Broad-temperature-range mechanically tunable hydrogel microcapsules for controlled active release.

Here, we report PNIPAm-co-PEGDA hydrogel shelled microcapsules with a thin oil layer to achieve tunable thermo-responsive release of the encapsulated small hydrophilic actives. We use a microfluidic device integrated with a temperature-controlled chamber for consistent and reliable production of the microcapsules by utilizing triple emulsion drops (W/O/W/O) with a thin oil layer as capsule templates. The interstitial oil layer between the aqueous core and the PNIPAm-co-PEGDA shell provides a diffusion barrier for the encapsulated active until the temperature reaches a critical point above which the destabilization of interstitial oil layer occurs. We find that the destabilization of the oil layer with temperature increase is caused by outward expansion of the aqueous core due to volume increase and the radial inward compression from the deswelling of the thermo-responsive hydrogel shell. The copolymerization of NIPAm with PEGDA increases the biocompatibility of the resulting microcapsule while offering the ability to alter the compressive modulus in broad ranges by simply varying crosslinker concentrations thereby to precisely tune the onset release temperature. Based on this concept, we further demonstrate that the release temperature can be enhanced up to 62 °C by adjusting the shell thickness even without varying the chemical composition of the hydrogel shell. Moreover, we incorporate gold nanorods within the hydrogel shell to spatiotemporally regulate the active release from the microcapsules by illuminating with non-invasive near infrared (NIR) light.

Grifola frondosa Extract Containing Bioactive Components Blocks Skin Fibroblastic Inflammation and Cytotoxicity Caused by Endocrine Disrupting Chemical, Bisphenol A.

Grifola frondosa (GF), a species of Basidiomycotina, is widely distributed across Asia and has been used as an immunomodulatory, anti-bacterial, and anti-cancer agent. In the present study, the pharmacological activity of the GF extract against an ecotoxicological industrial chemical, bisphenol A (BPA) in normal human dermal fibroblasts (NHDFs), was investigated. GF extract containing naringin, hesperidin, chlorogenic acid, and kaempferol showed an inhibitory effect on cell death and inflammation induced by BPA in the NHDFs. For the cell death caused by BPA, GF extract inhibited the production of reactive oxygen species responsible for the unique activation of the extracellular signal-regulated kinase. In addition, GF extract attenuated the expression of apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase-3) and the pro-inflammatory cytokine IL-1ß by the suppression of the redox-sensitive transcription factor, nuclear factor-kappa B (NF-κB) in BPA-treated NHDFs. For the inflammation triggered by BPA, GF extract blocked the inflammasome-mediated caspase-1 activation that leads to the secretion of IL-1ß protein. These results indicate that the GF extract is a functional antioxidant that prevents skin fibroblastic pyroptosis induced by BPA.

Oral delivery of stem-cell-loaded hydrogel microcapsules restores gut inflammation and microbiota.

Mesenchymal stem cells (MSCs) are an attractive candidate for the treatment of inflammatory bowel disease (IBD), but their poor delivery rate to an inflamed colon is a major factor hampering the clinical potential of stem cell therapies. Moreover, there remains a formidable hurdle to overcome with regard to survival and homing in to injured sites. Here, we develop a strategy utilizing monodisperse hydrogel microcapsules with a thin intermediate oil layer prepared by a triple-emulsion drop-based microfluidic approach as an in-situ oral delivering carrier. The oral delivery of stem-cell-loaded hydrogel microcapsules (SC-HM) enhances MSC survival and retention in the hostile stomach environment due to the intermediate oil layer and low value of the overall stiffness, facilitating programmable cell release during gastrointestinal peristalsis. SC-HM is shown to induce tissue repair, reduce the colonic macrophage infiltration responsible for the secretion of the pro-inflammatory factors, and significantly mitigate the severity of IBD in a mouse model, where MSCs released by SC-HM successfully accumulate at the colonic crypt. Moreover, a metagenomics analysis reveals that SC-HM ameliorates the dysbiosis of specific bacterial genera, including Bacteroides acidifaciens, Lactobacillus (L.) gasseri, Lactobacillus reuteri, and L. intestinalis, implying optimization of the microorganism's composition and abundance. These findings demonstrate that SC-HM is a potential IBD treatment candidate.

