Piperine as potential therapy of post-weaning porcine diarrheas: an in vitro study using a porcine duodenal enteroid model.

Post-weaning diarrhea in piglets is a major problem, resulting in a significant loss in pig production. This study aimed to investigate the effects of piperine, an alkaloid abundantly found in black peppers, on biological activities related to the pathogenesis of post-weaning diarrhea using a porcine duodenal enteroid model, a newly established intestinal stem cell-derived in vitro model recapitulating physiology of porcine small intestinal epithelia. Porcine duodenal enteroid models were treated with disease-relevant pathological inducers with or without piperine (8 µg/mL and/or 20 µg/mL) before measurements of oxidative stress, mRNA, and protein expression of proinflammatory cytokines, nuclear factor-kappa B (NF-κB) nuclear translocation, barrier leakage, and fluid secretion. We found that piperine (20 µg/mL) inhibited H2O2-induced oxidative stress, TNF-α-induced mRNA, and protein expression of proinflammatory cytokines without affecting NF-κB nuclear translocation, and prevented TNF-α-induced barrier leakage in porcine duodenal enteroid monolayers. Importantly, piperine inhibited fluid secretion induced by both forskolin and heat-stable toxins (STa) in a three-dimensional model of porcine duodenal enteroids. Collectively, piperine possesses both anti-inflammatory and anti-secretory effects in porcine enteroid models. Further research and development of piperine may provide novel interventions for the treatment of post-weaning porcine diarrhea.

A fungus-derived purpactin A as an inhibitor of TMEM16A chloride channels and mucin secretion in airway epithelial cells.

TMEM16A is a Ca2+-activated Cl- channel involved in mucus secretion in inflamed airways and proposed as a drug target for diseases associated with mucus hypersecretion including asthma. This study aimed to identify novel inhibitors of TMEM16A-mediated Cl- secretion in airway epithelial cells from a collection of compounds isolated from fungi indigenous in Thailand and examine its potential utility in mitigating airway mucus secretion using Calu-3 cells as a study model. Screening of > 400 fungal metabolites revealed purpactin A isolated from a soil-derived fungus Penicillium aculeatum PSU-RSPG105 as an inhibitor of TMEM16A-mediated Cl- transport with an IC50 value of ~2 µM. A consistent inhibitory effect of purpactin A on TMEM16A were observed regardless of TMEM16A activators or in the presence of an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a negative regulator of TMEM16A. In addition, purpactin A did not affect cell viability, epithelial barrier integrity and activities of membrane transport proteins essential for maintaining airway hydration including CFTR Cl- channels and apical BK K+ channels. Intriguingly, purpactin A prevented a Ca2+-induced mucin release in cytokine-treated airway cells. Taken together, purpactin A represents the first class of TMEM16A inhibitor derived from fungus, which may be beneficial for the treatment of diseases associated with mucus hypersecretion.

Chitin and Chitosan Derivatives as Biomaterial Resources for Biological and Biomedical Applications.

Chitin is a long-chain polymer of N-acetyl-glucosamine, which is regularly found in the exoskeleton of arthropods including insects, shellfish and the cell wall of fungi. It has been known that chitin can be used for biological and biomedical applications, especially as a biomaterial for tissue repairing, encapsulating drug for drug delivery. However, chitin has been postulated as an inducer of proinflammatory cytokines and certain diseases including asthma. Likewise, chitosan, a long-chain polymer of N-acetyl-glucosamine and d-glucosamine derived from chitin deacetylation, and chitosan oligosaccharide, a short chain polymer, have been known for their potential therapeutic effects, including anti-inflammatory, antioxidant, antidiarrheal, and anti-Alzheimer effects. This review summarizes potential utilization and limitation of chitin, chitosan and chitosan oligosaccharide in a variety of diseases. Furthermore, future direction of research and development of chitin, chitosan, and chitosan oligosaccharide for biomedical applications is discussed.

A prebiotic fructo-oligosaccharide promotes tight junction assembly in intestinal epithelial cells via an AMPK-dependent pathway.

