Cranberry Polyphenols and Prevention against Urinary Tract Infections: New Findings Related to the Integrity and Functionality of Intestinal and Urinary Barriers.

This work seeks to generate new knowledge about the mechanisms underlying the protective effects of cranberry against urinary tract infections (UTI). Using Caco-2 cells grown in Transwell inserts as an intestinal barrier model, we found that a cranberry-derived digestive fluid (containing 135 ± 5 mg of phenolic compounds/L) increased transepithelial electrical resistance with respect to control (ΔTEER = 54.5 Ω cm2) and decreased FITC-dextran paracellular transport by about 30%, which was related to the upregulation of the gene expression of tight junction (TJ) proteins (i.e., occludin, zonula occludens-1 [ZO-1], and claudin-2) (∼3-4-fold change with respect to control for claudin-2 and ∼2-3-fold for occludin and ZO-1). Similar protective effects, albeit to a lesser extent, were observed when Caco-2 cells were previously infected with uropathogenic Escherichia coli (UPEC). In a urinary barrier model comprising T24 cells grown in Transwell inserts and either noninfected or UPEC-infected, treatments with the cranberry-derived phenolic metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and phenylacetic acid (PAA) (250 µM) also promoted favorable changes in barrier integrity and permeability. In this line, incubation of noninfected T24 cells with these metabolites induced positive regulatory effects on claudin-2 and ZO-1 expression (∼3.5- and ∼2-fold change with respect to control for DOPAC and ∼1.5- and >2-fold change with respect to control for PAA, respectively). Overall, these results suggest that the protective action of cranberry polyphenols against UTI might involve molecular mechanisms related to the integrity and functionality of the urothelium and intestinal epithelium.

FXR1 stabilizes SNORD63 to regulate blood-tumor barrier permeability through SNORD63 mediated 2'-O-methylation of POU6F1.

How selectively increase blood-tumor barrier (BTB) permeability is crucial to enhance the delivery of chemotherapeutic agents to brain tumor tissues. In this study, we established in vitro models of the blood-brain barrier (BBB) and BTB using endothelial cells (ECs) co-cultured with human astrocytes (AECs) and glioma cells (GECs), respectively. The findings revealed high expressions of the RNA-binding protein FXR1 and SNORD63 in GECs, where FXR1 was found to bind and stabilize SNORD63. Knockdown of FXR1 resulted in decreased expression of tight-junction-related proteins and increased BTB permeability by down-regulating SNORD63. SNORD63 played a role in mediating the 2'-O-methylation modification of POU6F1 mRNA, leading to the downregulation of POU6F1 protein expression. POU6F1 showed low expression in GECs and acted as a transcription factor to regulate BTB permeability by binding to the promoter regions of ZO-1, occludin, and claudin-5 mRNAs and negatively regulating their expressions. Finally, the targeted regulation of FXR1, SNORD63, and POU6F1 expressions, individually or in combination, effectively enhanced doxorubicin passage through the BTB and induced apoptosis in glioma cells. This study aims to elucidate the underlying mechanism of the FXR1/SNORD63/POU6F1 axis in regulating BTB permeability, offering a novel strategy to improve the efficacy of glioma chemotherapy.

Mannose enhances intestinal immune barrier function and dextran sulfate sodium salt-induced colitis in mice by regulating intestinal microbiota.

