Junction Proteins Research Articles

Capsaicin attenuates the effect of inflammatory cytokines in a HaCaT cell model for basal keratinocytes.

The resolution of the skin's inflammatory response is only possible if its barrier function is restored. TRPV1 channel activation plays an important role during inflammation but the effect of this activation on the skin barrier under inflammatory conditions has not been clarified. We hypothesize that it could potentially aid the keratinocyte barrier by reducing inflammatory cytokine release and promoting tight junction development. To explore the role of TRPV1 activation in inflammation, we designed and optimized an in vitro model of keratinocytes with basal epidermal layer characteristics using HaCaT cells and TNFα to induce inflammation. TNFα increased the gene expression of tight junction protein claudin 1 (CLDN1) by at least 2.60 ± 0.16-fold, in a concentration-dependent manner, over a 48h period. The administration of a capsaicin pre-treatment reduced the CLDN1 expression to 1.51 ± 0.16-fold during the first 6h after TNFα induction, whereas IL-8 cytokine release was reduced 0.64 ± 0.17-fold. After 48h, CLDN1 protein levels increased by a factor of 6.57 ± 1.39 compared to cells only treated with TNFα. These results suggest that activation of TRPV1 by capsaicin can potentiate the increase in CLDN1 expression and CLDN1 protein synthesis induced by TNFα in cultured keratinocytes, while reducing the release of IL-8.

Robotic microinjection enables large-scale transgenic studies of Caenorhabditis elegans

The nematode Caenorhabditis elegans is widely employed as a model organism to study basic biological mechanisms. However, transgenic C. elegans are generated by manual injection, which remains low-throughput and labor-intensive, limiting the scope of approaches benefitting from large-scale transgenesis. Here, we report a robotic microinjection system, integrating a microfluidic device capable of reliable worm immobilization, transfer, and rotation, for high-speed injection of C. elegans. The robotic system provides an injection speed 2-3 times faster than that of experts with 7–22 years of experience while maintaining comparable injection quality and only limited trials needed by users to become proficient. We further employ our system in a large-scale reverse genetic screen using multiplexed alternative splicing reporters, and find that the TDP-1 RNA-binding protein regulates alternative splicing of zoo-1 mRNA, which encodes variants of the zonula occludens tight junction proteins. With its high speed, high accuracy, and high efficiency in worm injection, this robotic system shows great potential for high-throughput transgenic studies of C. elegans.

Oleanolic acid 28-O-β-D-glucopyranoside: A novel therapeutic agent against ulcerative colitis via anti-inflammatory, barrier-preservation, and gut microbiota-modulation

Ulcerative colitis (UC), an incurable and recurrent inflammatory bowel disease, presents a significant threat to health and highlights the need for novel therapeutic strategies. Oleanolic acid 28-O-β-D-glucopyranoside (OAG) is a naturally occurring pentacyclic triterpenoid found in ginseng. In this study, we demonstrated that OAG exhibited remarkable anti-UC activity in LPS-induced Caco-2 cells and DSS-induced model mice. First, OAG alleviated the symptoms of UC by mitigating weight loss, reducing the DAI score, and increasing colon length. Second, the inflammatory response was inhibited after OAG intervention, evidenced decreases in the spleen coefficient, cytokine levels, and inflammatory cell infiltration in colon tissue. Thirdly, OAG also enhanced intestinal epithelial barrier function, as evidenced by elevated TEER values, increased expression of tight junction proteins, diminished bacterial translocation, and maintained intact ultrastructure of colonic mucosal cells. Notably, compared with 5-aminosalicylic acid, OAG demonstrated superior efficacy in enhancing mucosal barrier function. Fourth, OAG increased microbial diversity, promoted the abundance of beneficial bacteria, reduced the abundance of harmful bacteria, and rebalanced the gut microbiome. Finally, the PI3K-AKT and MAPK signaling pathways were identified as crucial mechanisms underlying the therapeutic effects of OAG against UC through multi-omics. In summary, we identified OAG as a novel therapeutic agent against UC, demonstrating anti-inflammatory, barrier-preserving, and gut microbiota-modulating effects, highlighting its promising potential as a candidate UC drug.

