Epithelial Tight Junction Proteins Research Articles

A PROTEIN COMPLEX OF LIVER ORIGIN ACTIVATES A PRO-INFLAMMATORY PROGRAM THAT DRIVES HEPATIC AND INTESTINAL INJURY IN ALCOHOL-ASSOCIATED LIVER DISEASE

There is limited information on how the liver-to-gut axis contributes to alcohol-associated liver disease (AALD). We previously identified that high-mobility group box-1 (HMGB1) undergoes oxidation in hepatocytes and demonstrated elevated serum levels of oxidized HMGB1 ([O] HMGB1) in alcoholic patients. Since interleukin-1 beta (IL1B) increases in AALD, we hypothesized hepatocyte-derived [O] HMGB1 could interact with IL1B to activate a pro-inflammatory program that, besides being detrimental to the liver, drives intestinal barrier dysfunction. Alcohol-fed RageΔMye mice exhibited decreased NFκB signaling, a pro-inflammatory signature, and reduced total intestinal permeability, resulting in protection from AALD. Additionally, [O] HMGB1 bound and signaled through the receptor for advanced-glycation end-products (RAGE) in myeloid cells, driving hepatic inflammation, intestinal permeability, and increased portal blood lipopolysaccharide in AALD. We identified that [O] HMGB1 formed a complex with IL1B, which was found in the livers of patients with acute alcoholic hepatitis and mice with AALD. This complex originated from the liver, as it was absent in the intestine when hepatocytes did not produce [O] HMGB1. Mechanistically, the complex bound RAGE in Kupffer cells and macrophages induces a pro-inflammatory program. Moreover, it bound RAGE in intestinal macrophages and epithelial cells, leading to intestinal inflammation, altered intestinal epithelial cell tight junction protein expression, increased intestinal permeability, and elevated portal blood lipopolysaccharide, enhancing AALD pathogenesis. We identified a protein complex of liver origin that amplifies the pro-inflammatory feedback loop in AALD; therefore, targeting this complex could have significant therapeutic potential.

Microbe-Derived Antioxidants Protect IPEC-1 Cells from H2O2-Induced Oxidative Stress, Inflammation and Tight Junction Protein Disruption via Activating the Nrf2 Pathway to Inhibit the ROS/NLRP3/IL-1β Signaling Pathway.

Oxidative stress can induce inflammation and tight junction disruption in enterocytes. The initiation of inflammation is thought to commence with the activation of the ROS/NLRP3/IL-1β signaling pathway, marking a crucial starting point in the process. In our previous studies, we found that microbe-derived antioxidants (MAs) showed significant potential in enhancing both antioxidant capabilities and anti-inflammatory effects. The main aim of this research was to investigate the ability of MAs to protect cells from oxidative stress caused by H2O2, to reduce inflammatory responses, and to maintain the integrity of tight junction proteins by modulating the ROS/NLRP3/IL-1β signaling pathway. IPEC-1 cells (1 × 104 cells/well) were initially exposed to 100 mg/L of MAs for 12 h, after which they were subjected to 1 mM H2O2 treatment for 1 h. We utilized small interfering RNA (siRNA) to inhibit the expression of NLRP3 and Nrf2. Inflammatory factors such as IL-1β and antioxidant enzyme activity levels were detected by ELISA. Oxidative stress marker ROS was examined by fluorescence analysis. The NLRP3/IL-1β signaling pathway, Nrf2/HO-1 signaling pathway and tight junction proteins (ZO-1 and Occludin) were detected by RT-qPCR or Western blotting. In our research, it was observed that MA treatment effectively suppressed the notable increase in H2O2-induced inflammatory markers (TNF-α, IL-1β, and IL-18), decreased ROS accumulation, mitigated the expression of NLRP3, ASC, and caspase-1, and promoted the expression of ZO-1 and Occludin. After silencing the NLRP3 gene with siRNA, the protective influence of MAs was observed to be linked with the NLRP3 inflammasome. Additional investigations demonstrated that the treatment with MAs triggered the activation of Nrf2, facilitating its translocation into the nucleus. This process resulted in a notable upregulation of Nrf2, NQO1, and HO-1 expression, along with the initiation of the Nrf2-HO-1 signaling pathway. Consequently, there was an enhancement in the activities of antioxidant enzymes like SOD, GSH-Px, and CAT, which effectively mitigated the accumulation of ROS, thereby ameliorating the oxidative stress state. The antioxidant effectiveness of MAs was additionally heightened in the presence of SFN, an activator of Nrf2. The antioxidant and anti-inflammatory functions of MAs and their role in regulating intestinal epithelial tight junction protein disruption were significantly affected after siRNA knockdown of the Nrf2 gene. These findings suggest that MAs have the potential to reduce H2O2-triggered oxidative stress, inflammation, and disruption of intestinal epithelial tight junction proteins in IPEC-1 cells. This reduction is achieved by blocking the ROS/NLRP3/IL-1β signaling pathway through the activation of the Nrf2 pathway.