Background: Inflammatory bowel disease (IBD) greatly affects human quality of life. Mannose has been reported to be used to treat IBD, but the mechanism is currently unknown. Methods: C57/BL mice were used as research subjects, and the mouse acute colitis model was induced using dextran sulfate sodium salt (DSS). After oral administration of mannose, the body weights and disease activity index (DAI) scores of the mice were observed. The colon lengths, histopathological sections, fecal content microbial sequencing, colon epithelial inflammatory genes, and tight junction protein Occludin-1 expression levels were measured. We further used the feces of mice that had been orally administered mannose to perform fecal bacterial transplantation on the mice with DSS-induced colitis and detected the colitis-related indicators. Results: Oral administration of mannose increased body weights and colon lengths and reduced DAI scores in mice with DSS-induced colitis. In addition, it reduced the expression of colon inflammatory genes and the levels of serum inflammatory factors (TNF-α, IL-6, and IL-1ß), further enhancing the expression level of the colonic Occludin-1 protein and alleviating the toxic response of DSS to the intestinal epithelium of the mice. In addition, gut microbial sequencing revealed that mannose increased the abundance and diversity of intestinal flora. Additionally, after using the feces of the mannose-treated mice to perform fecal bacterial transplantation on the mice with DSS-induced colitis, they showed the same phenotype as the mannose-treated mice, and both of them alleviated the intestinal toxic reaction induced by the DSS. It also reduced the expression of intestinal inflammatory genes (TNF-α, IL-6, and IL-1ß) and enhanced the expression level of the colonic Occludin-1 protein. Conclusion: Mannose can treat DSS-induced colitis in mice, possibly by regulating intestinal microorganisms to enhance the intestinal immune barrier function and reduce the intestinal inflammatory response.

Transcriptional Levels of Intercellular Junction Proteins in an Alveolar Epithelial Cell Line Exposed to Irradiation or Bleomycin.

We performed a comparative study of the effects of X-ray irradiation and bleomycin on the mRNA levels of E-cadherin and tight junction proteins (claudin-3, claudin-4, claudin-18, ZO-2, and occludin) in an alveolar epithelial cell line L2. Irradiation decreased claudin-4 levels and increased occludin levels, while the levels of other mRNAs remained unchanged. Bleomycin increased the expression levels of all proteins examined except claudin-3. Irradiation and bleomycin have different effects on the expression level of intercellular junction proteins, indicating different reactions triggered in alveolar epithelial cells and a great prospects of further comparative studies.

Potassium diformate alleviated inflammation of IPEC-J2 cells infected with EHEC.

Potassium diformate (KDF) is a kind of formate, which possesses the advantages of antimicrobial activity, growth promotion and preventing diarrhea in weaned piglets. However, the researches of KDF in animal production mostly focused on apparent indexes such as growth performance and the mechanisms of KDF on intestinal health have not been reported. Thus, porcine small intestinal epithelial cells (IPEC-J2) infected with Enterohemorrhagic Escherichia coli (EHEC) was used to investigate the role of KDF on alleviating intestinal inflammation in this study. The 0.125 mg/mL KDF treated IPEC-J2 cells for 6 h and IPEC-J2 cells challenged with 5 × 107 CFU/mL EHEC for 4 h were confirmed as the optimum concentration and time for the following experiment. The subsequent experiment was divided into four groups: control group (CON), EHEC group, KDF group, KDF+EHEC group. The results showed that KDF increased the cell viability and the gene expression levels of SGLT3 and TGF-ß, while decreased the content of IL-1ß compared with the CON group. The cell viability and the gene expressions of SGLT1, SGLT3, GLUT2, Claudin-1, Occludin and TGF-ß, and the protein expression of ZO-1 in EHEC group were lower than those in CON group, whereas the gene expressions of IL-1ß, TNF, IL-8 and TLR4, and the level of phosphorylation NF-кB protein were increased. Pretreatment with KDF reduced the content of IgM and IL-1ß, the gene expressions of IL-1ß, TNF, IL-8 and TLR4 and the level of phosphorylation NF-кB protein, and increased the gene expression of TGF-ß and the protein expression of Occludin in IPEC-J2 cells infected EHEC. In conclusion, 0.125 mg/mL KDF on IPEC-J2 cells for 6 h had the beneficial effects on ameliorating the intestinal inflammation because of reduced pro-inflammatory cytokines and enhanced anti-inflammatory cytokines through regulating NF-кB signaling pathway under the EHEC challenge.

PI3K/Akt signaling pathway mediates the effect of low-dose boron on barrier function, proliferation and apoptosis in rat intestinal epithelial cells.

