ZO-1 Protein Expression Research Articles

Quercetin Attenuates the Combined Effects of Zearalenone and Lipopolysaccharide on IPEC-J2 Cell Injury through Activating the Nrf2 Signaling Pathway.

Zearalenone (ZEA) is a mycotoxin with an estrogen-like effect that is widely found in feed. Lipopolysaccharides (LPS) derived from Gram-negative bacteria are a common endotoxin, and both toxins have effects on human and livestock health. During animal feeding, ZEA as an exotoxin and LPS as an endotoxin have the potential to co-exist in organisms. At present, other studies have only focused on the hazards of single toxins, but there are fewer studies on the coexistence and interaction between ZEA and LPS. Therefore, a further study to investigate the combined toxic effects of different concentrations of ZEA and LPS is warranted. Quercetin (QUE) is a natural flavonoid compound with strong antioxidant and anti-inflammatory properties. It is unclear whether QUE can mitigate the combined effects of ZEA and LPS. IPEC-J2, isolated from the jejunum of non-breastfed neonatal piglets, is an ideal model for the study of epithelial cell transport, intestinal bacterial interactions, and the nutrient modulation of intestinal function. Therefore, the purpose of the present study was to demonstrate the effect of QUE in alleviating the combined toxic effect of ZEA and LPS on IPEC-J2 cell damage. Cell viability was measured after treating IPEC-J2 cells sequentially with 10, 20, 30, 40, 60, 80, and 100 μM ZEA, 1, 10, 50, and 100 μg/mL LPS, and 20, 40, 60, 80, 100, and 200 μM QUE for 24 h. Based on the cell viability results, 20 μM ZEA and 1 μg/mL LPS were selected as the most suitable concentrations for further analysis. For QUE, 20 μM increased the cell viability, while 40-200 μM QUE decreased the cell viability. Therefore, for the subsequent study, 20 μM QUE was selected in combination with 20 μM ZEA and 1 μg/mL LPS. The results showed that QUE increased the cellular viability and decreased the LDH content more compared to the effects of the ZEA+LPS group. At the gene level, QUE addition up-regulated the expression of Nrf2, HO-1, SOD2, and NQO1 at the gene or protein level compared to those of the ZEA+LPS group. The measurement of tight junction-related genes and proteins showed QUE up-regulated the expression of Claudin, ZO-1, and Occludin genes and proteins more than in the ZEA+LPS group. QUE addition reduced the rate of apoptosis more than that in the ZEA+LPS group. The expressions of Bcl-2 and Bax were examined at the gene level, and QUE addition significantly reduced the Bax gene expression level compared to that of the ZEA+LPS group, but there was no apparent variation in the expression level of Bcl-2. In summary, QUE can alleviate the combined toxic effects of ZEA and LPS on IPEC-J2 cells via modulating the Nrf2 signaling pathway, up-regulating the expression of antioxidative genes, and enhancing the intestinal barrier.

Agaricus blazei Polysaccharide Alleviates DSS-Induced Colitis in Mice by Modulating Intestinal Barrier and Remodeling Metabolism

Ulcerative colitis (UC) is a chronic noninfectious intestinal disease that severely affects patients’ quality of life. Agaricus blazei Murrill polysaccharide (ABP) is an effective active ingredient extracted from Agaricus blazei Murrill (ABM). It has good efficacy in inhibiting tumor cell growth, lowering blood pressure, and improving atherosclerosis. However, its effect on colitis is unclear. The aim of this study was to analyze the protective effects and potential mechanisms of ABP against dextran sulfate sodium (DSS)-induced acute colitis in mice. The results showed that dietary supplementation with ABP significantly alleviated DSS-induced colitis symptoms, inflammatory responses, and oxidative stress. Meanwhile, ABP intervention was able to maintain the integrity of the intestinal mechanical barrier by promoting the expression of ZO-1 and Occludin tight junction proteins and facilitating mucus secretion. Moreover, 16S rRNA sequencing results suggested that ABP intervention was able to alleviate DSS-induced gut microbiota disruption, and nontargeted metabolomics results indicated that ABP was able to remodel metabolism. In conclusion, these results demonstrate that dietary supplementation with ABP alleviated DSS-induced acute colitis by maintaining intestinal barrier integrity and remodeling metabolism. These results improve our understanding of ABP function and provide a theoretical basis for the use of dietary supplementation with ABP for the prevention of ulcerative colitis.

Abstract 12848: Edaravone Dexborneol Protects Against Cerebral Ischemia/Reperfusion -Induced Blood-Brain Barrier Dysfunction by Inhibiting Ferroptosis via Activation of Nrf-2/HO-1/GPX4 Signaling