Cell-Inspired Hydrogel Microcapsules with a Thin Oil Layer for Enhanced Retention of Highly Reactive Antioxidants.

In nature, individual cells are compartmentalized by a membrane that protects the cellular elements from the surrounding environment while simultaneously equipped with an antioxidant defense system to alleviate the oxidative stress resulting from light, oxygen, moisture, and temperature. However, this mechanism has not been realized in cellular mimics to effectively encapsulate and retain highly reactive antioxidants. Here, we report cell-inspired hydrogel microcapsules with an interstitial oil layer prepared by utilizing triple emulsion drops as templates to achieve enhanced retention of antioxidants. We employ ionic gelation for the hydrogel shell to prevent exposure of the encapsulated antioxidants to free radicals typically generated during photopolymerization. The interstitial oil layer in the microcapsule serves as an stimulus-responsive diffusion barrier, enabling efficient encapsulation and retention of antioxidants by providing an adequate pH microenvironment until osmotic pressure is applied to release the cargo on-demand. Moreover, addition of a lipophilic reducing agent in the oil layer induces a complementary reaction with the antioxidant, similar to the nonenzymatic antioxidant defense system in cells, leading to enhanced retention of the antioxidant activity. Furthermore, we show the complete recovery and even further enhancement in antioxidant activity by lowering the storage temperature, which decreases the oxidation rate while retaining the complementary reaction with the lipophilic reducing agent.

Kaempferol Blocks the Skin Fibroblastic Interleukin 1ß Expression and Cytotoxicity Induced by 12-O-tetradecanoylphorbol-13-acetate by Suppressing c-Jun N-terminal Kinase.

Kaempferol, a bioflavonoid present in fruits and vegetables, has a variety of antioxidant and anti-inflammatory capacities, but the functional role of kaempferol in oxidative skin dermal damage has yet to be well studied. In this study, we examine the role of kaempferol during the inflammation and cell death caused by 12-O-tetradecanoylphorbol-13-acetate (TPA) in normal human dermal fibroblasts (NHDF). TPA (5 µM) significantly induced cytotoxicity of NHDF, where a robust increase in the interleukin (IL)-1ß mRNA among the various pro-inflammatory cytokines. The skin fibroblastic cytotoxicity and IL-1ß expression induced by TPA were significantly ameliorated by a treatment with 100 nM of kaempferol. Kaempferol blocked the production of the intracellular reactive oxygen species (ROS) responsible for the phosphorylation of c-Jun N-terminal kinase (JNK) induced by TPA. Interestingly, we found that kaempferol inhibited the phosphorylation of nuclear factor-kappa B (NF-κB) and the inhibitor NF-κB (IκBα), which are necessary for the expression of cleaved caspase-3 and the IL-1ß secretion in TPA-treated NHDF. These results suggest that kaempferol is a functional agent that blocks the signaling cascade of the skin fibroblastic inflammatory response and cytotoxicity triggered by TPA.

Astaxanthin Inhibits Autophagic Cell Death Induced by Bisphenol A in Human Dermal Fibroblasts.

Astaxanthin, a natural antioxidant carotenoid, is a nutrient with diverse health benefits, given that it decreases the risk of oxidative stress-related diseases. In the present study, we investigate the functional role of astaxanthin during autophagic cell death induced by the estrogenic endocrine-disrupting chemical bisphenol A (BPA) in normal human dermal fibroblasts (NHDF). BPA significantly induced apoptotic cell death and autophagy in NHDF. Autophagic cell death evoked by BPA was significantly restored upon a treatment with astaxanthin (10 µM) via the inhibition of intracellular reactive oxygen species (ROS) production. Astaxanthin inhibited the phosphorylation of extracellular signal-regulated kinases (ERK) stimulated by ROS production, but it did not influence the activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) in BPA-treated NHDF. Astaxanthin abrogated the ERK-mediated activation of nuclear factor-kappa B (NF-κB), which is responsible for the mRNA expression of LC3-II, Beclin-1, Atg12, and Atg14 during apoptotic cell death induced by BPA. These results indicate that astaxanthin is a pharmacological and nutritional agent that blocks the skin fibroblastic autophagic cell death induced by BPA in human dermal fibroblasts.

Tart Cherry Extract Containing Chlorogenic Acid, Quercetin, and Kaempferol Inhibits the Mitochondrial Apoptotic Cell Death Elicited by Airborne PM10 in Human Epidermal Keratinocytes.