Bergapten enhances mitophagy to regulate intestinal barrier and Th17/Treg balance in mice with Crohn's disease-like colitis via PPARγ/NF-κB signaling pathway.

This study aimed to investigate whether bergapten (BG), a furanocoumarin phytohormone, holds promise for Crohn's disease (CD)-like colitis treatment and to preliminarily explore its potential mechanisms. 2,4,6-Trinitrobenzenesufonic acid (TNBS)-treated mice were applied to establish an in vivo research model, and BG was administered with different concentrations. The status of mice in each group was evaluated by disease activity index (DAI), and the severity was evaluated by pathological sections. The intestinal barrier was assessed by measuring in vivo intestinal permeability, peripheral blood intestinal fatty acid-binding protein (I-FABP) levels, epithelial resistance values, and tight junction protein levels. Markers were then used to assess Th17/Treg levels, mitophagy, and the peroxisome proliferator-activated receptor (PPAR)γ/ nuclear factor kappa B (NF-κB) signaling pathway. BG significantly reduced colon tissue damage in a concentration-dependent manner. DAI scores showed that the loose feces, occult blood, and weight loss of mice in the BG treatment were significantly reduced, and pathological section results revealed reduced inflammatory infiltration and fibrosis. Reduced serum FITC-dextran and I-FABP and increased levels of epithelial resistance and tight junction proteins support that the intestinal barrier was protected upon BG. The proportion of Th17 in mesenteric lymph nodes increased while Treg decreased in the model group. BG treatment effectively reduced the conversion of Treg to Th17. Additionally, BG was found to enhance mitophagy and activate the PPARγ/NF-κB signaling. BG demonstrates promising effects in ameliorating intestinal barrier damage and Th17/Treg imbalance in a murine model of CD-like colitis, while also promoting intracellular mitophagy. The PPARγ/NF-κB signaling pathway may serve as a key mediator of BG's regulatory mechanisms.

HUC-MSCs therapy for Crohn’s disease: efficacy in TNBS-induced colitis in rats and pilot clinical study

The use of mesenchymal stem cells (MSCs) has recently emerged as a promising new therapeutic strategy for many diseases including perianal fistulizing Crohn's disease (CD). Whether hUC-MSCs can promote the healing of luminal ulcer in CD has not been studied so far. The model of TNBS-induced colitis in rats was used to confirm the efficacy of hUC-MSCs in the treatment of CD. Then, seventeen CD patients refractory to or unsuitable for currently available therapies were enrolled and received once submucosal local injection through colonoscopy combined with once intravenous drip on the next day. All patients received a 24-week follow-up. Clinical and laboratory assessments were monitored at baseline, week 4, 8, 12, and 24. Endoscopic evaluations were conducted at baseline and week 12. Mucosal specimens were obtained at the margin of lesions by endoscopy biopsies and used for RNA sequencing. Two hUC-MSCs co-culture systems were established invitro, one with the mucosa specimens and the other with M1 macrophages induced from THP1. The expressions of genes representing inflammation (TNFα, IL-6, and IL-1β) and intestinal barrier function (ZO1, CLAUDIN1, and CDH1) were tested by RT-PCR. hUC-MSCs treatment increased body weight and decreased disease activity index (DAI), colon macroscopic damage index (CMDI), and histopathological score (HPS) of rats with TNBS-induced colitis. The results of the clinical study also showed that this mode of hUC-MSCs application was associated with regression of intestinal ulceration. Eight patients (47%) got endoscopic responses (SES-CD improvement of ≥50% from baseline) and three patients (17.65%) got mucosal healing (SES-CD is zero), with a parallel improvement of clinical and laboratory parameters without serious adverse events. RNA sequencing showed hUC-MSCs therapy was associated with an upregulation of transcripts linked to intestinal epithelial barrier integrity and a downregulation of inflammatory signaling pathways in the intestinal mucosa, especially the TNF signaling pathway, IL-17 signaling pathway, and TLR signaling pathway. RNA expression of intestinal epithelial tight junction protein (ZO1, CLAUDIN1, and CDH1), and the RNA expression of major intestinal inflammatory factors in CD (IL-1β, IL-6, and TNFα, p<0.001 for all) were improved significantly. Moreover, hUC-MSCs could attenuate the polarization of M1 macrophage induced from THP1, thereby decreasing the mRNA expression of IL-1β, IL-6, and TNFα significantly (p<0.05 for all). TSG-6 expression was evaluated in hUC-MSCs culture supernatant after treatment with TNFα, IFNγ, and LPS for 48h. And hUC-MSCs could inhibit the phosphorylation of JAK/STAT1 in the intestinal mucosa of CD patients. hUC-MSCs transplantation alleviated TNBS-induced colitis in rats. In this pilot clinical study, preliminary data suggested that this approach to administering hUC-MSCs might have potential for clinical efficacy and manageable safety in treating refractory CD, potentially providing hope for better outcomes. No serious adverse events were observed. This work was funded by General Program of National Natural Science Foundation of China (Grant No. 82270639), the Scientific research project of Shanghai Municipal Health Committee (Grant No. 202240001), Specialty Feature Construction Project of Shanghai Pudong New Area Health Commission (Grant No. PWZzb2022-05), Shanghai East Hospital Youth Research and Cultivation Foundation program (Grant No. DFPY2022015), Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai and Technology Development Project of Pudong Science, Technology and Economic Commission of Shanghai (Grant No. PKJ2021-Y08).