Metformin modulates the TXNIP-NLRP3-GSDMD pathway to improve diabetic bladder dysfunction

To validate the therapeutic efficacy of metformin on diabetic bladder dysfunction (DBD) and further elucidate whether the TXNIP-NLRP3-GSDMD axis serves as a target for metformin in ameliorating DBD. C57BL/6J mice were induced with diet-induced obesity by being fed a high-fat diet (HFD) for 16 weeks. After establishing the model, the mice were treated with metformin for 4 weeks, and their glucose metabolism-related parameters were assessed. Urine spot assays and urodynamic measurements were conducted to reflect the bladder function and urinary behavior in mice, while histological examination was performed to observe morphological changes. Western blot analysis was employed to measure the expression levels of pyroptotic factors such as TXNIP, NLRP3, GSDMD, and tight junction proteins. Metformin treatment significantly improved glucose tolerance and insulin sensitivity in mice. Moreover, it showed promise in decreasing urinary spot occurrence, reducing urination frequency, alleviating non-voiding contractions, and stabilizing peak urinary pressure. Following metformin therapy, mice displayed restored epithelial fold structure, increased thickness of the muscular layer, substantial decrease in muscle fiber content, notably reduced levels of TXNIP and GSDMD proteins in the metformin-treated group compared to the DBD group, and restored expression of tight junction proteins Zo-1, Claudin-1, and Occludin. Metformin ameliorates urothelial cells damage in DBD mice by inhibiting TXNIP generation and reducing NLRP3 and GSDMD production.

Chrysene contribution to bronchial asthma: Activation of TRPA1 disrupts bronchial epithelial barrier via ERK pathway

BackgroundElevated polycyclic aromatic hydrocarbon (PAH) levels are associated with exacerbation of asthma. Chrysene is one of the most prevalent unsubstituted PAHs in the environment. Transient receptor potential ankyrin 1 (TRPA1) can be used as a chemoreceptor to detect inhaled stimuli and plays an important role in the occurrence and deterioration of asthma. Whether exposure to a high concentration of chrysene in the environment can activate TRPA1 and contribute to the development of asthma, potentially through the dysfunction of the bronchial epithelial barrier, remains unclear. MethodsA cell-based assay was performed to verify the downregulation of the expression of E-cadherin and tight junction (TJ) proteins by chrysene in bronchial epithelial cells to explore the role of chrysene-mediated TRPA1 activation in the regulation of TJ protein expression through the extracellular signal-regulated protein kinase (ERK) pathway. Animal tests were conducted to determine whether chrysene could enhance airway hyperresponsiveness (AHR) induced by house dust mites (HDMs) and disrupt barrier function, thereby contributing to asthma. ResultsThe cell-based assay revealed that chrysene could disrupt the function of the bronchial epithelial barrier and decrease the expression levels of E-cadherin, zonula occludens-1 (ZO-1), occludin, and claudin-5 through the ERK pathway. Chrysene induced airway epithelial barrier dysfunction primarily through TRPA1 instead of transient receptor potential vanilloid 1. TRPA1 knockdown was able to attenuate chrysene-induced downregulation of TJ protein expression and downregulate ERK activation (p-ERK). Compared with exposure to HDM alone, coexposure to chrysene and HDM resulted in an increased incidence of AHR, disruption of barrier function, and eosinophilic inflammatory responses in a mouse model of asthma. Coexposure to chrysene and HDM increased TRPA1 expression. The animal test verified that the TRPA1 inhibitor HC030031 could suppress chrysene and HDM-induced asthma in mice. ConclusionsOur findings showed that chrysene contributed to the breakdown of the function of the bronchial epithelial barrier through the TRPA1–ERK axis and therefore acted as an adjuvant to contribute to asthma.

Cimicifuga heracleifolia kom. Attenuates ulcerative colitis through the PI3K/AKT/NF-κB signaling pathway

