Transcription and post-translational mechanisms: dual regulation of adiponectin-mediated Occludin expression in diabetes.

BACKGROUND: Occludin, a crucial component of tight junctions, has emerged as a promising biomarker for the diagnosis of acute ischemic disease, highlighting its significant potential in clinical applications. In the diabetes, Occludin serves as a downstream target gene intricately regulated by the adiponectin (APN) signaling pathway. However, the specific mechanism by which adiponectin regulates Occludin expression remains unclear. METHODS AND RESULTS: Endothelial-specific Ocln knockdown reduced APN-mediated blood flow recovery after femoral artery ligation and nullified APN's protection against high-fat diet (HFD)-triggered apoptosis and angiogenesis inhibition in vivo. Mechanically, we have meticulously elucidated APN's regulatory role in Occludin expression through a comprehensive analysis spanning transcriptional and post-translational dimensions. Foxo1 has been elucidated as a crucial transcriptional regulator of Occludin that is modulated by the APN/APPL1 signaling axis, as evidenced by validation through ChIP-qPCR assays and Western blot analysis. APN hindered Occludin degradation via the ubiquitin-proteasome pathway. Mass spectrometry analysis has recently uncovered a novel phosphorylation site, Tyr467, on Occludin. This site responds to APN, playing a crucial role in inhibiting Occludin ubiquitination by APN. The anti-apoptotic and pro-angiogenic effects of APN were attenuated in vitro and in vivo following Foxo1 knockdown or expression of a non-phosphorylatable mutant, OccludinY467A. Clinically, elevated plasma concentrations of Occludin were observed in patients with diabetes. A significant negative correlation was found between Occludin levels and APN concentrations. CONCLUSION: Our study proposes that APN modulates Occludin expression through mechanisms involving both transcriptional and post-translational interactions, thereby conferring a protective effect on endothelial integrity within diabetic vasculature.

Oncostatin M promotes epithelial barrier dysfunction in patients with eosinophilic chronic rhinosinusitis with nasal polyps.

BACKGROUND: Oncostatin M (OSM) may be involved in the promotion of mucosal epithelial barrier dysfunction in patients with eosinophilic chronic rhinosinusitis with nasal polyps (Eos CRSwNP) by inducing matrix metalloproteinase (MMP) -1 and -7. The aim was to evaluate the roles and mechanisms of action of OSM on MMP-1 and -7 synthesis from nasal epithelial cells (NECs). METHODS: OSM, OSM receptor (OSMR), MMP-1 and -7 expression was evaluated in nasal mucosa or primary NECs from scrapings by quantitative polymerase chain reaction (qPCR), immunofluorescence and immunohistochemistry. OSM and other cytokines were used to stimulate air-liquid interface (ALI) cultured NECs. qPCR, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence were used to evaluate the expression of OSMR, MMP-1, -7 and occludin in NECs. RESULTS: Elevated levels of OSMRß, MMP-1 and -7 were found in the tissues and scraped NECs of Eos CRSwNP in comparison to them obtained from the inferior turbinate (IT) and control subjects. The levels of OSM and OSMRß mRNA in tissues were positively correlated with the levels of MMP-1 and -7. OSM stimulation of NECs increased the expression of MMP-1 and -7, and the responses were suppressed by a STAT3 inhibitor, and a PI3K inhibitor respectively. In parallel studies, we found that stimulation with OSM disrupted the localization of occludin, a tight junction protein in NECs. The response was suppressed by a pan-MMP inhibitor. CONCLUSION: OSM induces the synthesis and release of MMP-1 and -7 in NECs. Furthermore, MMP-1 and -7 promote mucosal epithelial barrier dysfunction in patients with Eos CRSwNP.

The functional antagonist of sphingosine-1-phosphate, FTY720, impairs gut barrier function.

FTY720 or fingolimod is a known functional antagonist of sphingosine-1-phosphate (S1P), and it is effective in treating multiple sclerosis and preventing inflammatory bowel disease (IBD). Evidence shows that its use in mice can increase the susceptibility to mucosal infections. Despite the significant contribution of S1P to barrier function, the effect of the administration of FTY720 on the mucosal barrier has never been investigated. In this study, we looked into how FTY720 therapy affected the function of the gut barrier susceptibility. Administration of FTY720 to C57BL/6 mice enhances the claudin-2 expression and reduces the expression of claudin-4 and occludin, as studied by qPCR, Western blot, and immunofluorescence. FTY720 inhibits the Akt-mTOR pathway to decrease occludin and claudin-4 expression and increase claudin-2 expression. FTY720 treatment induced increased colonic inflammation, with notably greater immune cell infiltration, colon histopathology, and increased production of TNF-α, IFN-γ, CXCL-1, and CXCL-2 than that in control mice. Taking into account the close association of "the leaky gut" and gut dysbiosis among the major diseases, we therefore can infer that the vigilance of gut pathology should be maintained, where FTY720 is used as a treatment option.