Targeting microbiota–immune–synaptic plasticity to explore the effect of tea polyphenols on improving memory in the aged type 2 diabetic rat model

ABSTRACT Objectives The study aimed to explore whether TP could improve memory in the aged type 2 diabetic rat model by regulating microbiota-immune-synaptic plasticity axis. Methods The experiment was divided into two parts. Firstly, to investigate the effects of TP on the physiopathology of the aged T2DM model rats, rats were randomly divided into the Normal control group, the aged group, the Aged T2DM model group, the TP 75, 150, 300 mg/kg groups, the 150 mg/kg Piracetam group and the 3 mg/kg Rosiglitazone group. Then, to further verify whether TP improved memory in aged T2DM rat model by regulating intestinal flora, the fecal microbiota transplantation (FMT) from the rats in the 300 mg/kg TP group into the rats in the aged T2DM model group was carried out. Effects on gut microbiota, colonic integrity (epithelial tight junction proteins), and endotoxemia (serum LPS) were examined, along with synaptic structure, synaptic plasticity-related structural proteins and inflammation signaling of the hippocampus in our study. Results Our results demonstrated that TP alleviated memory impairments in the aged T2DM rat model. The specific outcomes were as follows: TP 300 mg/kg corrected the gut dysbacteriosis, alleviated intestinal permeability reduction and peripheral/central inflammation, inhibited the TLR4/NF-κB signaling pathway. Meanwhile, TP improved the synaptic plasticity in the hippocampus of the aged T2DM model rats, whose expressions of SYN, PSD 95, NMDAR1 and GluR1 in hippocampus were significantly up-regulated. Surprisingly, rats of the FMT group displayed the same changes. Discussion TP improves the memory in aged T2DM rat model. The mechanism may be related to the alteration of gut flora, which can inhibit hippocampal TLR4/NF-κB signaling to attenuate neuroinflammation, then improve synaptic plasticity. The study proposes that TP interventions aimed at manipulating the gut microbiota may hold great potential as an effective approach for preventing and treating this disease.

Short-term exposure to cigarette smoke upregulates cathepsin S and alters expression of tight junction ZO-1

A long-term exposure to cigarette smoke (CS) alters the integrity of airway epithelial barrier, contributes to lung dysfunction, and elicits the expression and activity of lung cathepsin S (CatS), a cysteine protease that participates in the remodeling of connective tissue and cell junctions. Here, we observed that a short-term (4 days) exposure of mice to CS increased the expression and activity of CatS, while the expression level of zonula occludens 1 (ZO-1), an epithelial tight junction protein that stabilizes barrier assembly, was reduced in lung tissue lysates. Present data support that proteolytically active CatS may contribute to the defect of ZO-1 in CS-exposed mice.

Pharmacologic therapeutics in sarcopenia with chronic kidney disease.

Inflammation, metabolic acidosis, renin-angiotensin system activation, insulin resistance, and impaired perfusion to skeletal muscles, among others, are possible causes of uremic sarcopenia. These conditions induce the activation of the nuclear factor-kappa B and mitogen-activated protein kinase pathways, adenosine triphosphate ubiquitin-proteasome system, and reactive oxygen species system, resulting in protein catabolism. Strategies for the prevention and treatment of sarcopenia in chronic kidney disease (CKD) are aerobic and resistance exercises along with nutritional interventions. Anabolic hormones have shown beneficial effects. Megestrol acetate increased weight, protein catabolic rate, and albumin concentration, and it increased intracellular water component and muscle mass. Vitamin D supplementation showed improvement in physical function, muscle strength, and muscle mass. Correction of metabolic acidosis showed an increase in protein intake, serum albumin levels, body weight, and mid-arm circumference. The kidney- gut-muscle axis indicates that dysbiosis and changes in gut-derived uremic toxins and short-chain fatty acids affect muscle mass, composition, strength, and functional capacity. Biotic supplements, AST-120 administration, hemodiafiltration, and preservation of residual renal function are alleged to reduce uremic toxins, including indoxyl sulfate (IS) and p-cresyl sulfate (PCS). Synbiotics reversed the microbiota change in CKD patients and decreased uremic toxins. AST-120 administration changed the overall gut microbiota composition in CKD. AST-120 prevented IS and PCS tissue accumulation, ameliorated muscle atrophy, improved exercise capacity and mitochondrial biogenesis, restored epithelial tight junction proteins, and reduced plasma endotoxin levels and markers of oxidative stress and inflammation. In a human study, the addition of AST-120 to standard treatment had modest beneficial effects on gait speed change and quality of life.