Background and aims: The blood-brain barrier (BBB) is a pivotal factor in the pathogenesis of cerebral ischemia-reperfusion (I/R) injury. Edaravone dexborneol (Eda.B) is a novel cytoprotective drug widely employed in ischemic stroke (IS). Recent research implicated ferroptosis in BBB disruption during cerebral ischemia. This study aimed to investigate the protective effects of Eda.B on the BBB in I/R from the standpoint of ferroptosis, exploring potential mechanisms and targets. Methods: An I/R model in Sprague-Dawley rats was established, and we assessed the effects of Eda.B by evaluating infarct volume, cellular apoptosis, and neurofunctional recovery through TTCstaining, TUNEL staining, and modified Garcia scoring system, respectively. The protective effects of Eda.B on BBB integrity was evaluated via Evans blue leakage measurement and protein expression of both ZO-1 and occludin. Additionally, we explored the correlation between ferroptosis and BBB integrity. Western blot analysis and immunohistochemical detection were used to detect changes in the expression levels of related proteins.Meanwhile the information of microscopic structural changes observation, GSH and MDA level measurements, and Fe 2+ tissue content determination were recorded. Bioinformatics analysis was also performed to probe the mechanisms of ferroptosis in BBB injury induced by cerebral ischemia, and we detected the expression of related proteins in the Nrf-2/HO-1 signaling pathway through western blot analysis. Results: Our findings revealed that Eda.B treatment enhanced neuro scores, diminished infarct volumes and cellular apoptosis, while ameliorated BBB integrity. Furthermore, expression of ZO-1, occludin, GPX4, and xCT protein expression was up-regulated, while 4-HNE expression was reduced in the Eda.B group. Eda.B also led to decreased MDA and Fe 2+ levels in the ipsilateral brain tissues, while GSH content was increased. Ultimately, an elevated expression of proteins associated with the Nrf-2/HO-1 signaling pathway has been discovered. Conclusions: Our study indicated that Eda.B safeguarded the blood-brain barrier (BBB) from cerebral I/R injury by inhibiting ferroptosis through the activation of the Nrf-2/HO-1/Gpx4 signaling pathway.

Apple Polyphenol Extract Ameliorates Atherosclerosis and Associated Cognitive Impairment through Alleviating Neuroinflammation by Weakening TLR4 Signaling and NLRP3 Inflammasome in High-Fat/Cholesterol Diet-Fed LDLR-/- Male Mice.

Although studies have supported the beneficial effects of the ingredients of apple polyphenol extract (APE), a polyphenol mixture being extracted from whole fresh apples, on neurodegenerative diseases, the role of APE in atherosclerosis-related cognitive impairment remains unclear. To clarify the role of APE in regulating cognitive dysfunction in mice with atherosclerosis and the underlying mechanisms, high-fat/cholesterol diet-fed male LDLR-/- mice were gavaged with 125 or 500 mg/(kg·bw·d) APE solution or sterile double-distilled water for consecutive 8 weeks, and age-matched C57BL/6 male mice were employed as normal control. APE intervention increased the serum concentration of high-density apolipoprotein cholesterol, improved atherosclerosis, and ameliorated cognitive function of mice by inhibiting the phosphorylation of tau protein, supporting with significantly reduced platform latency and obviously increased swimming distance in the target quadrant according to the Morris water maze test. APE intervention alleviated neuroinflammation by attenuating the activation of microglia and astrocytes and inhibiting TLR4 signaling with reduced protein expression of NF-κB, MyD88, TRIF, and IKKβ. Meanwhile, APE intervention inactivated NLRP3 inflammasome with downregulated protein expression of caspase-1, IL-18, and IL-1β. Additionally, APE intervention improved the damaged brain barrier structure by upregulating the protein expression of ZO-1 and occludin. Therefore, our research supplemented new data, supporting the potential of APE as an effective dietary bioactive ingredient to improve atherosclerosis and associated cognitive impairment.

Effects of Enteromorpha prolifera polysaccharides on growth performance, intestinal barrier function and cecal microbiota in yellow-feathered broilers under heat stress

BackgroundGlobal warming leading to heat stress (HS) is becoming a major challenge for broiler production. This study aimed to explore the protective effects of seaweed (Enteromorpha prolifera) polysaccharides (EPS) on the intestinal barrier function, microbial ecology, and performance of broilers under HS. A total of 144 yellow-feathered broilers (male, 56 days old) with 682.59 ± 7.38 g were randomly assigned to 3 groups: 1) TN (thermal neutral zone, 23.6 ± 1.8 °C), 2) HS (heat stress, 33.2 ± 1.5 °C for 10 h/d), and 3) HSE (HS + 0.1% EPS). Each group contained 6 replicates with 8 broilers per replicate. The study was conducted for 4 weeks; feed intake and body weights were measured at the end of weeks 2 and 4. At the end of the feeding trial, small intestine samples were collected for histomorphology, antioxidant, secretory immunoglobulin A (sIgA) content, apoptosis, gene and protein expression analysis; cecal contents were also collected for microbiota analysis based on 16S rDNA sequencing.ResultsDietary EPS promoted the average daily gain (ADG) of broilers during 3–4 weeks of HS (P < 0.05). At the end of HS on broilers, the activity of total superoxide dismutase (T-SOD), glutathione S-transferase (GST), and the content of sIgA in jejunum were improved by EPS supplementation (P < 0.05). Besides, dietary EPS reduced the epithelial cell apoptosis of jejunum and ileum in heat-stressed broilers (P < 0.05). Addition of EPS in HS group broilers' diet upregulated the relative mRNA expression of Occludin, ZO-1, γ-GCLc and IL-10 of the jejunum (P < 0.05), whereas downregulated the relative mRNA expression of NF-κB p65, TNF-α and IL-1β of the jejunum (P < 0.05). Dietary EPS increased the protein expression of Occludin and ZO-1, whereas it reduced the protein expression of NF-κB p65 and MLCK (P < 0.01) and tended to decrease the protein expression of TNF-α (P = 0.094) in heat-stressed broilers. Furthermore, the proportions of Bacteroides and Oscillospira among the three groups were positively associated with jejunal apoptosis and pro-inflammatory cytokine expression (P < 0.05) and negatively correlated with jejunal Occludin level (P < 0.05). However, the proportions of Lactobacillus, Barnesiella, Subdoligranulum, Megasphaera, Collinsella, and Blautia among the three groups were positively related to ADG (P < 0.05).ConclusionsEPS can be used as a feed additive in yellow-feathered broilers. It effectively improves growth performance and alleviates HS-induced intestinal injury by relieving inflammatory damage and improving the tight junction proteins expression. These beneficial effects may be related to inhibiting NF-κB/MLCK signaling pathway activation and regulation of cecal microbiota.