Ethnopharmacological relevanceCimicifuga heracleifolia Kom. (C. heracleifolia) has demonstrated efficacy in treating gastrointestinal disorders, including splenasthenic diarrhea. Ulcerative colitis (UC), a chronic inflammatory bowel disease, shares similarities with splenasthenic diarrhea. However, the pharmacological effects of C. heracleifolia on UC and the underlying mechanisms remain unexplored. Aim of the studyThe present study investigates the therapeutic potential and mechanisms of C. heracleifolia in UC. MethodsInitially, network pharmacology analysis, encompassing ingredient screening, target prediction, protein-protein interaction (PPI) network analysis, and enrichment analysis, was employed to predict the mechanisms of C. heracleifolia. The findings were further validated using transcriptomics and functional assays in a dextran sulfate sodium (DSS)-induced UC model. Additionally, bioactive compounds were identified through surface plasmon resonance (SPR) analysis, molecular docking, and cell-based assays. ResultsA total of 52 ingredients of C. heracleifolia were screened, and 32 key targets were identified within a PPI network comprising 285 potential therapeutic targets. Enrichment analysis indicated that the anti-UC effects of C. heracleifolia are mediated through immune response modulation and the inhibition of inflammatory signaling pathways. In vivo experiments showed that C. heracleifolia mitigated histological damage in the colon, reduced the expression of phosphorylated Akt1, nuclear factor-kappa B (NF-κB) p65, and inhibitor of Kappa B kinase α/β (IKKα/β), suppressed the content of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and enhanced the expression of tight junction proteins. Moreover, cimigenoside, caffeic acid, and methyl caffeate were identified as the bioactive constituents responsible for the UC treatment effects of C. heracleifolia. ConclusionsIn summary, this study is the first to demonstrate that C. heracleifolia exerts therapeutic effects on UC by enhancing the intestinal mucosal barrier and inhibiting the phosphatidylinositol 3-kinase (PI3K)/AKT/NF-κB signaling pathway. These findings offer valuable insights into the clinical application of C. heracleifolia for UC management.

Quinoa ameliorates polycystic ovary syndrome via regulating gut microbiota through PI3K/AKT/mTOR pathway and autophagy

BackgroundPolycystic ovary syndrome (PCOS) is a unity of endocrine and metabolic disorders, associated with PI3K/AKT/mTOR, autophagy, and gut microbiota. Quinoa is a valuable food source, which contains rich minerals, unsaturated fatty acids, and has a positive modulating effect on metabolic diseases. However, its effects and potential mechanisms on PCOS have not been reported yet. Therefore, the purpose of this study is to investigate the effect of quinoa on PCOS rats by regulating PI3K/AKT/mTOR, autophagy, and gut microbiota.MethodsTen–week-old female Sprague-Dawley (SD) rats have received letrozole for 24 days for induction of PCOS and subsequently were treated with a quinoa diet for 8 weeks. Vaginal smears were used to analyze the estrous cycle of rats. Hormone and biochemical indexes were analyzed by kit assays and glucometer. The pathological changes of ovary, pancreas, duodenum and colon were observed by HE staining. PI3K, AKT, mTOR and autophagy-related proteins in the ovary and colon were measured by western blot and immunohistochemistry staining. Tight junction proteins in colon were measured by immunohistochemistry staining. 16 s rDNA sequencing was used to detect the changes of intestinal microbiota in rats. Network pharmacology and molecular docking were used to study the possible targets and mechanisms of quinoa on PCOS. Spearman correlation analysis was used to study the relationship between intestinal microbial abundance and hormone levels of PCOS rats at the phylum and genus level.ResultsQuinoa significantly improved estrous cycle and biochemical parameters of PCOS-like rats, and the pathological state of ovary, pancreas, duodenum and colon tissues. Especially, quinoa significantly regulated the expression of PI3K, AKT, mTOR and autophagy-related proteins in the ovary. Quinoa may repair the intestinal barrier by upregulating the expression of tight junction proteins in the colon, and regulate autophagy-related factors in colon. Additionally, quinoa increased the abundance of Lactobacillu, Bacteroides and Oscillospira, and decreased the Firmicutes/Bacteroidetes ratio and the Blautia, and Prevotella, reversing the dysregulation of the gut microbiota. Correlation analysis showed that there is a strong correlation between gut microbiota with significant changes in abundance and hormone related to PCOS.ConclusionOur result indicated that effect of quinoa on PCOS maybe associated with activation of the PI3K/AKT/mTOR signaling pathway, inhibition of autophagy, and regulation of intestinal flora.

Lipid-lowering and antioxidant properties of probiotic Bifidobacterium animalis MSMC83 in rats on a high-fat diet.