Effect of supplementing a Bacillus subtilis-based probiotic on performance, intestinal integrity, and serum antioxidant capacity and metabolites concentrations of heat-stressed growing pigs.

Exposing pigs to heat stress (HS) seems to modify the intestinal microbiota which may compromise the integrity of the small intestine epithelia. Probiotics, live microorganisms, can help pigs to maintain a healthy intestinal environment. Eighty pigs (21.6 ± 3.4kg body weight) exposed to HS or thermal neutral (TN) conditions were used to evaluate the effect of a Bacillus subtilis-based probiotic on performance, body temperature, and intestinal integrity. Treatments were: TN pigs fed a control diet without (TN-C) or with 1 × 106 CFU probiotic/g of feed (TN-P), and HS pigs fed a control without (HS-C) or with probiotic (HS-P). The control diet was formulated with wheat, soybean meal, and free amino acids (AA). Feed and water were freely available during the 21-d study. At completion, samples from duodenum, jejunum, and ileum were collected to analyze epithelial histology and tight junction protein expression; antioxidant activity, and free AA and metabolites in serum. Relative abundance of Lactobacillus, Bifidobacterium, Escherichia coli, and Bacillus in ileal content was analyzed. Ambient temperature in the TN room ranged from 19 to 25 °C, and in HS room from 30 to 38.5 °C. Intestinal temperature in HS-P pigs was lower than in HS-C pigs. Weight gain and feed intake reduced, but feed:gain and respiration rate increased in HS compared to TN pigs, regardless of diet (P < 0.01). Probiotic increased weight gain and improved feed:gain (P < 0.05) in both TN and HS pigs, but feed intake did not differ. Heat stress decreased villi height in jejunum and villi height:crypt depth in duodenum and jejunum (P < 0.05). Probiotic increased villi height in duodenum and ileum, and villi height:crypt depth in all small intestine segments (P < 0.05). Relative abundance of Lactobacillus and Bifidobacterium tended to reduce, and E. coli tended to increase (P < 0.10) in ileal content of HS-C pigs. Ileal relative abundance of Bacillus was higher (P < 0.01) in HS-P pigs than in HS-C and TN-C pigs. Cystathionine, homocysteine, hydroxylysine, α-amino-adipic acid, citrulline, α-amino-n-butyric acid, P-Ser, and taurine were higher in HS than in TN pigs (P < 0.05). These data confirm the negative effect of HS on performance, body temperature, and intestinal integrity of pigs. These data suggest that supplementing 1 × 106 CFU probiotic/g of feed based on Bacillus subtilis DSM 32540 may help to counteract the negative effects of HS on the performance and intestinal integrity of pigs.

Scallop hydrolysates/κ-carrageenan hydrogels improve the alleviating effect of curcumin on DSS-induced colitis

Scallop (Patinopecten yessoensis) male gonad hydrolysates/κ-carrageenan (SK) double cross-linking network hydrogels were constructed as curcumin (Cur) delivery vehicles with good gel mechanical property and homogeneous honeycomb microstructures. Compared with Cur + DSS group, weight loss and colon length in SK-Cur + DSS group increased about 1.04 and 1.06 folds, while the disease active index (DAI) scores decreased about 13 %. Moreover, SK-Cur showed superior effect on intestinal barrier improvement, oxidative stress modulation, and inflammatory expression regulation, reflected by 1.1 folds increment of epithelial tight junction proteins, 16 % and 27 % decline in MPO and iNOS levels, 20 % decline in NF-κB expression, 4 %, 7 %, 6 % decline in TNF-α, IL-1β, IL-6 expression in serum, and 9 %, 20 %, 7 % decline in TNF-α, IL-1β, IL-6 expression in colon tissue. Furthermore, SK-Cur enriched alpha diversity and restored taxonomic composition with increasing Bacteroides and Verrucomicrobia abundance accompanied by decreasing Firmicutes in DSS-induced UC mice, roughly to a normal gut flora state.