Boron is an essential trace element with roles in growth, development, and physiological functions; however, its mechanism of action is still unclear. In this study, the regulatory roles of the PI3K/Akt signaling pathway on boron-induced changes in barrier function, proliferation, and apoptosis in rat intestinal epithelial cells were evaluated. Occludin levels, the proportion of cells in the G2/M phase, cell proliferation rate, and mRNA and protein expression levels of PCNA were higher, while the proportions of cells in the G0/G1 and S phases, apoptosis rate, and caspase-3 mRNA and protein expression levels were lower in cells treated with 0.8 mmol/L boron than in control IEC-6 cells (P < 0.01 or P < 0.05). However, 40 mmol/L boron decreased ZO-1 and Occludin levels, the proportion of cells in the G2/M phase, cell proliferation rate, and mRNA and protein levels of PCNA and increased the apoptosis rate and caspase-3 mRNA expression (P < 0.01 or P < 0.05). After specifically blocking PI3K and Akt signals (using LY294002 and MK-2206 2HCL), 0.8 mmol/L boron had no effects on Occludin, PCNA level, apoptosis rates, and caspase-3 levels (P < 0.05); however, the proliferation rate and PCNA levels decreased significantly (P < 0.01 or P < 0.05). The addition of 40 mmol/L boron did not affect ZO-1 and Occludin levels and did not affect the apoptosis rate or PCNA and caspase-3 levels. These results suggested that the PI3K/Akt signaling pathway mediates the effects of low-dose boron on IEC-6 cells.

A critical review on intestinal mucosal barrier protection effects of dietary polysaccharides.

Studies have shown that dietary polysaccharides, which are widely present in natural foods, have an important impact on the intestinal mucosal barrier. Dietary polysaccharides can maintain the intestinal barrier function through multiple mechanisms. The intestinal barrier is composed of mechanical, chemical, immune, and biological barriers, and dietary polysaccharides, as a bioactive component, can promote and regulate these four barriers. Dietary polysaccharides can enhance the expression of tight junction proteins and mucins such as occludin-1 and zonula occludens-1 (ZO-1) between intestinal epithelial cells, inhibit inflammatory response and oxidative stress, increase the growth of beneficial bacteria, produce beneficial metabolites such as short chain fatty acids (SCFAs), and promote the proliferation and metabolism of immune cells. Given the critical role of the intestinal mucosal system in health and disease, the protective effects of dietary polysaccharides may be potentially valuable for the prevention and treatment of gut-related diseases. Therefore, it is of great significance to further study the mechanism and application prospects of the intestinal mucosal barrier derived from plant, animal, fungal and bacterial sources.

Torreya grandis Kernel Oil Alleviates Loperamide-Induced Slow Transit Constipation via Up-Regulating the Colonic Expressions of Occludin/Claudin-1/ZO-1 and 5-HT3R/5-HT4R in BALB/c Mice.

SCOPE: Torreya grandis kernel has traditionally been used to remove intestinal parasites and increases intestinal motility. However, the effect of Torreya grandis kernel oil (TKO) on constipation has not yet been investigated. Therefore, mouse model is used to investigate the effect of TKO on slow transit constipation (STC) and its possible mechanism. METHODS AND RESULTS: The effects of TKO on intestinal motility of STC mice are evaluated by fecal weight, fecal water content, colon length, defecation test, and intestinal propulsion test. The mechanism of TKO alleviating STC is explored by detecting biochemical analysis, histological analysis, western blot, qRT-PCR, immunohistochemistry, and gut microbiota analysis. The results reveal that TKO effectively promotes defecation and intestinal motility, increases the level of endothelin-1, and restores the histopathological morphology of the colon under LOP pretreatment. The expression levels of occludin, claudin-1, and zonula occludens-1 (ZO-1) mRNA and protein are up-regulated in mice receiving TKO treatment. The colonic 5-hydroxytryptamine 3R/4R (5-HT3R/5-HT4R) expressions are also increased by TKO supplementation. Additionally, TKO rescues LOP-caused disorders of the gut microbiota. CONCLUSION: Consumption of TKO is beneficial to STC recovery, and it can alleviate LOP-induced STC by up-regulating the colonic expressions of Occludin/Claudin-1/ZO-1 and 5-HT3R/5-HT4R.