Hyperlipidaemia, the abnormally high concentration of lipids such as cholesterol in the body, has a series of deleterious effects on health that are least in part are due to increased inflammation and oxidative stress. Probiotics are living microorganisms that possess the efficacy to improve health. Among the many effects that have been ascribed to probiotics is the potential to lower the body lipid content. Here, we used a rat model of induced hyperlipidaemia to assess the lipid-lowering and antioxidant properties of the probiotic strain Bifidobacterium animalis MSMC83 as well as its impact on intestinal barrier immunity and the intestinal microbiota. Oral probiotic intake led to a reduction of body weight, fasting blood glucose, and lipid levels, and increased expression of cholesterol-7α-hydroxylase and antioxidant enzymes. Additionally, B. animalis MSMC83 decreased the levels of liver enzymes and pro-inflammatory cytokines, leading to reduced hepatic steatosis. Furthermore, it re-established intestinal barrier integrity as shown by restoration of the tight junction protein zonula occludens-1 amount and reduced pathogen-induced inflammation in the intestinal epithelium as shown by readjusted expression of toll-like receptors (TLRs). Moreover B. animalis MSMC83 contributed to the maintenance of a balanced, diverse microbiome. Thus, our results indicate that B. animalis MSMC83 alleviates risk factors associated with hyperlipidaemia, suggesting its use as a probiotic to counter the effects associated with unhealthy diets.

Fecal Microbiota Transplantation Induced by Wumei Pills Improves Chemotherapy-Induced Intestinal Mucositis in BALB/c Mice by Modulating the TLR4/MyD88/NF-κB Signaling Pathway.

Our previous studies have found that Wumei Pills can regulate the intestinal flora to inhibit chemotherapy-induced intestinal mucositis (CIM). However, there is still insufficient evidence to confirm that intestinal flora is the main link in the regulation of CIM by Wumei Pills, and its downstream mechanism is still unclear. We first obtained the signal pathway of the intervention of Wumei Pill on CIM through network pharmacological analysis and then transplanted the bacterial solution into CIM mice, combined with Western Blot, HE, ELISA and other biological technology-related proteins and inflammatory factors. It showed that 97 kinds of effective ingredients and 205 kinds of targets of Wumei pills were screened out and the potential mechanism of Wumei Pills on CIM may be the NF-κB signaling pathway. In contrast with the control group, the results displayed that the weight, food intake, and mice's colon length were apparently decreased in the 5-Fu group, while the diarrhea score was increased. However, FMT reversed this change, and the difference was statistically significant. Additionally, FMT could improve the pathological state of inflammatory cell infiltration in mice, reduce histopathological scores of colon and jejunum, decrease the expression levels of IL-1β, MPO, TNF-α, and IL-6, reverse the activation of signaling pathway named TLR4/Myd88/ NF-κB and down-regulate protein expression, thereby exerting its anti-inflammatory activities. Further experiments have found that FMT could reverse the decreasing of tight junction proteins and mucins caused by 5-Fu, thereby repairing the intestinal mucosal barrier, and FMT could also increase the content of acetic acid, propanoic acid, and butanoic acid in the feces of 5-Fu group. FMT can defend the intestinal mucosal barrier integrality by increasing the content of exercise fatty acids, and its mechanism may be in connection with its inhibition of TLR4/My- D88/NF-κB signal pathway to relieve inflammation.

Bacillus CotA laccase improved the intestinal health, amino acid metabolism and hepatic metabolic capacity of Pekin ducks fed naturally contaminated AFB1 diet

BackgroundAflatoxin B1 (AFB1) is a prevalent contaminant in agricultural products, presenting significant risks to animal health. CotA laccase from Bacillus licheniformis has shown significant efficacy in degrading mycotoxins in vitro test. The efficacy of Bacillus CotA laccase in animals, however, remains to be confirmed. A 2 × 2 factorial design was used to investigate the effects of Bacillus CotA laccase level (0 or 1 U/kg), AFB1 challenge (challenged or unchallenged) and their interactions on ducks. The purpose of this study was to evaluate the efficacy of Bacillus CotA laccase in alleviating AFB1 toxicosis of ducks.ResultsBacillus CotA laccase alleviated AFB1-induced declines in growth performance of ducks accompanied by improved average daily gain (ADG) and lower feed/gain ratio (F/G). Bacillus CotA laccase ameliorated AFB1-induced gut barrier dysfunctions and inflammation testified by increasing the jejunal villi height/crypt depth ratio (VH/CD) and the mRNA expression of tight junction protein 1 (TJP1) and zonula occluden-1 (ZO-1) as well as decreasing the expression of inflammation-related genes in the jejunum of ducks. Amino acid metabolome showed that Bacillus CotA laccase ameliorated AFB1-induced amino acid metabolism disorders evidenced by increasing the level of glutamic acid in serum and upregulating the expression of amino acid transport related genes in jejunum of ducks. Bacillus CotA laccase ameliorated AFB1-induced liver injury testified by suppressing oxidative stress, inhibiting apoptosis, and downregulating the expression of hepatic metabolic enzyme related genes of ducks. Moreover, Bacillus CotA laccase degraded AFB1 in digestive tract of ducks, resulting in the reduced absorption level of AFB1 across intestinal epithelium testified by the decreased level of AFB1-DNA adduct in the liver, and the reduced content of AFB1 residues in liver and feces of ducks.ConclusionsBacillus CotA laccase effectively improved the growth performance, intestinal health, amino acid metabolism and hepatic aflatoxin metabolism of ducks fed AFB1 diets, highlighting its potential as an efficient and safe feed enzyme for AFB1 degradation in animal production.Graphical