Tart cherry (Prunus cerasus L.), a medicinal food containing high concentrations of phytochemicals, has a variety of antioxidant activities and health benefits. Here, we investigate the functional effect of tart cherry during apoptotic cell death elicited by airborne particulate matter with a diameter of <10 µm (PM10) in human epidermal keratinocyte HaCaT cells. The PM10 particles significantly induced cytotoxicity in the HaCaT cells. The decrease in cell viability was restored upon treatment with tart cherry extract (200 µg/mL) containing chlorogenic acid, quercetin, and kaempferol. Tart cherry inhibited the intracellular reactive oxygen species (ROS) responsible for the distinctive activations of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in PM10-treated HaCaT cells. Interestingly, tart cherry significantly inhibited the expression of apoptosis-related genes (B-Cell Lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), and caspase-3) as regulated by the activation of transcription factor nuclear factor-kappa B (NF-κB). These results demonstrate that tart cherry is a medicinal food that blocks the mitochondrial pathway of apoptosis induced by PM10 in human epidermal keratinocytes.

A 75 kDa glycoprotein isolated from Cudrania tricuspidata Bureau induces colonic epithelial proliferation and ameliorates mouse colitis induced by dextran sulfate sodium.

Cudrania tricuspidata Bureau (CTB), a species of the Moraceae plant, has been used as a bruise recovery treatment. This study aimed to determine whether the 75 kDa phytoglycoprotein extracted from CTB has a regulatory effect on the proliferation of human colon epithelial cells and the pathological process of inflammatory bowel disease (IBD). We found that CTB glycoprotein significantly induces the proliferation of human colon epithelial HT-29 cells by activating protein kinase C. CTB glycoprotein stimulated the phosphorylation of c-Jun N-terminal kinase and transcription factor nuclear factor-κB, which are responsible for the expression of cell-cycle-related proteins (CDK2, CDK4, cyclin D1 and cyclin E) during its promotion of cell proliferation. Experimental colitis was induced in mice by adding dextran sulfate sodium to their drinking water at a concentration of 4% (W/V) for seven days. We found that CTB glycoprotein ameliorates the pathological process of IBD and lowers the disease activity index score, which was composed of body weight change, diarrhea, and hematochezia in ICR mice treated with dextran sulfate sodium. Hence, we suggest that CTB glycoprotein has the ability to prevent IBD by promoting cell proliferation signaling events via the activation of PKC, JNK and NF-κB in colon epithelial cells.

A screening study of high affinity peptide as molecular binder for AXL, tyrosine kinase receptor involving in Zika virus entry.

The recent extensive spread of Zika virus has led to increased interest in the development of early diagnostic tests. To the best of our knowledge, this is the first study to demonstrate the successful use of phage display to identify affinity peptides for quantitative analysis of AXL, a tyrosine kinase receptor involved in Zika virus entry. Biopanning of M13 phage library successfully identified a high affinity peptide, with the sequence AHNHTPIKQKYL. To study the feasibility of using free peptides for molecular recognition, we synthesized a series of amino acid-substituted peptides and examined their binding affinity for AXL using electrochemical impedance spectroscopy and square wave voltammetry. Most synthetic peptides had non-identical random coil structures based on circular dichroism spectroscopy. Of the peptides tested, AXL BP1 exhibited nanomolar binding affinity for AXL. To verify whether AXL BP1 could be used as a peptide inhibitor at the cellular level, two functional tests were carried out: a WST assay for cell viability and qRT-PCR for quantification of RNA levels in Zika virus-infected Huh7 cells. The results showed that AXL BP1 had low cytotoxicity and could block Zika virus entry. These results indicate that newly identified affinity peptides could potentially be used for the development of Zika virus entry inhibitors.

Nanospheres loaded with curcumin promote gut epithelial motility through F-actin-related migration signaling events.