Occludin is overexpressed in tubo-ovarian high-grade serous carcinoma compared to mesothelioma and is a marker of tumor progression and chemoresistance.

The objective of this study was to analyze the expression and prognostic role of the tight junction protein occludin in high-grade serous carcinoma (HGSC). Occludin protein expression by immunohistochemistry was analyzed in 602 HGSC (417 effusions, 185 surgical specimens). Expression in mesothelioma (n = 87; 45 effusions, 42 surgical specimens) was studied for comparative purposes. Occludin protein expression was found in 587/602 (98%) HGSC vs. 40/87 (46%) mesotheliomas and was predominantly limited to < 5% of cells in the latter (p < 0.001). Occludin was additionally overexpressed in HGSC effusions compared to surgical specimens (p < 0.001) and was overexpressed in post-chemotherapy effusions compared to chemo-naive effusions tapped at diagnosis (p = 0.015). Occludin expression in HGSC surgical specimens was associated with poor chemoresponse (p < 0.001) and primary resistance (p = 0.001). Expression in effusions and surgical specimens was unrelated to survival (p > 0.05). In conclusion, occludin expression is higher in HGSC compared to mesothelioma, and this protein is overexpressed in HGSC effusions, possibly reflecting changes in adhesion related to anchorage-independent growth in this microenvironment. Overexpression in post-chemotherapy compared to chemo-naïve effusions suggest a role in disease progression. Occludin expression in surgical specimens may be related to chemoresistance.

Aspirin Inhibits Brain Metastasis of Lung Cancer via Upregulation of Tight Junction Protein Expression in Microvascular Endothelial Cells.

BACKGROUND: The brain is one of the most vulnerable metastasis sites in lung cancer; approximately 40-50% of lung cancer patients develop brain metastasis during the disease course, contributing to the poor prognosis and high mortality of lung cancer patients. Therefore, it is important to clarify the molecular mechanism underlying brain metastasis of lung cancer for improving the overall survival of lung cancer patients. The present study aimed to investigate the potential role of blood-brain barrier (BBB) permeability in the development of brain metastasis of lung cancer and explore the effect of aspirin in an in-vitro BBB model. METHODS: An in-vitro BBB model was established. The expression of heat shock protein 70 (HSP 70), zonula occludens-1 (ZO-1), and occludin in rat brain microvascular endothelial cells was detected using Western blot at different time points following the administration of aspirin. RESULTS: HSP70, ZO-1, and occludin expressions did not show significant changes before aspirin administration, but increased noticeably after aspirin administration. Tumor necrosis factor-α (TNF-α) could significantly attenuate the increased expression of these proteins induced by aspirin. Additionally, TNF-α also significantly reversed the aspirin-induced decrease of BBB permeability. CONCLUSIONS: Aspirin may inhibit brain metastasis of lung cancer in a time-dependent manner via upregulating tight junction proteins to reduce BBB permeability, and this effect can be reversed by TNF-α.

Trichinella spiralis cathepsin L damages the tight junctions of intestinal epithelial cells and mediates larval invasion.