Oat-based postbiotics ameliorate high-sucrose induced liver injury and colitis susceptibility by modulating fatty acids metabolism and gut microbiota

High-sucrose (HS) consumption leads to metabolic disorders and increases susceptibility to colitis. Postbiotics hold great potentials in combating metabolic diseases and offer advantages in safety and processability, compared with living probiotics. We developed innovative oat-based postbiotics and extensively explored how they could benefit in rats with long-term high-sucrose consumption. The postbiotics fermented with Lactiplantibacillus plantarum (OF-1) and OF-5, the one fermented with the optimal selection of five probiotics (i.e., L. plantarum, Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus, Lactobacillus acidophilus, and Bifidobacterium lactis) alleviated HS induced liver injury, impaired fatty acid metabolism and inflammation through activating AMPK/SREBP-1c pathways. Moreover, oat-based postbiotics restored detrimental effects of HS on fatty acid profiles in liver, as evidenced by the increases in polyunsaturated fatty acids and decreases in saturated fatty acids, with OF-5 showing most pronounced effects. Furthermore, oat-based postbiotics prevented HS exacerbated susceptibility to dextran sodium sulfate caused colitis and reconstructed epithelial tight junction proteins in colons. Oat-based postbiotics, in particular OF-5 notably remodeled gut microbiota composition, e.g., enriching the relative abundances of Akkermansia, Bifidobacterium, Alloprevotella and Prevotella, which may play an important role in the liver-colon axis responsible for improvements of liver functions and reduction of colitis susceptibility. The heat-inactivated probiotics protected against HS-induced liver and colon damage, but such effects were less pronounced compared with oat-based postbiotics. Our findings emphasize the great value of oat-based postbiotics as nutritional therapeutics to combat unhealthy diet induced metabolic dysfunctions.

Phenotypic Characterization of 2D and 3D Prostate Cancer Cell Systems Using Electrical Impedance Spectroscopy.

Prostate cancer is the second leading cause of death in men. A challenge in treating prostate cancer is overcoming cell plasticity, which links cell phenotype changes and chemoresistance. In this work, a microfluidic device coupled with electrical impedance spectroscopy (EIS), an electrode-based cell characterization technique, was used to study the electrical characteristics of phenotype changes for (1) prostate cancer cell lines (PC3, DU145, and LNCaP cells), (2) cells grown in 2D monolayer and 3D suspension cell culture conditions, and (3) cells in the presence (or absence) of the anti-cancer drug nigericin. To validate observations of phenotypic change, we measured the gene expression of two epithelial markers, E-cadherin (CDH1) and Tight Junction Protein 1 (ZO-1). Our results showed that PC3, DU145, and LNCaP cells were discernible with EIS. Secondly, moderate phenotype changes based on differences in cell culture conditions were detected with EIS and supported by the gene expression of CDH1. Lastly, we showed that EIS can detect chemoresistant-related cell phenotypes with nigericin drug treatment. EIS is a promising label-free tool for detecting cell phenotype changes associated with chemoresistance. Further development will enable the detection and characterization of many other types of cancer cells.

CircMIRLET7BHG, upregulated in an m6A-dependent manner, induces the nasal epithelial barrier dysfunction in allergic rhinitis pathogenesis

ObjectiveAllergic rhinitis (AR) remains a frequent aspiratory allergic inflammatory disorder with a high incidence. Circular RNAs (circRNAs) have been revealed to participate in the pathogenesis of AR. This study investigated the biological function of circMIRLET7BHG (hsa_circ_0008668) in AR progression. MethodsOvalbumin (OVA)-exposed human nasal epithelial cell line (HNEpC) and mice were adopted as the in vitro and in vivo models of AR. Immunofluorescence staining was used to determine epithelial tight junction protein expression. Target molecule levels were assessed by RT-qPCR and Western blotting. Localization of circMIRLET7BHG and IGF2BP1 was observed by RNA-FISH and immunofluorescence. Epithelial barrier damage was determined by transepithelial electrical resistance and fluorescein isothiocyanate-dextran (FD4) permeability. Serum concentrations of IgE, sIgE, IFN-γ, IL-4, and IL-5 were detected by ELISA. Apoptosis, pathological changes, and eosinophil infiltration in nasal mucosa tissues were evaluated by TUNEL, H&E, and Sirius red staining, respectively. Molecular mechanism was analyzed by RNA pull-down, RIP, and MeRIP assays. ResultsAn increased expression of circMIRLET7BHG was found in AR patients and experimental models. Down-regulation of circMIRLET7BHG attenuated OVA-induced allergic symptoms via relieving epithelial thicknesses, eosinophil infiltration, apoptosis, and inflammatory response in mice. Subsequently, circMIRLET7BHG deficiency prevented OVA-induced epithelial barrier dysfunction by reducing epithelial permeability, and inhibiting tight junction proteins. Mechanistically, methyltransferase-like 3 (METTL3) enhanced circMIRLET7BHG expression via m6A methylation, which enhanced ADAM10 mRNA stability via interaction with IGF2BP1. ConclusionMETTL3-mediated m6A modification increased circMIRLET7BHG expression that consequently raised ADAM10 mRNA stability via interplay with IGF2BP1, thereby promoting AR by inducing epithelial barrier dysfunction.