NDMA enhances claudin-1 and -6 expression viaCYP2E1/ROS in AGS cells

Carcinogenic N-nitroso compounds, especially N-nitroso dimethylamine, increase the risk of gastric cancer development. Cytochrome P450-2E1 metabolizes this compound, thus generating an oxidant microenvironment. We aimed to evaluate in gastric adenocarcinoma cells if its effect on CYP2E1 and ROS affects signaling pathways associated with gastric cancer oncogenesis. The impact of N- nitroso dimethylamine upon CYP2E1 and ROS activation/secretion was evaluated by the DCFDA assay protocol, TER measurements, Stat3, pSTAT3, ERK1/2, and pERK1/2 expression, claudins-1 and -6 expression, and finally mRNA values of IL-1β IL-6, IL-8 and TNFα. Our results showed that exposure to N- N-nitroso dimethylamine disrupts the regulation of Stat3 and Erk1/2, alters the expression of claudin-1 and claudin-6 tight junction proteins, and increases the secretion of pro-inflammatory cytokines. These alterations induce a continuous local inflammatory process, an event identified as a gastric cancer promoter. In summary, N-nitroso dimethylamine can disrupt cell mechanisms associated with gastric cancer oncogenesis.

PM10 dysregulates epithelial barrier function in human corneal epithelial cells that is restored by antioxidant SKQ1

Exposure to airborne particulate <10 μm (PM10) adversely affects the ocular surface. This study tested PM10 on epithelial barrier integrity in immortalized human corneal epithelial cells (HCE-2) and mouse cornea, and whether antioxidant SKQ1 is restorative. HCE-2 were exposed to 100 μg/ml PM10 ± SKQ1 for 24 h. An Electric Cell-Substrate Impedance Sensing (ECIS) system monitored the impact of PM10. RT-PCR, western blotting and immunofluorescence measured levels of barrier and associated proteins, stanniocalcin 2 (STC2), and a kit measured total calcium. In vivo, female C57BL/6 mice were exposed to either control air or PM10 (±SKQ1) in a whole-body exposure chamber, and barrier associated proteins tested. Tight junction and mucins proteins in the cornea were tested. In HCE-2, PM0 vs control significantly reduced mRNA and protein levels of tight junction and adherence proteins, and mucins. ECIS data demonstrated that PM10 vs control cells exhibited a significant decrease in epithelial barrier strength at 4000 Hz indicated by reduced impedance and resistance. PM10 also upregulated STC2 protein and total calcium levels. In vivo, PM10 vs control reduced zonula occludens 1 and mucins. SKQ1 pre-treatment reversed PM10 effects both in vitro and in vivo. In conclusion, PM10 exposure reduced tight junction and mucin proteins, and compromised the seal between cells in the corneal epithelium leading to decreased epithelial barrier strength. This effect was reversed by SKQ1. Since the corneal epithelium forms the first line of defense against air pollutants, including PM10, preserving its integrity using antioxidants such as SKQ1 is crucial in reducing the occurrence of ocular surface disorders.

Ruhao Dashi granules exert therapeutic effects on H1N1 influenza virus infection by altering intestinal microflora composition.