BACKGROUND: Cathepsin L, a lysosomal enzyme, participates in diverse physiological processes. Recombinant Trichinella spiralis cathepsin L domains (rTsCatL2) exhibited natural cysteine protease activity and hydrolyzed host immunoglobulin and extracellular matrix proteins in vitro, but its functions in larval invasion are unknown. The aim of this study was to explore its functions in T. spiralis invasion of the host's intestinal epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: RNAi significantly suppressed the expression of TsCatL mRNA and protein with TsCatL specific siRNA-302. T. spiralis larval invasion of Caco-2 cells was reduced by 39.87% and 38.36%, respectively, when anti-TsCatL2 serum and siRNA-302 were used. Mice challenged with siRNA-302-treated muscle larvae (ML) exhibited a substantial reduction in intestinal infective larvae, adult worm, and ML burden compared to the PBS group, with reductions of 44.37%, 47.57%, and 57.06%, respectively. The development and fecundity of the females from the mice infected with siRNA-302-treated ML was significantly inhibited. After incubation of rTsCatL2 with Caco-2 cells, immunofluorescence test showed that the rTsCatL2 gradually entered into the cells, altered the localization of cellular tight junction proteins (claudin 1, occludin and zo-1), adhesion junction protein (e-cadherin) and extracellular matrix protein (laminin), and intercellular junctions were lost. Western blot showed a 58.65% reduction in claudin 1 expression in Caco-2 cells treated with rTsCatL2. Co-IP showed that rTsCatL2 interacted with laminin and collagen I but not with claudin 1, e-cadherin, occludin and fibronectin in Caco-2 cells. Moreover, rTsCatL2 disrupted the intestinal epithelial barrier by inducing cellular autophagy. CONCLUSIONS: rTsCatL2 disrupts the intestinal epithelial barrier and facilitates T. spiralis larval invasion.

Upregulation of occludin by cytolethal distending toxin facilitates Glaesserella parasuis adhesion to respiratory tract cells.

Virulent Glaesserella parasuis may engender systemic infection characterized by fibrinous polyserositis and pneumonia. G. parasuis causes systemic disease through upper respiratory tract infection, but the mechanism has not been fully characterized. Tight junction (TJ) proteins maintain the integrity and impermeability of the epithelial barriers. In this work, we applied the recombinant cytolethal distending toxin (CDT) holotoxin and cdt-deficient mutants to assess whether CDT interacted with TJ proteins of airway tract cells. Our results indicated that CDT induced the TJ occludin (OCLN) expression in newborn pig tracheal epithelial cells within the first 3 hours of bacterial infection, followed by a significant decrease. Overexpression of OCLN in target cells made them more susceptible to G. parasuis adhesion, whereas ablation of OCLN expression by CRISPR/Cas 9 gene editing technology in target cells decreased their susceptibility to bacterial adhesion. In addition, CDT treatment could upregulate the OCLN levels in the lung tissue of C57/BL6 mice. In summary, highly virulent G. parasuis strain SC1401 stimulated the tight junction expression, resulting in higher bacterial adhesion to respiratory tract cells, and this process is closely related to CDT. Our results may provide novel insights into G. parasuis infection and CDT-mediated pathogenesis.

PHD3 inhibits colon cancer cell metastasis through the occludin-p38 pathway.

Prolyl hydroxylase 3 (PHD3) hydroxylates HIFα in the presence of oxygen, leading to HIFα degradation. PHD3 inhibits tumorigenesis. However, the underlying mechanism is not well understood. Herein, we demonstrate that PHD3 inhibits the metastasis of colon cancer cells through the occludin-p38 MAPK pathway independent of its hydroxylase activity. We find that PHD3 inhibits colon cancer cell metastasis in the presence of the PHD inhibitor DMOG, and prolyl hydroxylase-deficient PHD3(H196A) suppresses cell metastasis as well. PHD3 controls the stability of the tight junction protein occludin in a hydroxylase-independent manner. We further find that PHD3-inhibited colon cancer cell metastasis is rescued by knockdown of occludin and that occludin acts as a negative regulator of cell metastasis, implying that PHD3 suppresses metastasis through occludin. Furthermore, knockdown of occludin induces phosphorylation of p38 MAPK, and the p38 inhibitor SB203580 impedes cell migration and invasion induced by occludin knockdown, indicating that occludin functions through p38. Moreover, knockdown of occludin enhances the expression of MKK3/6, the upstream kinase of p38, while overexpression of occludin decreases its expression. Our results suggest that PHD3 inhibits the metastasis of colon cancer cells through the occludin-p38 pathway independent of its hydroxylase activity. These findings reveal a previously undiscovered mechanism underlying the regulation of cancer cell metastasis by PHD3 and highlight a noncanonical hydroxylase-independent function of PHD3 in the suppression of cancer cells.

Colitis induced by PD-1 inhibitor combined with platinum-containing dual drug chemotherapy in Lewis mice and its mechanism.