Tight junctions play an important role in maintaining barrier integrity of intestinal epithelia. Activation of AMP-activated protein kinase (AMPK) promotes tight junction assembly in intestinal epithelial cells (IEC). Fructo-oligosaccharides (FOS), well-known prebiotics, have previously been shown to alleviate inflammation-associated intestinal epithelial disruption although the mechanisms were unclear. This study aimed to investigate any effect of FOS on AMPK activity and tight junction assembly under non-inflammatory and inflammatory conditions using T84 cells as an IEC model. As analyzed by western blot, FOS induced AMPK activation through a calcium sensing receptor (CaSR)-phospholipase C (PLC)- Ca2+/calmodulin-dependent protein kinase kinase-ß (CaMKKß) pathway. Calcium switch assays and immunofluorescence staining of zonula occludens-1 (ZO-1) revealed that FOS induced tight junction assembly via an CaMKKß-AMPK-dependent mechanism in IEC. Interestingly, FOS reversed the suppressive effect of lipopolysaccharide (LPS) on AMPK activity and tight junction assembly via a CaMKKß pathway. Taken together, these findings uncover a prebiotic-independent effect of FOS in promoting intestinal epithelial tight junction assembly through AMPK activation, which may have implications for the treatment of diseases whose pathogenesis involves impaired intestinal barrier function.

Inhibition of CFTR-mediated intestinal chloride secretion by a fungus-derived arthropsolide A: Mechanism of action and anti-diarrheal efficacy.

Secretory diarrhea is one of the most common types of diarrhea with high morbidity and mortality. Previous studies showed that inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels alleviated fluid loss in secretory diarrheas. This study aimed to identify novel CFTR inhibitors from fungal metabolites and explore its underlying mechanisms and potential utility in secretory diarrheas. Electrophysiological analyses in human intestinal epithelial (T84) cells were performed to investigate the effect and mechanism of fungal metabolites on CFTR-mediated Cl- secretion. Anti-diarrheal efficacy and the effect of compound on fluid absorption were investigated in mouse closed-loop models. We found that the screening identified arthropsolide A, a fungal metabolite from an endophytic fungus Roussoella sp. PSU-H51, as an inhibitor of CFTR-mediated Cl- secretion in T84 cells (IC50 ~0.8 µM). Arthropsolide A inhibited both CFTR and cAMP-activated basolateral K+ channels. Arthropsolide A had no effect on Na+-K+ ATPase activity. Interestingly, the inhibitory effect of arthropsolide A on CFTR was attenuated by cell depolarization and AMPK inhibition independent of multi-drug resistance protein 4, phosphodiesterases, and protein phosphatases. Importantly, arthropsolide A suppressed cholera toxin (CT)-induced Cl- secretion in T84 cells and CT-induced intestinal fluid secretion in mice by ~75% without affecting intestinal fluid absorption. Taken together, arthropsolide A represents a novel class of fungal metabolites that acts as a potent CFTR inhibitor. Further development of this class of compounds may provide a therapy for secretory diarrheas.

A fungal metabolite zearalenone as a CFTR inhibitor and potential therapy of secretory diarrheas.

Overstimulation of CFTR-mediated Cl- secretion plays an important role in the pathogenesis of secretory diarrheas, which remain an important global health problem. This study aimed to identify inhibitors of CFTR-mediated Cl- secretion from a library of fungus-derived compounds and to evaluate their pharmacological properties and anti-diarrheal utility. We identified zearalenone, 7'-dehydrozearalenone and 8'-hydroxyzearalenone isolated from the seagrass-derived fungus Fusarium sp. PSU-ES123 as inhibitors of CFTR-mediated Cl- secretion in human intestinal epithelial (T84) cells. Being the most potent fungal metabolite capable of inhibiting CFTR-mediated Cl- secretion, zearalenone reversibly inhibited CFTR Cl- channel activity in T84 cells with an IC50 of ∼0.5 µM. Functional and biochemical analyses and molecular docking studies indicate that zearalenone binds to the ß-estradiol binding sites in the ATP-binding pockets on NBD1 and NBD2 of CFTR. Mechanisms of CFTR inhibition by zearalenone do not involve activation of phosphodiesterases, protein phosphatases, multidrug-resistance protein 4 and AMP-activated protein kinases. Importantly, zearalenone significantly inhibited cholera toxin (CT)-induced Cl- secretion in T84 cells and blocked CT-induced intestinal fluid secretion in mice. Collectively, our study indicates that zearalenone represents the first class of fungus-derived CFTR inhibitors. Further development of this class of compounds may provide an effective treatment of secretory diarrheas.

Flufenamic acid protects against intestinal fluid secretion and barrier leakage in a mouse model of Vibrio cholerae infection through NF-κB inhibition and AMPK activation.