Antiviral medications for influenza could be ineffective due to the emergence of resistant influenza virus strains. Ruhao Dashi (RHDS) granules possess anti-inflammatory and antibacterial effects. The present study aimed to determine the efficacy of RHDS granules in treating influenza-infected mice and the mechanism underlying this treatment as well as its effect on the intestinal flora composition of the infected mice. The HPLC-UV method was used to identify the active components of RHDS granules. ICR mice were infected with influenza A virus (IAV) H1N1 subtype through a nasal drip. After the influenza mice model was successfully established, the pathological changes in the lungs were observed for 5 days after gavage treatment with 0.9% sterile saline and low, medium, and high doses (0.07, 0.14, and 0.28 g/mL, respectively) of RHDS granules. The serum levels of the cytokines IL-6 and TNF-α and sIgA were detected by ELISA. Real-time fluorescence quantitative PCR and western blotting assay were performed to determine the expression levels of the tight junction (TJ) proteins claudin-1, occludin, and zonula occludens-1 (ZO-1) in colon tissues. Furthermore, 16S rRNA gene sequencing of feces samples was conducted to assess the effect of RHDS granules on the gut microbiota. RHDS granules exerted a protective effect on the lung tissues of IAV-infected mice; moreover, the granules reduced the synthesis of proinflammatory cytokines and increased the relative expression levels of claudin-1, occludin, and ZO-1 in colon tissues. Furthermore, RHDS granule treatment increased the relative abundance of Lactobacillus, Akkermansia, and Faecalibaculum and decreased the relative abundance of Muribaculaceae; thus, RHDS granules could stabilize the intestinal microbiota to some extent. RHDS granules exert a therapeutic effect on IAV-infected mice probably by modifying the structural composition of their intestinal microbiota.

Laoxianghuang polysaccharide promotes the anti-inflammatory cytokine interleukin-10 in colitis via gut microbial linoleic acid

BackgroundOur previous study found that the polysaccharide from Laoxianghuang (LP), fermented fruit of bergamot (traditional Chinese medicine and food), can alter gut microbiota and regulate short-chain fatty acids (SCFAs) in vitro. Nevertheless, there is a paucity of reports on the impact of LP on gut microbiota in vivo. PurposeTo analyze the structures of LP, investigate the influence of LP on the damaged intestinal barrier in DSS-induced colitis mice, and further explore its potential mechanisms. MethodsWe analyzed the physicochemical properties of purified LP by HPLC, SEM, and FT-IR spectrum. Then, to assess the effect of LP in DSS-induced colitis mice, we observed the damage to the colon tissue, measured inflammatory cytokines and tight junction protein expression through RT-qPCR as well as immunofluorescent staining, and investigated the influence of LP on altering gut microbiota and metabolites using 16 s rRNA sequencing and HPLC-MS/MS. Ultimately, the impact of linoleic acid on inflammatory cytokines was confirmed by the LPS-induced RAW264.7 cells. ResultsLP, mainly galactoglucan, could inhibit weight loss and colon shortening, decrease levels of tumor necrosis factor-α (TNF-α), increase levels of interleukin-10 (IL-10) and the intestinal acetic acid and butyric acid, and promote the expression of tight junction proteins ZO-1 and Claudin-1. Meanwhile, LP enhanced the abundance of beneficial bacteria including Romboutsia, Eubacterium_coprostanoligenes_group, and Akkermansia, and regulated linoleic acid metabolism to increase the linoleic acid level. In vitro cell experiment proved that linoleic acid could elevate the level of IL-10 and inhibit inflammatory responses. ConclusionsOur results suggested that LP effectively alleviated colitis by promoting the anti-inflammatory cytokine interleukin-10 via gut microbiota-mediated linoleic acid metabolism.

Circ_0049979 ameliorates myocardial infarction through improving Cx43-mediated endothelial functions

Endothelial injury is a fundamental pathogenesis of coronary atherosclerotic heart disease (CHD). Circular RNAs (circRNAs) are important post-transcriptional regulators in many human major diseases, including CHD. The aim of the present study was to explore the role of circ_0049979, a novel identified circRNA from ANO8 gene locus, in endothelial injury during CHD. We found that expression of circ_0049979 was reduced by ox-LDL treatment in HUVECs in a dose-dependent manner. Loss- and gain-of-function experiments demonstrated that knockdown of circ_0049979 decreased the capacities of proliferation, migration and tube formation in normal HUVECs. While, overexpression of circ_0049979 improved these capacities in both normal and ox-LDL-incubated HUVECs. Then, the online bioinformatic tool Circinteractome was used to predicted the target miRNAs of circ_0049979, and miR-653 was selected as the candidate. We demonstrated that miR-653 directly interacted with and was negatively regulated by circ_0049979, and played a negative role in regulating proliferation, migration and tube formation of HUVECs. In terms of the mechanism, miR-653 post-transcriptionally suppressed the expression of the gap junction protein 43 (Cx43), a key protein of endothelial tight junction. Finally, we verified that overexpression of circ_0049979 was able to alleviate plaque formation, lipid deposition, and endothelial cell apoptosis, as well as myocardial infarction, in coronary atherosclerotic mice in vivo. In conclusion, circ_0049979 plays a protective role in coronary atherosclerotic myocardial infarction by improving miR-653/Cx43-mediated endothelial functions.