Aims: To explore the occurrence and possible mechanism of colitis in Lewis mice treated with PD-1 inhibitor combined with platinum-containing dual drug chemotherapy. Subjects and Methods: A Lewis lung cancer model of C57BL/6 mice was established, randomly divided into the treatment group (group C, PD-1 inhibitor + Carboplatin (CARB) + Pemetrexed (PEM)) and model group (group B, normal saline), and a control group (group A, normal saline) was set up. Observe the changes in tumor-free weight, tumor volume, disease activity index (DAI), colon histopathology, identify serum interleukin (IL)-10, interferon (IFN)-γ, the expression of claudin-1, and occludin mRNA in the colon in each animals. Results: Compared with group A, the tumor-free weight of mice in B decreased (P < 0.001), the content of IL-10 in serum increased (P < 0.01), the content of IFN-γ in serum decreased (P < 0.01). Compared with group B, the transplanted tumor volume in C was reduced (P < 0.05), DAI scores of D4 (P < 0.001), and D7 (P < 0.001) were increased, colonic histopathology analysis showed that colitis occurred, serum IL-10 content was decreased (P < 0.05), IFN-γ content was increased (P < 0.05), and the mRNA expression of claudin-1 (P < 0.05) and occludin (P < 0.05) was reduced. Conclusions: This treatment can inhibit the growth of transplanted tumors but will cause colitis in Lewis mice. The impairment of intestinal barrier function following administration cause an imbalance in the expression of pro-inflammatory and anti-inflammatory factors in the colon, thus causing colitis.

Regulation and function of adiponectin in the intestinal epithelial cells in response to Trichinella spiralis infection.

Besides metabolic homeostasis regulation, adipokines are recently emerged as important players in regulating immunity and inflammation. Helminth infection has known to modulate circulating adipokine secretion; however, the regulation and function of adipokines in response to helminth infection is still unclear. Here, we investigated the regulation and function of adiponectin during T. spiralis infection. While there was no change in circulating level of adiponectin, we found an increased adiponectin, but not leptin expression in the small intestine. Interestingly, the intestinal adiponectin expression was strongly associated with the expression of epithelial cell-derived cytokines IL-25, IL-33, and TSLP following infection. Indeed, mice deficiency of IL-25 receptor exhibited no intestinal adiponectin induction upon helminth infection. Interestingly, IL-25-induced adiponectin modulated intestinal epithelial cell responses by enhancing occludin and CCL17 expression. Using LPS-induced intestinal epithelial barrier dysfunctions in a Caco-2 cell monolayer model, adiponectin pretreatment enhanced a Transepithelial electrical resistance (TEER) and occludin expression. More importantly, adiponectin pretreatment of Caco2 cells prevented T. spiralis larval invasion in vitro and its administration during infection enhanced intestinal IL-13 secretion and worm expulsion in vivo. Altogether, our data suggest that intestinal adiponectin expression induced by helminth infection through the regulation of IL-25 promotes worm clearance and intestinal barrier function.

Bortezomib Increased Vascular Permeability by Decreasing Cell-Cell Junction Molecules in Human Pulmonary Microvascular Endothelial Cells.

Bortezomib (BTZ), a chemotherapeutic drug used to treat multiple myeloma, induces life-threatening side effects, including severe pulmonary toxicity. However, the mechanisms underlying these effects remain unclear. The objectives of this study were to (1) investigate whether BTZ influences vascular permeability and (2) clarify the effect of BTZ on the expression of molecules associated with cell-cell junctions using human pulmonary microvascular endothelial cells in vitro. Clinically relevant concentrations of BTZ induced limited cytotoxicity and increased the permeability of human pulmonary microvascular endothelial cell monolayers. BTZ decreased the protein expression of claudin-5, occludin, and VE-cadherin but not that of ZO-1 and ß-catenin. Additionally, BTZ decreased the mRNA expression of claudin-5, occludin, ZO-1, VE-cadherin, and ß-catenin. Our results suggest that BTZ increases the vascular permeability of the pulmonary microvascular endothelium by downregulating cell-cell junction molecules, particularly claudin-5, occludin, and VE-cadherin.