Fecal microbiota transplantation from HUC-MSC-treated mice alleviates acute lung injury in mice through anti-inflammation and gut microbiota modulation.

Acute lung injury (ALI) is a severe respiratory tract disorder facilitated by dysregulated inflammation, oxidative stress and intestinal ecosystem. Fecal microbiota transplantation (FMT) is a rapid method for gut microbiota (GM) reconstruction. Furthermore, our previous studies have confirmed that human umbilical cord mesenchymal stromal cells (HUC-MSCs) can alleviate ALI by improving GM composition. Therefore, we aimed to explore the efficacy and mechanism of FMT from HUC-MSCs-treated mice on ALI. In brief, fresh feces from HUC-MSCs-treated mice were collected for FMT, and the mice were randomly assigned into NC, FMT, LPS, ABX-LPS, and ABX-LPS-FMT groups (n = 12/group). Subsequently, the mice were administrated with antibiotic mixtures to deplete GM, and given lipopolysaccharide and FMT to induce ALI and rebuild GM. Next, the therapeutic effect was evaluated by bronchoalveolar lavage fluid (BALF) and histopathology. Immune cells in peripheral blood and apoptosis in lung tissues were measured. Furthermore, oxidative stress- and inflammation-related parameter levels were tested in BALF, serum, lung and ileal tissues. The expressions of apoptosis-associated, TLR4/NF-κB pathway-associated, Nrf2/HO-1 pathway related and tightly linked proteins in the lung and ileal tissues were assessed. Moreover, 16S rRNA was conducted to assess GM composition and distribution. Our results revealed that FMT obviously improved the pathological damage of lung and ileum, recovered the immune system of peripheral blood, decreased the cell apoptosis of lung, and inhibited inflammation and oxidative stress in BALF, serum, lung and ileum tissues. Moreover, FMT also elevated ZO-1, claudin-1, and occludin protein expressions, activating the Nrf2/HO-1 pathway but hindering the TLR4/NF-κB pathway. Of note, the relative abundances of Bacteroides, Christensenella, Coprococcus, and Roseburia were decreased, while the relative abundances of Xenorhabdus, Sutterella, and Acinetobacter were increased in the ABX-LPS-FMT group. FMT from HUC-MSCs-treated mice may alleviate ALI by inhibiting inflammation and reconstructing GM, additionally, we also found that the TLR4/NF-κB and Nrf2/HO-1 pathways may involve in the improvement of FMT on ALI, which offers novel insights for the functions and mechanisms of FMT from HUC-MSCs-treated mice on ALI.

Trans-Anethole Alleviates DSS-Induced Ulcerative Colitis by Remodeling the Intestinal Flora to Regulate Immunity and Bile Acid Metabolism.

Ulcerative colitis (UC) is the most common inflammatory bowel disease (IBD); it is incurable, and the treatment is expensive. Trans-anethole (TA), the main component of fennel, exhibits various biological activities. An increasing number of studies have demonstrated the efficacy of herbal active ingredients in the treatment of UC. This study aimed to investigate the effect and mechanism of TA in UC. In this study, we have experimented on mice with dextran sulfate sodium salt (DSS)-induced UC. The TA group was gavaged with 62.5 mg/kg TA by gavage once daily on days 8-14. To observe the effect of TA on the colon tissue, various investigations were performed, including western blot and immunohistochemistry for intestinal barrier protein expression, TUNEL staining for apoptosis, western blot, and ELISA for inflammation level, flow cytometry for Th17/Treg, LC-MS for blood bile acid content, GC-MS for blood fatty acid content, and 16s RNA for intestinal contents. TA alleviated weight loss in mice with UC; increased colon length; alleviated intestinal mucosal damage; upregulated claudin-1, occludin, and ZO-1 protein expression levels; reduced inflammatory factors in the colon and serum; and alleviated apoptosis. TA reduced fatty acid and bile acid levels by inhibiting colony abundance and reducing Th17/Treg cell differentiation in the colon. We found that TA alleviates DSS-induced UC by remodeling the intestinal flora to regulate immunity and bile acid metabolism.

Effect of TFF3 on tight junction protein in eosinophilic chronic sinusitis and its related mechanism