Curcumin, a hydrophobic polyphenol of turmeric, has a variety of biological functions as an herbal supplement, but its poor gastric absorption rate is one of the major factors limiting its oral bioavailability. In the present study, we investigated the functional role of nanospheres loaded with curcumin (nCur) with regard to the motility of gut epithelial HCT116 cells and enterocyte migration along the crypt-villus axis. nCur significantly increased the motility of HCT116 cells and showed much higher migration efficacy than the curcumin. nCur stimulated the small GTPases Rac1 and the phosphorylation of protein kinase C, responsible for the distinctive activation of the mitogen-activated protein kinases. Interestingly, nCur significantly induced the expression of α-actinin, profilin-1, and filamentous (F)-actin as regulated by the phosphorylation of nuclear factor-kappa B during its promotion of cell migration. In mouse models of gut epithelial migration, treatment with nCur had an enhancing effect on the movement of enterocytes along the crypt-villus axis and the expression of cytoskeletal reorganization-related factors. These results indicate that nCur is a functional agent that promotes gut epithelial motility through F-actin-related migration signaling events.

Urolithin A suppresses high glucose-induced neuronal amyloidogenesis by modulating TGM2-dependent ER-mitochondria contacts and calcium homeostasis.

Hyperglycemia in diabetes mellitus (DM) patients is a causative factor for amyloidogenesis and induces neuropathological changes, such as impaired neuronal integrity, neurodegeneration, and cognitive impairment. Regulation of mitochondrial calcium influx from the endoplasmic reticulum (ER) is considered a promising strategy for the prevention of mitochondrial ROS (mtROS) accumulation that occurs in the Alzheimer's disease (AD)-associated pathogenesis in DM patients. Among the metabolites of ellagitannins that are produced in the gut microbiome, urolithin A has received an increasing amount of attention as a novel candidate with anti-oxidative and neuroprotective effects in AD. Here, we investigated the effect of urolithin A on high glucose-induced amyloidogenesis caused by mitochondrial calcium dysregulation and mtROS accumulation resulting in neuronal degeneration. We also identified the mechanism related to mitochondria-associated ER membrane (MAM) formation. We found that urolithin A-lowered mitochondrial calcium influx significantly alleviated high glucose-induced mtROS accumulation and expression of amyloid beta (Aß)-producing enzymes, such as amyloid precursor protein (APP) and ß-secretase-1 (BACE1), as well as Aß production. Urolithin A injections in a streptozotocin (STZ)-induced diabetic mouse model alleviated APP and BACE1 expressions, Tau phosphorylation, Aß deposition, and cognitive impairment. In addition, high glucose stimulated MAM formation and transglutaminase type 2 (TGM2) expression. We first discovered that urolithin A significantly reduced high glucose-induced TGM2 expression. In addition, disruption of the AIP-AhR complex was involved in urolithin A-mediated suppression of high glucose-induced TGM2 expression. Markedly, TGM2 silencing inhibited inositol 1, 4, 5-trisphosphate receptor type 1 (IP3R1)-voltage-dependent anion-selective channel protein 1 (VDAC1) interactions and prevented high glucose-induced mitochondrial calcium influx and mtROS accumulation. We also found that urolithin A or TGM2 silencing prevented Aß-induced mitochondrial calcium influx, mtROS accumulation, Tau phosphorylation, and cell death in neuronal cells. In conclusion, we suggest that urolithin A is a promising candidate for the development of therapies to prevent DM-associated AD pathogenesis by reducing TGM2-dependent MAM formation and maintaining mitochondrial calcium and ROS homeostasis.

Phytoglycoprotein isolated from Dioscorea batatas Decne promotes intestinal epithelial wound healing.

Dioscorea batatas Decne (DBD) has been used to heal various illnesses of the kidney and intestine as an herbal medicine in Asia. As a source of therapeutic agents, many glycoproteins have been isolated from mushrooms and plants, but the functional role of glycoprotein in intestinal epithelial wound healing has not been understood yet. In the present study, we investigated the wound healing potentials of the 30 kDa glycoprotein (DBD glycoprotein) isolated from DBD in human intestinal epithelial (INT-407) cells. We found that DBD glycoprotein (100 µg·mL-1) significantly increased the motility of INT-407 cells for 24 h by activating protein kinase C (PKC). DBD glycoprotein stimulated the activation of p38 mitogen-activated protein kinase (MAPK), which is responsible for the phosphorylation of NF-κB inhibitor α (IκBα). DBD glycoprotein increased the level of profilin-1 (PFN1), α-actinin and F-actin expression via activation of transcription factor, nuclear factor-kappa B (NF-κB) during its promotion of cell migration. Experimental mouse colitis was induced by adding dextran sulfate sodium (DSS) to the drinking water at a concentration of 4% (W/V) for 7 days. We figured out that administration of DBD glycoprotein (10 and 20 mg·kg-1) lowers the levels of disease activity index and histological inflammation in DSS-treated ICR mice. In this regard, we suggest that DBD glycoprotein has ability to promote the F-actin-related migration signaling events via activation of PKC and NF-κB in intestinal epithelial cells and prevent inflammatory bowel disease.

