mM C10 for 60min followed by incubation in fresh buffer led to (i) recovery in epithelial permeability (i.e. transepithelial electrical resistance (TEER) and apparent permeability coefficient (Papp) of [(14)C]-mannitol), (ii) recovery of cell viability parameters (monolayer morphology, plasma membrane potential, mitochondrial membrane potential, and intracellular calcium) and (iii) reduction in mRNA expression associated with inflammation (IL-8). Pre-incubation of monolayers with a mucosal prostaglandin cytoprotectant was attempted in order to further decipher the mechanism of C10. Misoprostol (100nM), inhibited C10-induced changes in monolayer parameters, an effect that was partially attenuated by the EP1 receptor antagonist, SC51322. In rat isolated intestinal tissue mucosae and in situ loop instillations, C10-induced respective increases in the [(14)C]-mannitol Papp and the AUC of FITC-dextran 4000 (FD-4) were similarly inhibited by misoprostol, with accompanying morphological damage spared. These data support a temporary membrane perturbation effect of C10, which is linked to its capacity to mainly increase paracellular flux, but which can be prevented by pre-exposure to misoprostol.

Electroacupuncture activates enteric glial cells and protects the gut barrier in hemorrhaged rats.

AIM: To investigate whether electroacupuncture ST36 activates enteric glial cells, and alleviates gut inflammation and barrier dysfunction following hemorrhagic shock. METHODS: Sprague-Dawley rats were subjected to approximately 45% total blood loss and randomly divided into seven groups: (1) sham: cannulation, but no hemorrhage; (2) subjected to hemorrhagic shock (HS); (3) electroacupuncture (EA) ST36 after hemorrhage; (4) vagotomy (VGX)/EA: VGX before hemorrhage, then EA ST36; (5) VGX: VGX before hemorrhage; (6) α-bungarotoxin (BGT)/EA: intraperitoneal injection of α-BGT before hemorrhage, then EA ST36; and (7) α-BGT group: α-BGT injection before hemorrhage. Morphological changes in enteric glial cells (EGCs) were observed by immunofluorescence, and glial fibrillary acidic protein (GFAP; a protein marker of enteric glial activation) was evaluated using reverse transcriptase polymerase chain reaction and western blot analysis. Intestinal cytokine levels, gut permeability to 4-kDa fluorescein isothiocyanate (FITC)-dextran, and the expression and distribution of tight junction protein zona occludens (ZO)-1 were also determined. RESULTS: EGCs were distorted following hemorrhage and showed morphological abnormalities. EA ST36 attenuated the morphological changes in EGCs at 6 h, as compared with the VGX, α-BGT and HS groups. EA ST36 increased GFAP expression to a greater degree than in the other groups. EA ST36 decreased intestinal permeability to FITC-dextran (760.5 ± 96.43 ng/mL vs 2466.7 ± 131.60 ng/mL, P < 0.05) and preserved ZO-1 protein expression and localization at 6 h after hemorrhage compared with the HS group. However, abdominal VGX and α-BGT treatment weakened or eliminated the effects of EA ST36. EA ST36 reduced tumor necrosis factor-α levels in intestinal homogenates after blood loss, while vagotomy or intraperitoneal injection of α-BGT before EA ST36 abolished its anti-inflammatory effects. CONCLUSION: EA ST36 attenuates hemorrhage-induced intestinal inflammatory insult, and protects the intestinal barrier integrity, partly via activation of EGCs.

Fungicidal effect of isoquercitrin via inducing membrane disturbance.

Isoquercitrin is a flavonoid isolated from Aster yomena, which has been used as a traditional medicinal herb. In the present study, we investigated the antifungal activity and the underlying mechanism of isoquercitrin. Isoquercitrin had a potent effect in the susceptibility test against pathogenic fungi and almost no hemolysis. Propidium iodide and potassium release assays were conducted in Candida albicans, and these studies confirmed that isoquercitrin induced membrane damage, thereby, increasing permeability. Membrane potential was analyzed using 3,3'-dipropylthiacarbocyanine iodide [DiSC3(5)], and the transition of membrane potential was indicated by an increased fluorescence intensity. To further analyze these results using model membranes, giant unilamellar vesicles and large unilamellar vesicles that encapsulated calcein were prepared and the detection of calcein leakage from liposomes indicated that membrane was disturbed. We further verified membrane disturbance by observing the disordered status of the lipid bilayer with 1,6-diphenyl-1,3,5-hexatriene fluorescence. Moreover, changes in size and granularity of the cell were revealed in flow cytometric analysis. All these results suggested the membrane disturbance and the degree of disturbance was estimated to be within a range of 2.3 nm to 3.3 nm by fluorescein isothiocyanate-dextran analysis. Taken together, isoquercitrin exerts its fungicidal effect by disturbing the membrane of cells.

Delivery of berberine using chitosan/fucoidan-taurine conjugate nanoparticles for treatment of defective intestinal epithelial tight junction barrier.

Bacterial-derived lipopolysaccharides (LPS) can cause defective intestinal barrier function and play an important role in the development of inflammatory bowel disease. In this study, a nanocarrier based on chitosan and fucoidan was developed for oral delivery of berberine (Ber). A sulfonated fucoidan, fucoidan-taurine (FD-Tau) conjugate, was synthesized and characterized by Fourier transform infrared (FTIR) spectroscopy. The FD-Tau conjugate was self-assembled with berberine and chitosan (CS) to form Ber-loaded CS/FD-Tau complex nanoparticles with high drug loading efficiency. Berberine release from the nanoparticles had fast release in simulated intestinal fluid (SIF, pH 7.4), while the release was slow in simulated gastric fluid (SGF, pH 2.0). The effect of the berberine-loaded nanoparticles in protecting intestinal tight-junction barrier function against nitric oxide and inflammatory cytokines released from LPS-stimulated macrophage was evaluated by determining the transepithelial electrical resistance (TEER) and paracellular permeability of a model macromolecule fluorescein isothiocyanate-dextran (FITC-dextran) in a Caco-2 cells/RAW264.7 cells co-culture system. Inhibition of redistribution of tight junction ZO-1 protein by the nanoparticles was visualized using confocal laser scanning microscopy (CLSM). The results suggest that the nanoparticles may be useful for local delivery of berberine to ameliorate LPS-induced intestinal epithelia tight junction disruption, and that the released berberine can restore barrier function in inflammatory and injured intestinal epithelial.