GLUT1 contributes to impaired epithelial tight junction in the late phase of acute lung injury

Dysfunction of epithelial barrier is crucial for the development of acute lung injury (ALI). This study was aimed to evaluate the role of glucose transporter 1 (GLUT1) in dysregulation of epithelial tight junction in ALI. GLUT1 was inhibited with specific antagonists WZB117 or BAY876 to see the effects on epithelial tight junction in a well-established LPS-induced mouse ALI model as well as in vitro cultured epithelial cells. Pharmacological inhibition of GLUT1 with WZB117 at either a low or high dose had no effects on lung injury and inflammation 24 h after LPS challenge, but significantly decreased the pulmonary inflammatory responses induced by LPS at 72 h with a high dose, which was verified by treatment with BAY876. WZB117 or BAY876 also recovered the expression of epithelial tight junction proteins ZO-1 and occludin. In cultured BEAS-2B and A549 cells, LPS induced increased GLUT1 expression, accompanied by decreased expression of tight junction protein ZO-1 and occludin. Blockade of GLUT1 restored LPS-induced disruption of ZO-1 and occludin in BEAS-2B rather than A549. Taken together, our results showed that GLUT1 is responsible for dysfunction of epithelial tight junctions in the late phase of LPS-induced ALI.

A Novel Combination Therapy Tβ4/VIP Protects against Hyperglycemia-Induced Changes in Human Corneal Epithelial Cells.

Despite the prevalence of diabetic retinopathy, the majority of adult diabetic patients develop visually debilitating corneal complications, including impaired wound healing. Unfortunately, there is limited treatment for diabetes-induced corneal damage. The current project investigates a novel, peptide-based combination therapy, thymosin beta-4 and vasoactive intestinal peptide (Tβ4/VIP), against high-glucose-induced damage to the corneal epithelium. Electric cell-substrate impedance sensing (ECIS) was used for real-time monitoring of barrier function and wound healing of human corneal epithelial cells maintained in either normal glucose (5 mM) or high glucose (25 mM) ± Tβ4 (0.1%) and VIP (5 nM). Barrier integrity was assessed by resistance, impedance, and capacitance measurements. For the wound healing assay, cell migration was also monitored. Corneal epithelial tight junction proteins (ZO-1, ZO-2, occludin, and claudin-1) were assessed to confirm our findings. Barrier integrity and wound healing were significantly impaired under high-glucose conditions. However, barrier function and cell migration significantly improved with Tβ4/VIP treatment. These findings were supported by high-glucose-induced downregulation of tight junction proteins that were effectively maintained similar to normal levels when treated with Tβ4/VIP. These results strongly support the premise that Tβ4 and VIP work synergistically to protect corneal epithelial cells against hyperglycemia-induced damage. In addition, this work highlights the potential for significant translational impact regarding the treatment of diabetic patients and associated complications of the cornea.

Green rooibos (Aspalathus linearis) promotes gut health: insight into mechanisms

Ethnopharmacological relevanceParalleling the increasing incidence of gastrointestinal disorders world-wide, therapeutic investigations of nutraceuticals to promote gastrointestinal health are gaining popularity. Although anecdotally well-known for its gut health promoting potential, sparse scientific evidence supports this action of Aspalathus linearis (Burm.f.) R. Dahlgren - or rooibos - at the gastrointestinal epithelial level. Aim of the studyTraditionally, rooibos is considered to exert antispasmodic, anti-inflammatory, and anti-nociceptive effects in the gut. However, the direct effect on intestinal epithelium is unknown. Thus, to assess the validity of anecdotal claims, two larval zebrafish models were utilized to evaluate effects of rooibos on intestinal health. Materials and methodsFirstly, a larval zebrafish model of gastrointestinal inflammation (2-day TNBS-exposure) was employed. Co-administration of 6α-methylprednisolone served as an internal treatment control. Assessments included live imaging techniques and post-mortem immunofluorescent staining of epithelial tight junction proteins. In addition, whole body H2O2 and prostaglandin E2 assays were performed. Secondly, a gastrointestinal motility assay was performed, with known pro- and anti-kinetic mediators to assess the effect of rooibos to alter functional outcome in vivo. ResultsAqueous and ethanol extracts of green rooibos rescued TNBS-induced reductions in neutral red stained length of larval mid-intestines. Subsequent experiments confirmed the rescue capacity of the aqueous green rooibos extract regarding whole body oxidative and inflammatory status. Concerning tight junction proteins, only the aqueous green rooibos extract – and not prednisolone – normalized both zona occludens-1 and occludin expression levels when compared the TNBS group. In terms of gastrointestinal motility, the aqueous green rooibos extract significantly reduced the extent of gut motility dysregulation achieved by kinetic modulators. ConclusionsData indicates the potential of a 2 mg/ml aqueous extract of green rooibos to improve gastrointestinal integrity and functionality in vivo, suggesting beneficial effects of rooibos may already occur at the level of the gut. This provides some evidence to support indigenous knowledge.