Nanospheres Loaded with Curcumin Improve the Bioactivity of Umbilical Cord Blood-Mesenchymal Stem Cells via c-Src Activation During the Skin Wound Healing Process.

Curcumin, a hydrophobic polyphenol derived from turmeric, has been used a food additive and as a herbal medicine for the treatment of various diseases, but the clinical application of curcumin is restricted by its poor aqueous solubility and its low permeability and bioavailability levels. In the present study, we investigate the functional role of a nanosphere loaded with curcumin (CN) in the promotion of the motility of human mesenchymal stem cells (MSCs) during the skin wound healing process. CN significantly increased the motility of umbilical cord blood (UCB)-MSCs and showed 10000-fold greater migration efficacy than curcumin. CN stimulated the phosphorylation of c-Src and protein kinase C which are responsible for the distinctive activation of the MAPKs. Interestingly, CN significantly induced the expression levels of α-actinin-1, profilin-1 and filamentous-actin, as regulated by the phosphorylation of nuclear factor-kappa B during its promotion of cell migration. In a mouse skin excisional wound model, we found that transplantation of UCB-MSCs pre-treated with CN enhanced wound closure, granulation, and re-epithelialization at mouse skin wound sites. These results indicate that CN is a functional agent that promotes the mobilization of UCB-MSCs for cutaneous wound repair.

Nanosphere Loaded with Curcumin Inhibits the Gastrointestinal Cell Death Signaling Pathway Induced by the Foodborne Pathogen Vibrio vulnificus.

Curcumin, a hydrophobic polyphenol of turmeric, has a variety of biological functions as a herbal supplement, but its poor gastric absorption rate is one of the major factors limiting its oral bioavailability. In the present study, we have investigated the functional role of a nanosphere loaded with curcumin (CN) during host cell death elicited by the Gram-negative bacterium V. vulnificus in human gastrointestinal epithelial HT-29 cells and an ileal-ligated mouse model. The recombinant protein (r) VvhA produced by V. vulnificus significantly reduced the viability of HT-29 cells. The cytotoxic effect of rVvhA was restored upon a treatment with CN (100 ng/mL), which had shown 1000-fold higher anti-apoptotic efficacy than curcumin. CN inhibited the phosphorylation of c-Src and PKC mediated by intracellular ROS responsible for the distinctive activation of the MAPKs in rVvhA-treated HT-29 cells. Interestingly, CN significantly restored the expression of Bax, Bcl-2, and cleaved caspase-3 as regulated by the phosphorylation of NF-κB. In mouse models of V. vulnificus infection, treatment with CN had a blocking effect that elevated the levels of TUNEL-positive DNA fragmentation and apoptosis-related proteins. These results indicate that CN is a functional agent that manipulates the V. vulnificus VvhA signaling pathway responsible for gastrointestinal cell death.

Melatonin restores Muc2 depletion induced by V. vulnificus VvpM via melatonin receptor 2 coupling with Gαq.

BACKGROUND: Melatonin (5-methoxy-N-acetyltryptamine), a hormone produced in the pineal gland, has a variety of biological functions as an antioxidant, but a functional role of melatonin in the regulation of intestinal mucin (Muc) production during bacterial infection has yet to be described in detail. In this study, we investigate the effects of melatonin during Muc2 repression elicited by the Gram-negative bacterium V. vulnificus. METHODS: Mucus-secreting human HT29-MTX cells were used to study the functional role of melatonin during Muc2 depletion induced by the recombinant protein (r) VvpM produced by V. vulnificus. The regulatory effects of melatonin coupling with melatonin receptor 2 (MT2) on the production of reactive oxygen species (ROS), the activation of PKCδ and ERK, and the hypermethylation of the Muc2 promoter as induced by rVvpM were examined. Experimental mouse models of V. vulnificus infection were used to study the role of melatonin and how it neutralizes the bacterial toxin activity related to Muc2 repression. RESULTS: Recombinant protein (r) VvpM significantly reduced the level of Muc2 in HT29-MTX cells. The repression of Muc2 induced by rVvpM was significantly restored upon a treatment with melatonin (1 µM), which had been inhibited by the knockdown of MT2 coupling with Gαq and the NADPH oxidase subunit p47 phox. Melatonin inhibited the ROS-mediated phosphorylation of PKCδ and ERK responsible for region-specific hypermethylation in the Muc2 promoter in rVvpM-treated HT29-MTX cells. In the mouse models of V. vulnificus infection, treatment with melatonin maintained the level of Muc2 expression in the intestine. In addition, the mutation of the VvpM gene from V. vulnificus exhibited an effect similar to that of melatonin. CONCLUSIONS: These results demonstrate that melatonin acting on MT2 inhibits the hypermethylation of the Muc2 promoter to restore the level of Muc2 production in intestinal epithelial cells infected with V. vulnificus.