A 3D in-vitro biomimicking Caco-2 intestinal permeability model-based assessment of physically modified telmisartan towards an alkalizer-free formulation development.

Efficient telmisartan delivery for hypertension management requires the incorporation of meglumine and/or sodium hydroxide as an alkalizer in the formulation. Long-term use of powerful alkalis with formulation as part of chronic therapy can cause metabolic alkalosis, ulcers, diarrhea, and body pain. Here, we aimed to design a telmisartan formulation without alkalizers. Telmisartan properties were tailor-made by microfluidizer-based physical modification. After microfluidization, telmisartan nanosuspension was lyophilized to obtain telmisartan premix powder. The optimized telmisartan nanosuspension had an average particle size of 579.85 ± 32.14 nm. The lyophilized premix was characterized by FT-IR, DSC, and PXRD analysis to ensure its physicochemical characteristics. The solubility analysis of premix showed 2.2 times, 2.3 times, and 6 times solubility improvement in 0.1 N HCl, phosphate buffer pH 7.5, and pH 6.8 compared to pure telmisartan. A 3D in-vitro Caco-2 model was developed to compare apparent permeability of API and powder premix. It showed that the powder premix was more permeable than pure API. The tablet formulation prepared from the telmisartan premix showed a dissolution profile comparable to that of the marketed formulation. The technique present herein can be used as a platform technology for solubility and permeability improvement of similar classes of molecules.

Evaluation of serum zonulin and occludin levels in obsessive-compulsive disorder and the effect of major depressive disorder comorbidity.

Objective: The aim of this study is to determine whether the levels of zonulin and occludin, tight junctions (TJ) proteins in the intestinal epithelium, will differ between obsessive compulsive disorder (OCD) patients and healthy controls. We also intended to investigate whether these would vary in OCD patients with and without major depressive disorder (MDD) comorbidity and in comparison with healthy controls. Methods: Sixty patients diagnosed with OCD and 30 healthy controls were included in the study. The cases were administered the Yale-Brown Obsessive Compulsive Scale (Y-BOCS) and the Hamilton Depression Rating Scale (HDRS). The patients were divided into two subgroups based on their HDRS scores and presence of MDD comorbidity. Zonulin and occludin levels were measured using the ELISA method. The research was carried out between April 2021 and October 2021. Results: Zonulin and occludin levels were significantly higher in the OCD patient group than in the control group (p<0.001). The levels of both were also significantly higher in the OCD patients with MDD comorbidity (OCD+MDD) compared to those without MDD (OCD-MDD) (p<0.001). Zonulin and occludin levels also rose significantly as disease severity increased in the OCD patient group (respectively; p<0.001, p=0.001). The levels of both increased in line with the severity of depression based on HDRS scores in the OCD+MDD group (p<0.001). A positive correlation was determined between the duration of OCD and zonulin and occludin levels. Evaluation of the entire OCD group revealed a moderate positive correlation between Y-BOCS and HDRS scores and zonulin and occludin. Conclusions: Zonulin and occludin levels in this research were significantly higher in the patients with OCD than in the healthy controls. That elevation was positively correlated with disease duration and severity, and the increase was significantly more pronounced in OCD with MDD comorbidity. These findings point to a possible disorder in the intestinal barrier and blood-brain barrier in OCD patients.

Evaluation of the Antifibrotic Effects of Drugs Commonly Used in Inflammatory Intestinal Diseases on In Vitro Intestinal Cellular Models.

The mechanism underlying intestinal fibrosis, the main complication of inflammatory bowel disease (IBD), is not yet fully understood, and there is no therapy to prevent or reverse fibrosis. We evaluated, in in vitro cellular models, the ability of different classes of drugs currently used in IBD to counteract two pivotal processes of intestinal fibrosis, the differentiation of intestinal fibroblasts to activated myofibroblasts using CCD-18Co cells, and the epithelial-to-mesenchymal transition (EMT) of intestinal epithelial cells using Caco-2 cells (IEC), both being processes induced by transforming growth factor-ß1 (TGF-ß1). The drugs tested included mesalamine, azathioprine, methotrexate, prednisone, methylprednisolone, budesonide, infliximab, and adalimumab. The expression of fibrosis and EMT markers (collagen-I, α-SMA, pSmad2/3, occludin) was assessed by Western blot analysis and by immunofluorescence. Of the drugs used, only prednisone, methylprednisolone, budesonide, and adalimumab were able to antagonize the pro-fibrotic effects induced by TGF-ß1 on CCD-18Co cells, reducing the fibrosis marker expression. Methylprednisolone, budesonide, and adalimumab were also able to significantly counteract the TGF-ß1-induced EMT process on Caco-2 IEC by increasing occludin and decreasing α-SMA expression. This is the first study that evaluates, using in vitro cellular models, the direct antifibrotic effects of drugs currently used in IBD, highlighting which drugs have potential antifibrotic effects.