Plant-derived squalene supplementation improves growth performance and alleviates acute oxidative stress-induced growth retardation and intestinal damage in piglets

Piglets are particularly susceptible to oxidative stress, which causes inferior growth performance and intestinal damage. Squalene (SQ), a natural bioactive substance enriched in shark liver oil, shows excellent antioxidant properties and can currently be obtained at a low cost from deodorizer distillate during the production of plant oil. This study aimed to evaluate the effects of plant-derived SQ supplementation on the growth performance of piglets and explore the beneficial roles of SQ against oxidative stress and intestinal injury in diquat-challenged piglets. Forty piglets were randomly divided into five groups and fed a basal diet supplemented with SQ at 0, 500, 1000, or 2000 mg/kg for 5 weeks. Acute oxidative stress was induced in the piglets with diquat (10 mg/kg BW) at the fourth week of the experiment, followed by a 7-d recovery period. Results showed that before the diquat challenge, SQ supplementation significantly improved growth performance (average daily gain and feed conversion ratio) and serum antioxidant status, and after the diquat challenge, SQ supplementation significantly mitigated diquat-induced growth arrest, intestinal villous atrophy, intestinal epithelial cell apoptosis, intestinal hyperpermeability, and deficiency of intestinal epithelial tight junction proteins (zonula occludens-1, occludin, and claudin-3). Under oxidative stress induced by diquat, SQ supplementation consistently improved the antioxidant status of the small intestine, liver, and muscle. In vitro, SQ was shown to alleviate hydrogen peroxide (H2O2)-induced increase of the levels of intracellular reactive oxygen species and apoptosis of porcine intestinal epithelial cells. Taken together, SQ supplementation improves growth performance and effectively alleviates acute oxidative stress-induced growth retardation and intestinal injury via improving antioxidant capacity in piglets. Our findings may provide an efficient strategy for alleviating oxidative stress-induced inferior growth performance and intestinal damage in piglets.

Effect of Vitamin D on the Proliferation and Barrier of Atrophic Vaginal Epithelial Cells.

Atrophic vaginitis is very common in postmenopausal women due to declining estrogen levels. Vitamin D plays an important role in promoting epithelial cell proliferation, migration and adhesion. We established a rat model of ovariectomy (OVX) induced atrophic vaginitis with the aim of investigating the effects of Vitamin D supplementation on the vaginal epithelial barrier. The results showed that ovariectomised rats had significantly higher vaginal pH, reduced Lactobacillus, significantly lower uterine and vaginal weights, and lower vaginal epithelial PCNA, occludin, and E-cadherin mRNA expression compared with sham-operated rats. Vitamin D supplementation could reduce the vaginal pH, promote the proliferation and keratinization of vaginal epithelial cells, enhance the expression of PCNA mRNA in vaginal tissues, and improve the vaginal and uterine atrophy. Vitamin D can also increase the expression of E-cadherin and occludin proteins in vaginal tissues, maintain the integrity of the vaginal epithelium, increase the number of Lactobacillus, and reduce pathogenic bacterial infections. In vitro experiments demonstrated that 1,25(OH)2D3 could promote the proliferation and migration of VK2/E6E7 vaginal epithelial cells and increase the expression of E-cadherin protein. In conclusion, we demonstrated that Vitamin D can regulate the expression of vaginal epithelial tight junction proteins, promotes cell proliferation, and improves vaginal atrophy due to estrogen deficiency.

Expression of tight junction proteins in smokers and non-smokers with generalized Stage III periodontitis.

This study aims to evaluate the gingival crevicular fluid (GCF) levels of tumor necrosis factor-α (TNF-α), zonula occludens-1 (ZO-1), occludin (Occ), and tricellulin (Tric) in periodontitis, as well as their alterations due to smoking. Tight junctions (TJ), which consist of transmembrane and cytoplasmic scaffolding proteins, connect the epithelial cells of the periodontium. Occ, claudins, junctional adhesion molecules, and Tric are transmembrane TJ proteins found at the cell membrane. The transmembrane TJ proteins and the intracellular cytoskeleton are directly linked by cytoplasmic scaffolding proteins such as ZO-1. Although the functions and locations of these molecules have been defined, their behavior in periodontal inflammation is unknown. The study included four groups: individuals with periodontal health without smoking (C; n = 31), individuals with generalized Stage III periodontitis without smoking (P; n = 28), individuals with periodontal health while smoking (CS; n = 22), and individuals with generalized Stage III periodontitis while smoking (PS; n = 18). Clinical periodontal parameters were recorded, and enzyme-linked immunosorbent assay (ELISA) was used to examine ZO-1, Occ, Tric, and TNF-α levels in GCF. In the periodontitis groups, clinical parameters were significantly higher (p < .001). The site-specific levels of TNF-α, ZO-1, Tric, and Occ in the P group were statistically higher than those in the other groups (p < .05). TNF-α, probing pocket depth (PPD), and bleeding on probing (BOP) exhibited positive correlations with all TJ proteins (p < .005). Smoking could potentially affect the levels of epithelial TJ proteins in the GCF, thereby potentially playing a significant role in the pathogenesis of the periodontal disease.