Hepatitis C Virus Disrupts Annexin 5-Mediated Occludin Integrity through Downregulation of Protein Kinase Cα (PKCα) and PKCη Expression, Thereby Promoting Viral Propagation.

Annexins (ANXs) comprise a family of calcium- and phospholipid-binding proteins and are implicated in the hepatitis C virus (HCV) life cycle. Here, we demonstrate a novel role of ANX5 in the HCV life cycle. Comparative analysis by quantitative PCR in human hepatoma cells revealed that ANX2, ANX4, and ANX5 were highly expressed among the ANX family proteins. Knockdown of ANX5 mRNA resulted in marked enhancement of HCV RNA replication but had no effect on either HCV translation or assembly. Using the HCV pseudoparticle (HCVpp) system, we observed enhancement of HCVpp infectivity in ANX5 knockdown Huh-7OK1 cells, suggesting that ANX5 is involved in suppression of HCV entry. Additionally, we observed that subcellular localizations of tight-junction proteins, such as claudin 1 (CLDN1) and occludin (OCLN), were disrupted in the ANX5 knockdown cells. It was reported that HCV infection was facilitated by disruption of OCLN distribution and that proper distribution of OCLN was regulated by its phosphorylation. Knockdown of ANX5 resulted in a decrease of OCLN phosphorylation, thereby disrupting OCLN distribution and HCV infection. Further analysis revealed that protein kinase C (PKC) isoforms, including PKCα and PKCη, play important roles in the regulation of ANX5-mediated phosphorylation and distribution of OCLN and in the restriction of HCV infection. HCV infection reduced OCLN phosphorylation through the downregulation of PKCα and PKCη expression. Taken together, these results suggest that ANX5, PKCα, and PKCη contribute to restriction of HCV infection by regulating OCLN integrity. We propose a model that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting HCV propagation. IMPORTANCE Host cells have evolved host defense machinery to restrict viral infection. However, viruses have evolved counteracting strategies to achieve their infection. In the present study, we obtained results suggesting that ANX5 and PKC isoforms, including PKCα and PKCη, contribute to suppression of HCV infection by regulating the integrity of OCLN. The disruption of OCLN integrity increased HCV infection. We also found that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting viral infection. We propose that HCV disrupts ANX5-mediated OCLN integrity to establish a persistent infection. The disruption of tight-junction assembly may play important roles in the progression of HCV-related liver diseases.

Diosmetin Maintains Barrier Integrity by Reducing the Expression of ABCG2 in Colonic Epithelial Cells.

Crohn's disease (CD) is a relapsing and chronic inflammatory bowel disease. Recent advances have highlighted that dysfunction of the barrier function formed by a polarized monolayer of columnar epithelial cells plays a crucial role in the pathophysiology of CD. At present, we reported that diosmetin increased cell viability by reducing the levels of TNFα and IL-6 in lipopolysaccharide (LPS)-treated colonic epithelial Caco-2 cells. Meanwhile, diosmetin conferred a direct effect on maintaining barrier integrity by reducing epithelial permeability and increasing the expression of proteins associated with tight junctions, including zonula occludens-l (ZO-1), occludin, and claudin-1, in LPS-treated Caco-2 cells and in 2,4,6-trinitrobenzene sulfonic acid-induced CD mice. Additionally, diosmetin decreased the protein content of adenosine triphosphate-binding cassette efflux transporter G2 (ABCG2) in vitro and in vivo. Over-expression of ABCG2 had an important impact on the epithelial permeability and barrier-related protein levels induced by LPS in Caco-2 cells. At the same time, Ko143, a specific ABCG2 inhibitor, dramatically enhanced the role of diosmetin in ZO-1 and occludin proteins in LPS-treated Caco-2 cells. Mechanically, diosmetin significantly attenuated the role of LPS in the phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), phosphatidylinositol-3-kinase (PI3K)/protein kinase B (PKB/AKT), and cAMP-response element binding protein (CREB) in Caco-2 cells. The AMPK inhibitor Compound C obviously prevented the effect of diosmetin on ZO-1 and occludin expression in LPS-treated Caco-2 cells. Taken together, the results of this study suggest that AMPK/AKT/CREB-mediated ABCG2 expression plays a crucial role in diosmetin, improving the barrier dysfunction in CD.