Development of peptide biosensor for the detection of dengue fever biomarker, nonstructural 1.

Dengue virus (DENV) nonstructural 1 (NS1) protein is a specific and sensitive biomarker for the diagnosis of dengue. In this study, an efficient electrochemical biosensor that uses chemically modified affinity peptides was developed for the detection of dengue virus NS1. A series of amino acid-substituted synthetic peptides was rationally designed, chemically synthesized and covalently immobilized to a gold sensor surface. The sensor performance was monitored via square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS). Potential affinity peptides specific for NS1 were chosen according to the dynamic current decrease in SWV experiments. Using circular dichroism, the molar ellipticity of peptides (DGV BP1-BP5) was determined, indicating that they had a mostly similar in random coil structure, not totally identical. Using SWV, DGV BP1 was selected as a promising recognition peptide and limit of detection for NS1 was found to be 1.49 µg/mL by the 3-sigma rule. DGV BP1 showed good specificity and stability for NS1, with low signal interference. The validation of the sensor to detect NS1 proteins was confirmed with four dengue virus culture broth (from serotype 1 to 4) as proof-of-concept. The detection performance of our sensor incorporating DGV BP1 peptides showed a statistically significant difference. These results indicate that this strategy can potentially be used to detect the dengue virus antigen, NS1, and to diagnosis dengue fever within a miniaturized portable device in point-of-care testing.

BICD1 mediates HIF1α nuclear translocation in mesenchymal stem cells during hypoxia adaptation.

Hypoxia inducible factor 1α (HIF1α) is a master regulator leading to metabolic adaptation, an essential physiological process to maintain the survival of stem cells under hypoxia. However, it is poorly understood how HIF1α translocates into the nucleus in stem cells under hypoxia. Here, we investigated the role of a motor adaptor protein Bicaudal D homolog 1 (BICD1) in dynein-mediated HIF1α nuclear translocation and the effect of BICD1 regulation on hypoxia adaptation and its therapeutic potential on human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). In our results, silencing of BICD1 but not BICD2 abolished HIF1α nuclear translocation and its activity. BICD1 overexpression further enhanced hypoxia-induced HIF1α nuclear translocation. Hypoxia stimulated direct bindings of HIF1α to BICD1 and the intermediate chain of dynein (Dynein IC), which was abolished by BICD1 silencing. Akt inhibition reduced the binding of BICD1 to HIF1α and nuclear translocation of HIF1α. Conversely, Akt activation or GSK3ß silencing further enhanced the hypoxia-induced HIF1α nuclear translocation. Furthermore, BICD1 silencing abolished hypoxia-induced glycolytic reprogramming and increased mitochondrial ROS accumulation and apoptosis in UCB-MSCs under hypoxia. In the mouse skin wound healing model, the transplanted cell survival and skin wound healing capacities of hypoxia-pretreated UCB-MSCs were reduced by BICD1 silencing and further increased by GSK3ß silencing. In conclusion, we demonstrated that BICD1-induced HIF1α nuclear translocation is critical for hypoxia adaptation, which determines the regenerative potential of UCB-MSCs.

Intestinal epithelial cell apoptosis due to a hemolytic toxin from Vibrio vulnificus and protection by a 36 kDa glycoprotein from Rhus verniciflua Stokes.