Nuclear factor kappa B (NF-κB)-mediated inflammatory responses play crucial roles in the pathogenesis of diarrhea caused by the Vibrio cholerae El Tor variant (EL), which is a major bacterial strain causing recent cholera outbreaks. Flufenamic acid (FFA) has previously been demonstrated to be a potent activator of AMP-activated protein kinase (AMPK), which is a negative regulator of NF-κB signaling. This study aimed to investigate the anti-diarrheal efficacy of FFA in a mouse model of EL infection and to investigate the mechanisms by which FFA activates AMPK in intestinal epithelial cells (IEC). In a mouse closed loop model of EL infection, FFA treatment (20mg/kg) significantly abrogated EL-induced intestinal fluid secretion and barrier disruption. In addition, FFA suppressed NF-κB nuclear translocation and expression of proinflammatory mediators and promoted AMPK phosphorylation in the EL-infected mouse intestine. In T84 cells, FFA induced AMPK activation. Furthermore, FFA promoted tight junction assembly and prevented interferon gamma (IFN-γ)-induced barrier disruption in an AMPK-dependent manner. Biochemical and molecular docking analyses indicated that FFA activates AMPK via a direct stimulation of calcium/calmodulin-dependent protein kinase kinase beta (CaMKKß) activity. Collectively, our data indicate that FFA represents a class of existing drugs that may be of potential utility in the treatment of cholera caused by EL infection via AMPK-mediated suppression of NF-κB signaling in IEC.

Pathophysiological mechanisms of diarrhea caused by the Vibrio cholerae O1 El Tor variant: an in vivo study in mice.

Cholera is caused by infection with Vibrio cholerae. This study aimed to investigate the pathophysiology of diarrhea caused by the V. cholerae O1 El Tor variant (EL), a major epidemic strain causing severe diarrhea in several regions. In the ligated ileal loop model of EL-induced diarrhea in the ICR mice, a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and a calcium-activated chloride channel (CaCC) inhibitor similarly inhibited intestinal fluid secretion. In addition, barrier disruption and NF-κB-mediated inflammatory responses, e.g., iNOS and COX-2 expression, were observed in the infected ileal loops. Interestingly, intestinal fluid secretion and barrier disruption were suppressed by NF-κB and COX-2 inhibitors, whereas an iNOS inhibitor suppressed barrier disruption without affecting fluid secretion. Furthermore, EP2 and EP4 PGE2 receptor antagonists ameliorated the fluid secretion in the infected ileal loops. The amount of cholera toxin (CT) produced in the ileal loops by the EL was ∼2.4-fold of the classical biotype. The CT transcription inhibitor virstatin, a toll-like receptor-4 (TLR-4) antibody and a CT antibody suppressed the EL-induced intestinal fluid secretion, barrier disruption and COX-2 expression. The CT at levels detected during EL infection induced mild intestinal barrier disruption without inducing inflammatory responses in mouse intestine. Collectively, this study indicates that CT-induced intestinal barrier disruption and subsequent TLR-4-NF-κB-mediated COX-2 expression are involved in the pathogenesis of EL-induced diarrhea and represent promising novel therapeutic targets of cholera.

Activation of AMPK by chitosan oligosaccharide in intestinal epithelial cells: Mechanism of action and potential applications in intestinal disorders.

Chitosan oligosaccharide (COS), a biomaterial derived from chitin, is absorbed by intestinal epithelia without degradation and has diverse biological activities including intestinal epithelial function. However, the mode of action is still unclear. This study aimed to investigate the effect of COS on AMP-activated protein kinase (AMPK) in intestinal epithelial cells (IEC) and its potential applications in the intestinal diseases benefited from AMPK activation. COS with molecular weights (MW) from 5,000Da to 14,000Da induced AMPK activation in T84 cells. That with MW of 5,000-Da was the most potent polymer and was used in the subsequent experiments. COS also activated AMPK in other IEC including HT-29 and Caco-2 cells. Mechanism of COS-induced AMPK activation in T84 cells involves calcium-sensing receptor (CaSR)-phospholipase C (PLC)-IP3 receptor channel-mediated calcium release from endoplasmic reticulum (ER). In addition, COS promoted tight junction assembly in T84 cells in an AMPK-dependent manner. COS also inhibited NF-κB transcriptional activity and NF-κB-mediated inflammatory response and barrier disruption via AMPK-independent mechanisms. Interestingly, luminal exposure to COS suppressed cholera toxin-induced intestinal fluid secretion by ∼30% concurrent with AMPK activation in a mouse closed loop model. Importantly, oral administration of COS prevented the development of aberrant crypt foci in a mouse model of colitis-associated colorectal cancer (CRC) via a mechanism involving AMPK activation-induced ß-catenin suppression and caspase-3 activation. Collectively, this study reveals a novel action of COS in activating AMPK via CaSR-PLC-IP3 receptor channel-mediated calcium release from ER. COS may be beneficial in the treatment of secretory diarrheas and CRC chemoprevention.