Objective: To study the effect of trefoil factor family (TFF) 3 on the expression of tight junctions (TJs) in the nasal mucosa epithelium of eosinophilic chronic rhinosinusitis (eCRS) and its mechanism. Methods: From September to December 2020, eligible patients from the Department of Otorhinolaryngology of the First Affiliated Hospital of Nanchang University were recruited, including 11 control patients and 37 patients with chronic rhinosinusitis with nasal polyps (CRSwNP), from whom nasal mucosa and nasal polyp tissue samples were collected. Immunohistochemistry (IHC) was used to detect the localization and expression intensity of TFFs (TFF1, TFF2 and TFF3) and TJs (occudin, claudin-1 and ZO-1) in nasal mucosa. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and western blot (WB) were used to detect the mRNA and protein expression. A cell model of tight junction injury in human nasal epithelial cells (HNECs) through stimulation with interleukin (IL)-13 was also established. The optimal modeling concentration and time for HNECs were determined, which were subsequently treated with TFF3 and/or a phosphoinositide 3-kinase (PI3K)-specific inhibitor (LY294002). Finally, RT-qPCR and WB were used to assess the effects of TFF3 on tight junctions and the PI3K/serine/threonine kinase (Akt) signaling pathway. Data were analyzed statistically using GraphPad Prism 7 software. Results: IHC results showed that the expression of TFF1 and TFF3 in nasal mucosa of eCRS group was significantly higher than that of control group (t=4.62, P=0.002; t=5.89, P<0.001), respectively, mainly expressed in goblet cell. The expression of occludin, claudin-1 and ZO-1 in the nasal mucosa of the eCRS group was lower than that of the control group (occludin t=3.98, P=0.019; claudin-1 t=5.15, P=0.002; ZO-1 t=5.42, P=0.001), respectively. WB results showed that the expression of TFF3 in non-eosinophilic chronic sinusitis (Non-eCRS) group and eCRS group was higher than that in the control group (t=3.62, P=0.036; t=5.93, P<0.001). The expression of occludin, claudin-1 and ZO-1 in eCRS group was lower than that in the control group (occludin t=5.14, P=0.002; claudin-1 t=6.35, P<0.001; ZO-1 t=6.64, P<0.001), respectively. The RT-qPCR results showed that compared with the control group, the levels of TFF1 and TFF3 mRNA were increased in the nasal mucosal epithelium of the Non-eCRS and eCRS groups (TFF1 t=3.98, P=0.046, t=4.89, P=0.002; TFF3 t=3.50, P=0.044, t=6.78, P<0.001). There was no statistically significant difference in TFF2 mRNA levels between the Non-eCRS and eCRS groups (t=1.34, P=0.061; t=3.37, P=0.055). Compared with the control group, Non-eCRS and eCRS groups showed a decrease in the mRNA levels of occludin, claudin-1 and ZO-1 (occludin t=4.27, P=0.011, t=5.61, P=0.007; claudin-1 t=3.62, P=0.036, t=6.80, P<0.001; ZO-1 t=3.47, P=0.047, t=7.86, P<0.001). The mRNA levels of TFF3 and TJs in eCRS nasal mucosa tissue showed a moderate positive correlation (occludin r=0.661, claudin-1 r=0.614, ZO-1 r=0.548, all P<0.001); TFF1 showed a low degree of positive correlation with the expression of occludin, claudin-1 and ZO-1 (occludin r=0.467, P=0.040; claudin-1 r=0.362, P=0.012; ZO-1 r=0.425, P=0.025). The establishment of cell models showed that compared with normal HNECs, the mRNA expression of TFF3 was most significantly increased at a concentration of 50 ng/ml stimulated by IL-13 (t=3.72, P=0.013); The mRNA expression of occludin, claudin-1 and ZO-1 decreased (occludin t=3.18, P=0.031; claudin-1 t=3.86, P=0.010; ZO-1 t=5.16, P=0.002). The expression of TFF3 mRNA increased most significantly after 15 hours of IL-13 stimulation (t=3.14, P=0.034); The mRNA expression of occludin, claudin-1 and ZO-1 decreased (occludin t=3.97, P=0.010; claudin-1 t=4.78, P=0.004; ZO-1 t=5.16, P=0.004). TJs damage model could be established by treating HNECs with 50 ng/ml IL-13 for 15 hours. Intervention experiments showed that compared with the IL-13 group, the IL-13+TFF3 group showed an increase in TJs mRNA expression (occludin t=6.10, P=0.009; claudin-1 t=5.90, P=0.013; ZO-1 t=9.44, P=0.007). Compared with the IL-13 group, the expression of TJs protein in the IL-13+TFF3 group increased (occludin t=3.23, P=0.013; claudin-1 t=9.40, P=0.017; ZO-1 t=2.23, P=0.032); The expression of TJs protein decreased in the IL-13+TFF3+LY294002 group (occludin t=4.73, claudin-1 t=8.77, ZO-1 t=3.51, all P<0.001). Compared with the IL-13+TFF3 group, the IL-3+TFF3+LY294002 group showed a decrease in PI3K and p-Akt/Akt protein expression (PI3K t=13.29, p-Akt/Akt t=10.30, all P<0.001). The increased mRNA and protein expression of occludin, claudin-1 and ZO-1 induced by TFF3 were also inhibited by LY294002. Conclusion: TFF3 can up-regulate the expression of occludin, claudin-1, and ZO-1 through PI3K/Akt pathway, and has a certain protective effect on the nasal mucosal epithelial barrier, providing a new idea for treating eCRS.

Antibacterial diarrhea effect and action mechanism of Portulaca oleracea L. water extract based on the regulation of gut microbiota and fecal metabolism.

Portulaca oleracea has served as food and folk medicine in many parts of the world for thousands of years. Portulaca oleracea extract (POE) was prepared from fresh plants. This study aims to evaluate the antibacterial diarrhea effect and explore the possible mechanism. POE was effective in reducing diarrhea rate, improving intestinal tissue, and reducing cytokines concentrations of interleukin (IL)-6, IL-10, IL-12 p40 and TNF-α in blood. Besides, the result of histological observation showed that the mucus layer thickness and crypt length in the POE-treated group was higher than that in the model group. The POE could significantly upregulate the protein expression of MUC2, occludin and ZO-1. 16S rRNA sequencing analysis showed that Parabacteroides, Clostridium and Muribaculaceae may be the key functional microflora of POE. The non-targeted metabolomics also suggested that the antibacterial diarrheal effects of P. oleracea may be attributed to the regulation of amino acid metabolism and composition of the gut microbiota. Portulaca oleracea has definite clinical efficacy against bacterial diarrhea and anti-inflammatory effects. Its regulation of gut microbiota and fecal metabolism may account for its antibacterial diarrhea and anti-inflammatory effects. © 2023 Society of Chemical Industry.