Rhus verniciflua stokes (RVS) has been used as a functional food to cure inflammatory diseases in Korea. In the present study, we carry out an investigation of the cellular mechanism of a 36 kDa glycoprotein isolated from RVS fruit (RVS glycoprotein) during the apoptosis of human gastrointestinal epithelial HCT116 cells induced by the hemolytic toxin (VvhA) produced by V. vulnificus. Recombinant protein (r) VvhA produced by V. vulnificus stimulated apoptosis by activating the phosphorylation of protein kinase C (PKC) through the production of intracellular reactive oxygen species (ROS). However, RVS glycoprotein significantly inhibited the level of ROS production and PKC activation in rVvhA-stimulated HCT116 cells. Interestingly, we found that RVS glycoprotein has inhibitory effects on the phosphorylation of c-Jun N-terminal kinase (JNK) and nuclear factor-kappa B (NF-κB), which are responsible for the expression of Bax and cleaved caspase-3 in HCT116 cells treated with rVvhA, respectively. On the basis of these results, we suggest that RVS glycoprotein blocks mitochondrial apoptotic cell death induced by rVvhA via the inhibition of ROS-mediated signaling events in HCT116 cells.

Melatonin inhibits apoptotic cell death induced by Vibrio vulnificus VvhA via melatonin receptor 2 coupling with NCF-1.

Melatonin, an endogenous hormone molecule, has a variety of biological functions, but a functional role of melatonin in the infection of Gram-negative bacterium Vibrio vulnificus has yet to be described. In this study, we investigated the molecular mechanism of melatonin in the apoptosis of human intestinal epithelial (HCT116) cells induced by the hemolysin (VvhA) produced by V. vulnificus. Melatonin (1 µM) significantly inhibited apoptosis induced by the recombinant protein (r) VvhA, which had been inhibited by the knockdown of MT2. The rVvhA recruited caveolin-1, NCF-1, and Rac1 into lipid rafts to facilitate the production of ROS responsible for the phosphorylation of PKC and JNK. Interestingly, melatonin recruited NCF-1 into non-lipid rafts to prevent ROS production via MT2 coupling with Gαq. Melatonin inhibited the JNK-mediated phosphorylation of c-Jun responsible for Bax expression, the release of mitochondrial cytochrome c, and caspase-3/-9 activation during its promotion of rVvhA-induced apoptotic cell death. In addition, melatonin inhibited JNK-mediated phosphorylation of Bcl-2 responsible for the release of Beclin-1 and Atg5 expression during its promotion of rVvhA-induced autophagic cell death. These results demonstrate that melatonin signaling via MT2 triggers recruitment of NCF-1 into non-lipid rafts to block ROS production and JNK-mediated apoptotic and autophagic cell deaths induced by rVvhA in intestinal epithelial cells.

Lysophosphatidic Acid Receptor 1 Is Important for Intestinal Epithelial Barrier Function and Susceptibility to Colitis.

Intestinal epithelial cells form a barrier that is critical in protecting the host from the hostile luminal environment. Previously, we showed that lysophosphatidic acid (LPA) receptor 1 regulates proliferation of intestinal epithelial cells, such that the absence of LPA1 mitigates the epithelial wound healing process. This study provides evidence that LPA1 is important for the maintenance of epithelial barrier integrity. The epithelial permeability, determined by fluorescently labeled dextran flux and transepithelial resistance, is increased in the intestine of mice with global deletion of Lpar1, Lpar1-/- (Lpa1-/-). Serum liposaccharide level and bacteria loads in the intestinal mucosa and peripheral organs were elevated in Lpa1-/- mice. Decreased claudin-4, caudin-7, and E-cadherin expression in Lpa1-/- mice further suggested defective apical junction integrity in these mice. Regulation of LPA1 expression in Caco-2 cells modulated epithelial permeability and the expression levels of junctional proteins. The increased epithelial permeability in Lpa1-/- mice correlated with increased susceptibility to an experimental model of colitis. This resulted in more severe inflammation and increased mortality compared with control mice. Treatment of Caco-2 cells with tumor necrosis factor-α and interferon-γ significantly increased paracellular permeability, which was blocked by cotreatment with LPA, but not LPA1 knockdown cells. Similarly, orally given LPA blocked tumor necrosis factor-mediated intestinal barrier defect in mice. LPA1 plays a significant role in maintenance of epithelial barrier in the intestine via regulation of apical junction integrity.