Therapeutic mechanism of Liangxue-Guyuan-Yishen decoction on intestinal stem cells and tight junction proteins in gastrointestinal acute radiation syndrome rats.

On the basis of the previous research, the Traditional Chinese Medicine theory was used to improve the drug composition for gastrointestinal acute radiation syndrome (GI-ARS). The purpose of this study was to study the therapeutic mechanism of Liangxue-Guyuan-Yishen decoction (LGYD) on GI-ARS and to provide a new scheme for the treatment of radiation injury. Here, we investigated the effects of LGYD on intestinal stem cells (ISCs) in a GI-ARS rat model. Rat health and survival and the protective efficacy of LGYD on the intestines were analyzed. The active principles in LGYD were detected using liquid chromatography-mass spectrometry (LC-MS). ISC proliferation, intestinal epithelial tight junction (TJ) protein expression and regulatory pathways were explored using immunohistochemistry, western blotting (WB) and reverse transcription quantitative polymerase chain reaction (RT-qPCR), respectively. Involvement of the WNT and MEK/ERK pathways in intestinal recovery was screened using network pharmacology analysis and validated by WB and RT-qPCR. LGYD administration significantly improved health and survival in GI-ARS rats. Pathological analysis showed that LGYD ameliorated radiation-induced intestinal injury and significantly promoted LGR5+ stem cell regeneration in the intestinal crypts, upregulated TJ protein, and accelerated crypt reconstruction in the irradiated rats. LC-MS revealed ≥13 constituents that might contribute to LGYD's protective effects. Collectively, LGYD can promote crypt cell proliferation and ISCs after radiation damage, the above effect may be related to WNT and MEK/ERK pathway.

Paeonol alleviates ulcerative colitis in mice by increasing short-chain fatty acids derived from Clostridium butyricum

BackgroundIncreasing evidence suggests that repairing the damaged intestinal epithelial barrier and restoring its function is the key to solving the problem of prolonged ulcerative colitis. Previous studies have shown that paeonol (pae) can alleviate colitis by down-regulating inflammatory pathways. In addition, pae also has a certain effect on regulating intestinal flora. However, it remains unclear whether pae can play a role in repairing the intestinal barrier and whether there is a relationship between the therapeutic effect and the gut microbiota. PurposesThe aim of this study is to investigate the effect of pae on intestinal barrier repair in UC mice and how the gut microbiota plays a part in it. Study Design and MethodsThe therapeutic effect of pae was evaluated in a 3% DSS-induced UC mouse model. The role of pae in repairing the intestinal barrier was evaluated by detecting colonic cupped cells by Alcian blue staining, the expression of colonic epithelial tight junction protein by immunofluorescence and western blot, and the proportion of IL-22+ILC3 cells in the lamina propria lymphocytes by flow cytometry. Subsequently, 16S rRNA sequencing was used to observe the changes in intestinal flora, GC-MS was used to detect the level of SCFAs, and qPCR was used to identify the abundance of Clostridium butyricum in the intestine to evaluate the effect of pae on the gut microbiota. The antibiotic-mediated depletion of the gut flora was then used to verify that pae depends on C. butyricum to play a healing role. Finally, non-targeted metabolomics was employed to investigate the potential pathways of pae regulating C. butyricum. ResultsPae could improve intestinal microecological imbalance and promote the production of short-chain fatty acids (SCFAs). Most importantly, we identified C. butyricum as a key bacterium responsible for the intestinal barrier repair effect of pae in UC mice. Eradication of intestinal flora by antibiotics abolished the repair of the intestinal barrier and the promotion of SCFAs production by pae, while C. butyricum colonization could restore the therapeutic effects of pae in UC mice, which further confirmed that C. butyricum was indeed the “driver bacterium” of pae in UC treatment. Untargeted metabolomics showed that pae regulated some amino acid metabolism and 2-Oxocarboxylic acid metabolism in C. butyricum. ConclusionsOur study showed that the restoration of the impaired intestinal barrier by pae to alleviate colitis is associated with increased C. butyricum and SCFAs production, which may be a promising strategy for the treatment of UC.