Oral supplementation with lactic acid bacteria improve the intestinal epithelial barrier and gut microbiota of broiler chicks to alleviate Salmonella Enteritidis infection

Lactic acid bacteria (LAB) play a key role in regulating the balance of gut microbiota and serve as a suitable alternative to antibiotics. This study aims to evaluate the characteristics of 2 LAB isolates Lactiplantibacillus plantarum Lp71 (L. plantarum Lp71) and Enterococcus faecium Ef72 (E. faecium Ef72), and their roles in alleviating Salmonella Enteritidis infection. Sixty 1-day-old chicks were randomly divided into 4 groups which treated with or without L. plantarum Lp71 and E. faecium Ef72 mixture for 21 d, and then intestinal samples were collected for gut microbiota analysis, pathological and immunohistochemical analysis at 24 h post infection with or without Salmonella Enteritidis on the 22nd d. The results showed that L. plantarum Lp71 and E. faecium Ef72 had the ability to anti-acid and anti-bile salt. Salmonella Enteritidis infection damaged the intestinal epithelial barrier and reduced the expression level of tight junction proteins (ZO-1, Claudin-1, Occludin). Oral supplementation with L. plantarum Lp71 and E. faecium Ef72 mixture could alleviated the damages to intestinal epithelial barrier by Salmonella Enteritidis infection. Salmonella Enteritidis could cause abnormal Akkermansia muciniphila proliferation and decrease the diversity of cecal microbiota in chicks. These conditions could have further led to reduce gut microbiota health index (GMHI), and improve microbial dysbiosis index (MDI). Moreover, oral supplementation with L. plantarum Lp71 and E. faecium Ef72 mixture could effectively prevent the aforementioned infection outcomes and increase the abundance proportions of the several key functions in metabolic pathways metabolic pathways such as transcription and signal transduction mechanisms. In summary, L. plantarum Lp71 and E. faecium Ef72 could be the probiotics candidates that used to prevent the damage from enteric pathogens such as Salmonella Enteritidis in broiler chicks.

GAP JUNCTION FUNCTION IS ESSENTIAL FOR SURVIVAL OF ACUTE LYMPHOBLASTIC LEUKEMIA CELLS.

Acute lymphoblastic leukemia has an intimate physical relationship with nonmalignant bone marrow stromal cells. We have recently demonstrated that stromal cells contribute to the survival of leukemia cells and that there is a bidirectional transfer of intracellular material between them. Understanding the mechanisms of stromal support of leukemia may provide insights into new therapies. To test the hypothesis that gap junctions are formed between acute lymphoblastic leukemia cells and nonmalignant stromal cells, and that gap junction function is essential for the survival of leukemia cells. We employed a well-characterized in vitro model of human bone marrow stromal cells and primary human B lymphoblastic leukemia cells and measured leukemia cell survival in coculture using flow cytometry. We measured the effects of gap junction antagonist peptides, carbenoxolone (a drug known to interfere with the gap junction function), and several leukemia chemotherapy drugs including methotrexate upon leukemia cell survival. We demonstrated that stromal cells need to be alive and metabolically active to keep leukemia cells alive. Physical contact between stromal and leukemia cells leads to an increase in gap junction proteins in leukemia cells. Gap junction inhibitory peptides impaired leukemia cell survival as did carbenoxolone, a nonpeptide inhibitor of the gap junction function. Stromal cell survival was not affected. We observed a very modest enhancement of methotrexate antileukemia activity by low-dose carbenoxolone but no significant interactions with dexamethasone, vincristine, mercaptopurine, or doxorubicin. These studies demonstrate that acute lymphoblastic cell survival is impaired by interference with the gap junction function. The development of drugs targeting gap junctions may provide a novel approach to the therapy of acute lymphoblastic leukemia.