Preventing occludin tight-junction disruption via inhibition of microRNA-193b-5p attenuates viral load and influenza-induced lung injury.

Virus-induced lung injury is associated with loss of pulmonary epithelial-endothelial tight junction integrity. While the alveolar-capillary membrane may be an indirect target of injury, viruses may interact directly and/or indirectly with miRs to augment their replication potential and evade the host antiviral defense system. Here, we expose how the influenza virus (H1N1) capitalizes on host-derived interferon-induced, microRNA (miR)-193b-5p to target occludin and compromise antiviral defenses. Lung biopsies from patients infected with H1N1 revealed increased miR-193b-5p levels, marked reduction in occludin protein, and disruption of the alveolar-capillary barrier. In C57BL/6 mice, the expression of miR-193b-5p increased, and occludin decreased, 5-6 days post-infection with influenza (PR8). Inhibition of miR-193b-5p in primary human bronchial, pulmonary microvascular, and nasal epithelial cells enhanced antiviral responses. miR-193b-deficient mice were resistant to PR8. Knockdown of occludin, both in vitro and in vivo, and overexpression of miR-193b-5p reconstituted susceptibility to viral infection. miR-193b-5p inhibitor mitigated loss of occludin, improved viral clearance, reduced lung edema, and augmented survival in infected mice. Our results elucidate how the innate immune system may be exploited by the influenza virus and how strategies that prevent loss of occludin and preserve tight junction function may limit susceptibility to virus-induced lung injury.

Limosilactobacillus johnsoni and Limosilactobacillus mucosae and Their Extracellular Vesicles Alleviate Gut Inflammatory Injury by Mediating Macrophage Polarization in a Lipopolysaccharide-Challenged Piglet Model.

BACKGROUND: Limosilactobacillus johnsoni (L. j) and Limosilactobacillus mucosae (L. m) can alleviate the inflammatory response. OBJECTIVES: This study aimed to elucidate the underlying mechanisms by which L. j- and L. m-derived extracellular vesicles (EVs) mitigate lipopolysaccharide (LPS)-induced intestinal injury. METHODS: Piglets were assigned to 4 groups: oral phosphate-buffered saline inoculation for 2 wk prior to intraperitoneal injection of physiological saline or LPS, and oral L. j/L. m inoculation for 2 wk prior to intraperitoneal injection of LPS. The intestinal integrity, macrophage markers, cytokine levels, and microbiota were determined. The cytokine levels and macrophage phenotype were detected after L. j/L. m and their EVs were coincubated with macrophages. The levels of cytokines, tight junction proteins, and apoptosis were measured after intestinal epithelial cells were cocultured with macrophages. RESULTS: LPS challenge decreased jejunal villus length; expression levels of zonula occludens-1 (ZO-1), occludin, arginase-1 (Arg1), and interleukin (IL)-10; and number of CD163+ cells and increased the expression levels of inducible nitric oxide synthase (iNOS), IL-1ß, IL-6, and tumor necrosis factor (TNF)-α compared with that in the control. L. j and L. m pretreatment rescued the aforementioned indicators compared with LPS challenge. Pretreatment of L. j and L. m and their EVs reversed the levels of IL-1ß, IL-6, TNF-α, and IL-10 and the gene expression of iNOS and Arg1 in the LPS group in macrophages. Pretreatment with L. j and L. m-derived EVs increased ZO-1 and occludin mRNA expression and reduced IL-1ß, caspase-3, and bax gene expression in intestinal epithelial cells of the coculture system. Enzyme-treated EVs were less effective than native EVs. CONCLUSIONS: This study suggests that EVs secreted by L. j and L. m control inflammation by modulating macrophage polarization, thereby improving intestinal barrier function.