The study on telomere length for age estimation in a Thai population.

Age is one of the key parameters in establishing a physical characteristic profile of an individual. For biological evidence left in crime scenes such as blood, saliva, hair, etc, the evidence owner's age can be determined only by DNA extracted from these materials. Previous researches have found that there are certain DNA regions with specialized characteristic and function called telomere being able to predict age. The present study was to determine the correlation between telomere length and age as well as the effect of sex on the correlation and to create linear regression equation for age estimation in Thai population for forensic purposes. Blood samples obtained from unrelated healthy Thai fresh cadavers without anatomical organ abnormalities were used in this study. All cadaver subjects underwent the postmortem examination in jurisdiction of the Department of Forensic Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, and Institute of Forensic Medicine, Police General Hospital. Fifty blood samples from both sexes of all ages divided into 6 groups for equal age distribution (0-11, 12-23, 24-35, 36-47, 48-59, and 60 years old and older) were collected for a total of 100 samples. The extracted genomic DNA samples were then subjected to telomere length estimation by terminal restriction fragment (TRF) assay. The results showed that the mean TRF length was inversely correlated with age (r = -0.625), and sex did not have a statistically significant influence on the association between age and mean TRF length (P > 0.05). The obtained linear regression equation was y = 113.538 ± 9.604 - 0.012 × (R = 0.391; P < 0.001). However, the correlation was too low to be used for age estimation with high certainty and a possible reason for this in part would be the postmortem DNA degradation at some level, even using fresh cadaver blood, and other biological factors such as ethnicity and DNA sources. Roughly, those individuals who had a mean TRF length longer than 6.3 kilobase (kb), between 5.5 and 6.3 kb, and shorter than 5.5 kb aged younger than 28 years, 30 to 44 years, and older than 46 years, respectively (P < 0.01). As a preliminary study, this study highlighted that telomere length could act as a useful biomarker of aging in human population and might be used for rough age estimation in a Thai population. However, further studies with a larger sample size and/or in living human bloods as well as other cell types are recommended to support the results of this study.

Protection against oxidative stress in beta thalassemia/hemoglobin E erythrocytes by inhibitors of glutathione efflux transporters.

In beta thalassemia/hemoglobin E (Hb E), abnormally high levels of oxidative stress account for accelerated senescence and increased destruction of erythrocytes. The present study aimed to investigate the role of glutathione efflux transporters, namely cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance-associated protein 1 (MRP1), in the control of glutathione levels and protection against oxidative challenges in beta thalassemia/Hb E erythrocytes. We found that CFTR protein was expressed in the erythrocytes of beta thalassemia/Hb E patients. Treatments with GlyH-101 (50 µM), a small molecule CFTR inhibitor, and MK571 (50 µM), an MRP1 inhibitor, reduced H(2)O(2)-induced free radical generation in the erythrocytes by ∼80% and 50%, respectively. Furthermore, combined treatment with GlyH-101 and MK571 completely abolished the induction of reactive oxygen radicals. Increased oxidative stress in the erythrocytes following H(2)O(2) challenges was accompanied by a decrease in intracellular level of reduced glutathione (GSH), which was prevented by treatments with GlyH-101 and MK571. CMFDA-based assays revealed that GlyH-101 and MK571 reduced H(2)O(2)-induced glutathione efflux from the erythrocytes by 87% and 66%, respectively. Interestingly, H(2)O(2)-induced osmotic tolerance of erythrocytes, a sign of erythrocyte aging, was ameliorated by treatment with GlyH-101. Our study indicates that oxidative stress induces glutathione efflux via CFTR and MRP1 in beta thalassemia/Hb E erythrocytes. Pharmacological inhibition of glutathione efflux represents a potential therapy to delay aging and premature destruction of erythrocytes in beta thalassemia/Hb E.