Ifit2 restricts murine coronavirus spread to the spinal cord white matter and its associated myelin pathology.

Interferon-induced protein with tetratricopeptide repeats 2, Ifit2, is critical in restricting neurotropic murine-β-coronavirus, RSA59 infection. RSA59 intracranial injection of Ifit2-deficient (-/-) compared to wild-type (WT) mice results in impaired acute microglial activation, reduced CX3CR1 expression, limited migration of peripheral lymphocytes into the brain, and impaired virus control followed by severe morbidity and mortality. While the protective role of Ifit2 is established for acute viral encephalitis, less is known about its influence during the chronic demyelinating phase of RSA59 infection. To understand this, RSA59 infected Ifit2-/- and Ifit2+/+ (WT) were observed for neuropathological outcomes at day 5 (acute phase) and 30 post-infection (chronic phase). Our study demonstrates that Ifit2 deficiency causes extensive RSA59 spread throughout the spinal cord gray and white matter, associated with impaired CD4+ T and CD8+ T cell infiltration. Further, the cervical lymph nodes of RSA59 infected Ifit2-/- mice showed reduced activation of CD4+ T cells and impaired IFNγ expression during acute encephalomyelitis. Interestingly, BBB integrity was better preserved in Ifit2-/- mice, as evidenced by tight junction protein Claudin-5 and adapter protein ZO-1 expression surrounding the meninges and blood vessels and decreased Texas red dye uptake, which may be responsible for reduced leukocyte infiltration. In contrast to sparse myelin loss in WT mice, the chronic disease phase in Ifit2-/- mice was associated with severe demyelination and persistent viral load, even at low inoculation doses. Overall, our study highlights that Ifit2 provides antiviral functions by promoting acute neuroinflammation and thereby aiding virus control and limiting severe chronic demyelination. IMPORTANCE Interferons execute their function by inducing specific genes collectively termed as interferon-stimulated genes (ISGs), among which interferon-induced protein with tetratricopeptide repeats 2, Ifit2, is known for restricting neurotropic viral replication and spread. However, little is known about its role in viral spread to the spinal cord and its associated myelin pathology. Toward this, our study using a neurotropic murine β-coronavirus and Ifit2-deficient mice demonstrates that Ifit2 deficiency causes extensive viral spread throughout the gray and white matter of the spinal cord accompanied by impaired microglial activation and T cell infiltration. Furthermore, infected Ifit2-deficient mice showed impaired activation of T cells in the cervical lymph node and relatively intact blood-brain barrier integrity. Overall, Ifit2 plays a crucial role in mounting host immunity against neurotropic murine coronavirus in the acute phase while preventing mice from developing viral-induced severe chronic neuroinflammatory demyelination, the characteristic feature of human neurological disease multiple sclerosis (MS).

CircRNA SCAR Improves High-Glucose-Induced Mitochondrial Dysfunction and Permeability Damage in Retinal Microvascular Endothelial Cells.

This study was designed to assess the role and mechanism of circRNA SCAR in human retinal microvascular endothelial cells (hRMVECs) treated with high glucose. Quantitative real-time polymerase chain reaction (qRT-PCR) and cell counting kit 8 (CCK-8) were used to detect the effects of different concentrations of glucose on circRNA SCAR expression and cell proliferation in hRMVECs. Cell viability, levels of oxygen species (ROS), malondialdehyde (MDA) and adenosine triphosphate (ATP), as well as activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) in the transfected hRMVECs in each group were detected using CCK-8 and their corresponding detection kits. Changes in mtDNA copy number in high-glucose-induced hRMVECs were observed by qRT-PCR. Additionally, western blot was applied to detect effect of overexpressing circRNA SCAR on the expression levels of mitochondrial function-related proteins (Drp1 and Fis1) and cell permeability-related proteins (claudin-5, occludin and ZO-1) in hRMVECs under high-glucose concentration. According to experimental results, high glucose significantly downregulated circRNA SCAR expression and inhibited cell proliferation in hRMVECs. Instead, overexpression of this circRNA SCAR promoted cell proliferation, reduced levels of ROS, MDA and ATP, and increased SOD and CAT activities in hRMVECs under high-glucose concentration. Also, circRNA SCAR overexpression reversed the high-glucose-induced decrease in mtDNA copy number as well as, high-glucose-induced upregulation of Drp1 and Fis1 protein expression and downregulation of claudin-5, occludin and ZO-1 protein expression in hRMVECs. In summary, circRNA SCAR promotes the proliferation of hRMVECs under high-glucose concentration, alleviates oxidative stress induced by high glucose, and improves mitochondrial function and permeability damage.

Kunxian capsule alleviates podocyte injury and proteinuria by inactivating β-catenin in db/db mice.