AHR activation relieves deoxynivalenol-induced disruption of porcine intestinal epithelial barrier functions

Mycotoxins are ubiquitous natural pollutants that pose a serious threat to public health. Deoxynivalenol (DON) as one of the most prominent mycotoxins has a noticeable adverse effect on intestinal barrier function, which depends on the intestinal barrier integrity. However, the potential mechanisms and effective therapeutic strategies remain unclear. Aryl hydrocarbon receptor (AHR) has been implicated in the modulation of intestinal barrier function and inflammation. The study aims to investigate the unique role of AHR in mediating DON-induced intestinal epithelial barrier function. In the current study, we revealed that DON triggered mitochondrial structural damage and functional impairment, leading to oxidative stress and apoptosis in porcine jejunal epithelial cells (IPEC-J2). DON altered the integrity of IPEC-J2 cells by disrupting the distribution and function of tight junction proteins. Additionally, DON activated TNF-α/NF-κB/MLCK signaling pathway, thereby eliciting inflammatory response. Notably, DON inhibited AHR nuclear translocation and attenuated xenobiotic response element promoter activity and its target genes. However, overexpression of AHR mitigated DON-induced disruption of intestinal epithelial barrier functions by suppressing TNF-α/NF-κB/MLCK pathway in IPEC-J2 cells. Our findings indicate that AHR regulates intestinal epithelial barrier function and therefore is a novel therapeutic molecule for intestinal disorders.

Real meat shows good performance in maintaining colonic barrier function at a recommended amount of intake in mice

AbstractThis study aimed to investigate the effects and underlying mechanisms of real meat and plant‐based meat analogs on intestinal barrier function. Methods including PacBio sequencing, HPLC, histological observation, western blotting, and RT‐qPCR were jointly applied. The results indicated that the 20‐day intake of real meat and plant‐based meat analogs did not induce pathological changes in the colon of mice. However, when the feeding period was extended to 68 days, inflammatory infiltration was observed in the colon tissue of mice fed plant‐based meat analogs. In contrast, the intake of real meat for 68 days performed better in maintaining colonic barrier function by reducing cell apoptosis, increasing the number of goblet cells and mucin secretion, and upregulating the expression of tight junction proteins. High‐throughput sequencing revealed that the intake of real meat significantly altered the composition of the gut microbiota, reducing pro‐inflammatory bacteria (such as Bacteroides, Desulfovibrio, or Colidextribacter) or pathogenic bacteria (such as Mucispirillum and Helicobacter) while increasing beneficial bacteria (such as Lactobacillus, Bifidobacterium, or Roseburia). Additionally, the consumption of real meat markedly decreased protein fermentation in the colon, leading to lower levels of cadaverine, putrescine, and spermine. These changes associated with real meat consumption reduced the activation of the pro‐inflammatory pathway (lipopolysaccharide/Toll‐like receptor 4/nuclear factor kappa B), ultimately maintaining colonic homeostasis in mice. Collectively, the findings suggest that the proper intake of real meat is more beneficial for colon health in mice compared to plant‐based meat analogs.

Morphological, electrophysiological, and molecular alterations in foetal noncompacted cardiomyopathy induced by disruption of ROCK signalling.

Left ventricular noncompaction cardiomyopathy is associated with heart failure, arrhythmia, and sudden cardiac death. The developmental mechanism underpinning noncompaction in the adult heart is still not fully understood, with lack of trabeculae compaction, hypertrabeculation, and loss of proliferation cited as possible causes. To study this, we utilised a mouse model of aberrant Rho kinase (ROCK) signalling in cardiomyocytes, which led to a noncompaction phenotype during embryogenesis, and monitored how this progressed after birth and into adulthood. The cause of the early noncompaction at E15.5 was attributed to a decrease in proliferation in the developing ventricular wall. By E18.5, the phenotype became patchy, with regions of noncompaction interspersed with thick compacted areas of ventricular wall. To study how this altered myoarchitecture of the heart influenced impulse propagation in the developing and adult heart, we used histology with immunohistochemistry for gap junction protein expression, optical mapping, and electrocardiography. At the prenatal stages, a clear reduction in left ventricular wall thickness, accompanied by abnormal conduction of the ectopically paced beat in that area, was observed in mutant hearts. This correlated with increased expression of connexin-40 and connexin-43 in noncompacted trabeculae. In postnatal stages, left ventricular noncompaction was resolved, but the right ventricular wall remained structurally abnormal through to adulthood with cardiomyocyte hypertrophy and retention of myocardial crypts. Thus, this is a novel model of self-correcting embryonic hypertrabeculation cardiomyopathy, but it highlights that remodelling potential differs between the left and right ventricles. We conclude that disruption of ROCK signalling induces both morphological and electrophysiological changes that evolve over time, highlighting the link between myocyte proliferation and noncompaction phenotypes and electrophysiological differentiation.