Preclinical Evaluation of Sodium Butyrate's Potential to Reduce Alcohol Consumption: A Dose-Escalation Study in C57BL/6J Mice in Antibiotic-Enhanced Binge-Like Drinking Model.

INTRODUCTION: In our earlier efforts to establish gut-brain axis during alcohol use disorder (AUD), we have demonstrated that supplementation of C57BL/6J male mice with 8 mg/mL sodium butyrate, a major short-chain fatty acid, in drinking water reduced ethanol intake and neuroinflammatory response in antibiotic (ABX)-enhanced voluntary binge-like alcohol consumption model, drinking in the dark (DID). METHODS: To further evaluate the preclinical potential of SB, we have set a dose-escalation study in C57BL/6J male mice to test effects of ad libitum 20 mg/mL SB and 50 mg/mL SB and their combinations with ABX in the DID procedure for 4 weeks. Effects of these SB concentrations on ethanol consumption and bodily parameters were determined for the duration of the treatments. At the end of study, blood, liver, and intestinal tissues were collected to study any potential adverse effects ad to measure blood ethanol concentrations. RESULTS: Increasing SB concentrations in the drinking water caused a loss in the protective effect against ethanol consumption and produced adverse effects on body and liver weights, reduced overall liquid intake. The hypothesis that these effects were due to aversion to SB smell/taste at these high concentrations were further tested in a follow up proof-of-concept study with intragastric gavage administration of SB. The higher gavage dose (320 mg/kg) caused reduction in ethanol consumption without any adverse effects. CONCLUSION: Overall, these findings added more support for the therapeutic potential of SB in management of AUD, given a proper form of administration.

Role of tight junction proteins in shaping the immune milieu of malignancies.

INTRODUCTION: Tight junctions (TJs) and their constituent proteins play pivotal roles in cellular physiology and anatomy by establishing functional boundaries within and between neighboring cells. While the involvement of TJ proteins, such as claudins, in cancer is extensively studied, studies highlighting their interaction with immune system are still meager. Studies indicate that alterations in cytokines and immune cell populations can affect TJ proteins, compromising TJ barrier function and exacerbating pro-inflammatory conditions, potentially leading to epithelial cell malignancy. Disrupted TJs in established tumors may foster a pro-tumor immune microenvironment, facilitating tumor progression, invasion, epithelial-to-mesenchymal transition and metastasis. Although previous literature contains many studies describing the involvement of TJs in pathogenesis of malignancies their role in modulating the immune microenvironment of tumors is just beginning to be unleashed. AREAS COVERED: This article for the first time attempts to discern the importance of interaction between TJs and immune microenvironment in malignancies. To achieve the above aim a thorough search of databases like PubMed and Google Scholar was conducted to identify the recent and relevant articles on the topic. EXPERT OPINION: Breaking the vicious cycle of dysbiosis/infections/chemical/carcinogen-induced inflammation-TJ remodeling-malignancy-TJ dysregulation-more inflammation can be used as a strategy to complement the effect of immunotherapies in various malignancies.

The Basic Requirement of Tight Junction Proteins in Blood-Brain Barrier Function and Their Role in Pathologies.

This review addresses the role of tight junction proteins at the blood-brain barrier (BBB). Their expression is described, and their role in physiological and pathological processes at the BBB is discussed. Based on this, new approaches are depicted for paracellular drug delivery and diagnostics in the treatment of cerebral diseases. Recent data provide convincing evidence that, in addition to its impairment in the course of diseases, the BBB could be involved in the aetiology of CNS disorders. Further progress will be expected based on new insights in tight junction protein structure and in their involvement in signalling pathways.

MITOCHONDRIAL ANTIVIRAL PATHWAYS CONTROL ANTI-HIV RESPONSES AND ISCHEMIC STROKE OUTCOMES VIA THE RIG-1 SIGNALING AND INNATE IMMUNITY MECHANISMS.

Occludin (ocln) is one of the main regulatory cells of the blood-brain barrier (BBB). Ocln silencing resulted in alterations of the gene expression signatures of a variety of genes of the innate immunity system, including IFN-stimulated genes (ISGs) and the antiviral retinoic acid-inducible gene-1 (RIG-1) signaling pathway, which functions as a regulator of the cytoplasmic sensors upstream of the mitochondrial antiviral signaling protein (MAVS). Indeed, we observed dysfunctional mitochondrial bioenergetics, dynamics, and autophagy in our system. Alterations of mitochondrial bioenergetics and innate immune protection translated into worsened ischemic stroke outcomes in EcoHIV-infected ocln deficient mice. Overall, these results allow for a better understanding of the molecular mechanisms of viral infection in the brain and describe a previously unrecognized role of ocln as a key factor in the control of innate immune responses and mitochondrial dynamics, which affect cerebral vascular diseases such as ischemic stroke.