Diabetic kidney disease (DKD) remains the primary cause of end-stage renal disease (ESRD) globally, but treatment options are limited. Kunxian capsule (KXC) has been utilized for the treatment of autoimmune diseases and IgA nephropathy in China. However, its effect on DKD remains poorly investigated. Therefore, this study aimed to explore the protective effect of KXC in db/db mice and elucidate its underlying mechanism. The renoprotective effects of KXC were assessed in a DKD mouse model using male BKS db/db diabetic mice. After 8 weeks of treatment, the urinary albumin-to-creatinine ratio (UACR), blood biochemical parameters, renal histopathological manifestation, and podocyte ultrastructural changes were evaluated. Additionally, the expression of podocyte epithelial-to-mesenchymal transition (EMT) markers [WT1, ZO-1, and collogen I (Col1a1)] was quantitatively analyzed. Furthermore, we explored the role of KXC in the β-catenin signaling pathway to elucidate the underlying mechanism of KXC's renoprotective effect. KXC treatment effectively reduced albuminuria and attenuated renal structural abnormalities in db/db mice. Additionally, KXC restored the protein and mRNA expression of WT1 and ZO-1 while suppressing the expression of Col1a1 in db/db mice, indicating its ability to alleviate podocyte EMT. Mechanistically, KXC exerted a significant suppressive effect on the activation of β-catenin signaling in diabetic kidneys. KXC has the potential to protect podocytes during DKD by alleviating podocyte EMT through inactivating β-catenin signaling.

Chlorogenic Acid Alleviates Chronic Stress-Induced Intestinal Damage by Inhibiting the P38MAPK/NF-κB Pathway.

Chronic stress can cause intestinal barrier damage. MAPK and NF-κB are closely related to it. Chlorogenic acid (CGA), a dietary polyphenol, has been shown to have intestinal protective effects, but whether by regulating MAPK and NF-κB is not known. Therefore, in this experiment, 24 Wistar rats were randomly divided into 4 groups (C group, CS group, CS + SB203580, and CS + CGA group). Rats in the CS group were restrained stress for 6 h per day for 21 days. Rats in the CS + SB203580 group were given SB203582 (0.5 mg/kg, intraperitoneal injection) 1 h before restraint stress every other day. Rats in the CS + CGA group were given CGA (100 mg/kg, gavage) 1 h before restraint stress. In chronic stress, intestinal barrier damage was evident, while being restored after CGA treatment. After chronic stress, the levels of p-P38 were increased (P < 0.01), while the levels of p-JNK and p-ERK were not changed. The levels of p-p38 were elevated after CGA treatment (P < 0.01). These results suggested that p38MAPK played an important role in chronic stress-induced intestinal injury, and CGA could inhibit p38MAPK activity. Therefore, we chose SB203582 (P38MAPK inhibitor) to elucidate the role of p38. After chronic stress, intestinal tight junction key proteins Occludin, ZO-1, and Claudin3 protein and gene expression were reduced (P < 0.01), while being elevated after CGA or SB203582 intervention (P < 0.05). After CGA treatment, the levels of p-IκB, p-p65, p-p38, and TNF-α were reduced (P < 0.01). SB203582 intervention reduced p-p65 and TNF-α levels significantly (P < 0.01). These results suggested that CGA could inhibit the NF-κB pathway by suppressing p38MAPK, thereby alleviating chronic stress-induced intestinal damage.

Glycyrrhinic acid and probiotics alleviate deoxynivalenol-induced cytotoxicity in intestinal epithelial cells

Deoxynivalenol (DON) is one of the most prevalent mycotoxin contaminants, which posing a serious health threat to animals and humans. Previous studies have found that individually supplemented probiotic or glycyrrhinic acid (GA) could degrade DON and alleviate DON-induced cytotoxicity. The present study investigated the effect of combining GA with Saccharomyces cerevisiae (S. cerevisiae) and Enterococcus faecalis (E. faecalis) using orthogonal design on alleviating IPEC-J2 cell damage induced by DON. The results showed that the optimal counts of S. cerevisiae and E. faecalis significantly promoted cell viability. The optimal combination for increasing cell viability was 400 µg/mL GA, 1 × 106 CFU/mL S. cerevisiae and 1 × 106 CFU/mL E. faecalis to make GAP, which not only significantly alleviated the DON toxicity but also achieved the highest degradation rate of DON (34.7%). Moreover, DON exposure significantly increased IL-8, Caspase3 and NF-κB contents, and upregulated the mRNA expressions of Bax, Caspase 3, NF-κB and the protein expressions of Bax, TNF-α and COX-2. However, GAP addition significantly reduced aforementioned genes and proteins. Furthermore, GAP addition significantly increased the mRNA expressions of Claudin-1, Occludin, GLUT2 and ASCT2, and the protein expressions of ZO-1, Claudin-1 and PePT1. It was inferred that the combination of GA, S. cerevisiae, and E. faecalis had the synergistic effect on enhancing cell viability and DON degradation, which could protect cells from DON-induced damage by reducing DON cytotoxicity, alleviating cell apoptosis and inflammation via inhibiting NF-κB signaling pathway, improving intestinal barrier function, and regulating nutrient absorption and transport. These findings suggest that GAP may have potential as a dietary supplement for livestock or humans exposed to DON-contaminated food or feed.

Baicalin Attenuates H2O2-Induced Oxidative Stress by Regulating the AMPK/Nrf2 Signaling Pathway in IPEC-J2 Cells.

Oxidative stress can adversely affect the health status of the body, more specifically by causing intestinal damage by disrupting the permeability of the intestinal barrier. This is closely related to intestinal epithelial cell apoptosis caused by the mass production of reactive oxygen species (ROS). Baicalin (Bai) is a major active ingredient in Chinese traditional herbal medicine that has antioxidant, anti-inflammatory, and anti-cancer properties. The purpose of this study was to explore the underlying mechanisms by which Bai protects against hydrogen peroxide (H2O2)-induced intestinal injury in vitro. Our results indicated that H2O2 treatment caused injury to IPEC-J2 cells, resulting in their apoptosis. However, Bai treatment attenuated H2O2-induced IPEC-J2 cell damage by up-regulating the mRNA and protein expression of ZO-1, Occludin, and Claudin1. Besides, Bai treatment prevented H2O2-induced ROS and MDA production and increased the activities of antioxidant enzymes (SOD, CAT, and GSH-PX). Moreover, Bai treatment also attenuated H2O2-induced apoptosis in IPEC-J2 cells by down-regulating the mRNA expression of Caspase-3 and Caspase-9 and up-regulating the mRNA expression of FAS and Bax, which are involved in the inhibition of mitochondrial pathways. The expression of Nrf2 increased after treatment with H2O2, and Bai can alleviate this phenomenon. Meanwhile, Bai down-regulated the ratio of phosphorylated AMPK to unphosphorylated AMPK, which is indicative of the mRNA abundance of antioxidant-related genes. In addition, knockdown of AMPK by short-hairpin RNA (shRNA) significantly reduced the protein levels of AMPK and Nrf2, increased the percentage of apoptotic cells, and abrogated Bai-mediated protection against oxidative stress. Collectively, our results indicated that Bai attenuated H2O2-induced cell injury and apoptosis in IPEC-J2 cells through improving the antioxidant capacity through the inhibition of the oxidative stress-mediated AMPK/Nrf2 signaling pathway.

Regulation of Gut Microflora by Lactobacillus casei Zhang Attenuates Liver Injury in Mice Caused by Anti-Tuberculosis Drugs.

The gut-liver axis may provide a new perspective for treating anti-tuberculosis drug-induced liver injury (ATDILI). Herein, the protective effect of Lactobacillus casei (Lc) was investigated by modulating gut microflora (GM) and the toll like receptor 4 (TLR4)-nuclear factor (NF)-κB-myeloiddifferentiationfactor 88 (MyD88) pathway. C57BL/6J mice were given three levels of Lc intragastrically for 2 h before administering isoniazid and rifampicin for 8 weeks. Blood, liver, and colon tissues, as well as cecal contents, were collected for biochemical and histological examination, as well as Western blot, quantitative real time polymerase chain reaction (qRT-PCR), and 16S rRNA analyses. Lc intervention decreased alkaline phosphatase (ALP), superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), and tumor necrosis factor (TNF)-α levels (p < 0.05), recovered hepatic lobules, and reduced hepatocyte necrosis to alleviate liver injury induced by anti-tuberculosis drugs. Moreover, Lc also increased the abundance of Lactobacillus and Desulfovibrio and decreased Bilophila abundance, while enhancing zona occludens (ZO)-1 and claudin-1 protein expression compared with the model group (p < 0.05). Furthermore, Lc pretreatment reduced the lipopolysaccharide (LPS) level and downregulated NF-κB and MyD88 protein expression (p < 0.05), thus restraining pathway activation. Spearman correlation analysis indicated that Lactobacillus and Desulfovibrio were positively correlated with ZO-1 or occludin protein expression and negatively correlated with pathway protein expression. Desulfovibrio had significant negative relationships with alanine aminotransferase (ALT) and LPS levels. In contrast, Bilophila had negative associations with ZO-1, occludin, and claudin-1 protein expressions and positive correlations with LPS and pathway proteins. The results prove that Lactobacillus casei can enhance the intestinal barrier and change the composition of the gut microflora. Moreover, Lactobacillus casei may also inhibit TLR4-NF-κB-MyD88 pathway activation and alleviate ATDILI.

Daphnetin attenuates intestinal inflammation, oxidative stress, and apoptosis in ulcerative colitis via inhibiting REG3A-dependent JAK2/STAT3 signaling pathway.

Daphnetin is a natural coumarin compound with anti-inflammatory, anti-oxidant, and anti-apoptotic effects, which has been previously demonstrated to ameliorate DSS-induced ulcerative colitis (UC). However, the molecular mechanism involved in the daphnetin-mediated pathological process of UC remains unclarified. The current study used DSS-induced mice and LPS-challenged Caco-2 cells as UC models. Bodyweight, disease activity index (DAI) score, and colon length were used to evaluate the severity of colitis. The histological changes in colon tissues were observed using H&E and PAS staining. Protein levels were detected by western blot. The malondialdehyde (MDA) and superoxide dismutase (SOD) activities were used to assess oxidative stress. Inflammatory responses were evaluated by detecting the levels of inflammatory cytokines (IFN-r, IL-1β, IL-6, and TNF-α) using flow cytometry. CCK-8 and TUNEL assay were employed to determine cell growth and cell death, respectively. The results showed that daphnetin could ameliorate the severity of colitis and attenuate the damage to intestinal structure in DSS-induced mice. Compared with the DSS group, the expression of ZO-1, occludin, and anti-apoptotic protein (BCL-2) was increased while the level of pro-apoptotic proteins (Bax and cleaved caspase 3) was decreased in DSS + daphnetin group. The activity of MDA and SOD, as well as the levels of inflammatory cytokines were substantially suppressed by daphnetin. In consistency, in vitro assays indicated that daphnetin protected Caco-2 cells from LPS-stimulated viability impairment, apoptosis, oxidative stress, and inflammation. Furthermore, daphnetin suppressed the activity of JAK2/STAT signaling in LPS-induced Caco-2 cells in a REG3A-dependent manner. REG3A overexpression abated the ameliorative effects of daphnetin while JAK2/STAT signaling inhibition functioned synergically with daphnetin in LPS-stimulated Caco-2 cells. Collectively, this study deepened the understanding of the therapeutic effects of daphnetin on UC and uncovered for the first time that daphnetin functioned through REG3A-activated JAK2/STAT3 signaling in UC, which may provide novel insights for the treatment of UC.

Choline chloride:glycerol deep eutectic solvents assist in the permeation of daptomycin across Caco-2 cells mimicking intestinal bilayer

Daptomycin is a cyclic lipopeptide antibiotic used for complicated skin infections and right-sided endocarditis. The oral absorption of daptomycin is limited because of its restricted permeability across the intestinal epithelium, low lipophilicity, low bioavailability due to enzymatic degradation and high molecular weight. This study investigates the enhancement in intestinal absorption of daptomycin using a new class of permeation enhancers. Caco-2 cell-based model, mimicking the intestinal bilayer, was used as an in-vitro mucosal barrier to examine the diffusion of daptomycin. The study explored the potential of deep eutectic solvents (DESs) as oral permeation enhancers, in comparison with sodium caprate, a well-known intestinal permeation enhancer. Safe and non-toxic concentrations of DESs were determined using an MTT assay. Accordingly, a safe and effective concentration range was employed for enhancing the permeation of daptomycin. Diffusion studies performed using RP-HPLC indicated an increase in permeation of daptomycin in the presence of sodium caprate and DESs. Confocal microscopy enabled elucidation of the mechanism of permeation enhancement via reduced expression of cell junction proteins. Both, sodium caprate and DESs lowered the expression of ZO-1 protein, due to the loosening of tight junctions between Caco-2 cells and thus enhanced permeation of daptomycin. Therefore, DESs can be used as safe and effective permeation enhancers for daptomycin and explored for other oral peptide-based therapeutics .

Hexavalent chromium disrupts the skin barrier by targeting ROS-mediated mitochondrial pathway apoptosis in keratinocytes

Hexavalent chromium (Cr(VI)), a toxic heavy metal, is ubiquitous in daily life. Exposure to this toxic substance in occupational settings can cause dermatitis and cancer. As the body's largest organ, the skin plays a crucial role in protecting the organism against external aggressions. While previous studies have focused on the effects of Cr(VI) on skin inflammation, this study investigates the potential toxicity of Cr(VI) from the skin barrier and integrity perspective. The in vivo results of this study showed that mice exposed to Cr(VI) experienced skin deterioration and hemorrhaging, as well as a reduction in the thickness of the collagen fiber layer. TUNEL and Occludin staining results revealed that Cr(VI)'s toxicity primarily targeted keratinocytes. Experiments in vitro demonstrated that Cr(VI) treatment decreased the activity of HaCaT cells, altered cell morphology, and increased LDH secretion. Further research revealed that Cr(VI) could modify membrane permeability, impair membrane integrity, and reduce the protein expression of ZO-1 and Occludin. In addition, it was discovered that Cr(VI) promoted cell apoptosis and inhibited AKT activation. However, the addition of a caspase inhibitor and an AKT activator prevented Cr(VI)-induced injury to the cell membrane barrier, indicating that apoptosis plays a crucial role in this process. The addition of three apoptotic pathway inhibitors, confirmed that Cr(VI) damaged the cell barrier through ROS-mediated mitochondrial pathway apoptosis. Moreover, the use of a ROS inhibitor significantly reduced Cr(VI)-induced apoptosis and cell barrier injury. In conclusion, this study provides an experimental foundation for the treatment of skin injury caused by Cr(VI).

Matrine provides a protective effect against Eimeria tenella challenge by alleviating intestinal barrier damage

As a virulent and harmful protozoan, Eimeria tenella (E.tenella) causes harmful chicken coccidiosis, inducing high economic losses in the chicken industry. The management of the coccidial disease has been greatly hampered by drug resistance. Matrine is an active ingredient from Ku Shen (Radix Sophorae Flavescentis), a typical pesticide in chinese medicine. The aim of this study was to examine matrine’s possible effectiveness in the treatment of coccidiosis and its protective function on the intestinal barrier. The anticoccidial index (ACI), the levels of anti-oxidant indexes, and secretory immunoglobulin A (sIgA) were detected. The levels of mRNA and protein expression of Occludin, ZO-1, and Claudin-1 were determined through quantitative real-time PCR (RT-qPCR) and immunohistochemistry (IHC) analysis. Matrine exhibited a moderate ACI value, and ACI values of 122.51 and 143.42 corresponded to 5 and 10 mg/kg of matrine, respectively. Compared to the infective control group, the expression of tight junction proteins significantly increased in the matrine-treatment group by RT-PCR and IHC analysis, which are essential for the mucosal immune system and the intestinal barrier. Besides, the matrine-treatment group showed a more complete intestinal structure, fewer bleeding spots, and coccidian by histopathology analysis. We also found that, matrine significantly enhanced the antioxidant ability and significantly increased the content of sIgA. Above all, matrine was considered an efficient drug against E.tenella by the anti-oxidant efficacy, and the ability to protect the composition and function of the intestinal barrier.