Expression Levels Of Zonula Occludens-1 Research Articles

Biochanin A inhibits endothelial dysfunction induced by IL‑6‑stimulated endothelial microparticles in Perthes disease via the NFκB pathway.

Endothelial dysfunction caused by the stimulation of endothelial microparticles (EMPs) by the inflammatory factor IL-6 is one of the pathogenic pathways associated with Perthes disease. The natural active product biochanin A (BCA) has an anti-inflammatory effect; however, whether it can alleviate endothelial dysfunction in Perthes disease is not known. The present in vitro experiments on human umbilical vein endothelial cells showed that 0-100 pg/ml IL-6-EMPs could induce endothelial dysfunction in a concentration-dependent manner, and the results of the Cell Counting Kit 8 assay revealed that, at concentrations of <20 µM, BCA had no cytotoxic effect. Reverse transcription-quantitative PCR demonstrated that BCA reduced the expression levels of the endothelial dysfunction indexes E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) in a concentration-dependent manner. Immunofluorescence and western blotting illustrated that BCA increased the expression levels of zonula occludens-1 and decreased those of ICAM-1. Mechanistic studies showed that BCA inhibited activation of the NFκB pathway. In vivo experiments demonstrated that IL-6 was significantly increased in the rat model of ischemic necrosis of the femoral head, whereas BCA inhibited IL-6 production. Therefore, in Perthes disease, BCA may inhibit the NFκB pathway to suppress IL-6-EMP-induced endothelial dysfunction, and could thus be regarded as a potential treatment for Perthes disease.

Dietary sodium acetate and sodium butyrate attenuate intestinal damage and improve lipid metabolism in juvenile largemouth bass (Micropterus salmoides) fed a high carbohydrate diet by reducing endoplasmic reticulum stress

High-carbohydrate (HC) diets decrease the intestinal levels of sodium acetate (SA) and sodium butyrate (SB) and impair the gut health of largemouth bass; however, SA and SB have been shown to enhance immunity and improve intestinal health in farmed animals. Thus, the present study was to investigate the effects of dietary SA and SB on HC diet-induced intestinal injury and the potential mechanisms in juvenile largemouth bass. The experiment set five isonitrogenous and isolipidic diets, including a low-carbohydrate diet (9% starch) (LC), a high carbohydrate diet (18% starch) (HC), and the HC diet supplemented with 2 g/kg SA (HCSA), 2 g/kg SB (HCSB) or a combination of 1 g/kg SA and 1 g/kg SB (HCSASB). The feeding experiment was conducted for 8 weeks. A total of 525 juvenile largemouth bass with an initial body weight of 7.00 ± 0.20 g were used. The results showed that dietary SA and SB improved the weight gain rate and specific growth rate (P < 0.05) and ameliorated serum parameters (alkaline phosphatase, acid phosphatase, glutamate transaminase, and glutamic oxaloacetic transaminase) (P < 0.05). And, importantly, dietary SA and SB repaired the intestinal barrier by increasing the expression levels of zonula occludens-1, occludin, and claudin-7 (P < 0.05), reduced HC-induced intestinal damage, and alleviated intestinal inflammation and cell apoptosis by attenuating HC-induced intestinal endoplasmic reticulum stress (P < 0.05). Further results revealed that dietary SA and SB reduced HC-induced intestinal fat deposition by inhibiting adipogenesis and promoting lipolysis (P < 0.05). In summary, this study demonstrated that dietary SA and SB attenuated HC-induced intestinal damage and reduced excessive intestinal fat deposition in largemouth bass.

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

BackgroundLimosilactobacillus johnsoni (L. j) and Limosilactobacillus mucosae (L. m) can alleviate the inflammatory response. ObjectivesThis study aimed to elucidate the underlying mechanisms by which L. j- and L. m-derived extracellular vesicles (EVs) mitigate lipopolysaccharide (LPS)-induced intestinal injury. MethodsPiglets 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. ResultsLPS 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-1β, 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-1β, 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-1β, caspase-3, and bax gene expression in intestinal epithelial cells of the coculture system. Enzyme-treated EVs were less effective than native EVs. ConclusionsThis study suggests that EVs secreted by L. j and L. m control inflammation by modulating macrophage polarization, thereby improving intestinal barrier function.

Bile acids supplementation modulates lipid metabolism, intestinal function, and cecal microbiota in geese.

Bile acids(BAs) are important components of bile and play a significant role in fat metabolism. However, there is currently no systematic evaluation of the use of BAs as feed additives for geese.This study aimed to investigate the effects of adding BAs to goose feed on growth performance, lipid metabolism, intestinal morphology, mucosal barrier function, and cecal microbiota. A total of 168 28-day-old geese were randomly assigned to four treatment groups and fed diets supplemented with 0, 75, 150, or 300 mg/kg of BAs for 28 days. The addition of 75 and 150 mg/kg of BAs significantly improved the feed/gain (F/G) (p < 0.05).The addition of BAs decreased abdominal fat percentage and serum total cholesterol (TC) levels, with 150 mg/kg of BAs significantly reducing serum triglyceride levels and increased expression of Farnesoid X Receptor (FXR) mRNA in the liver(p < 0.05), 300 mg/kg of BAs significantly increasing the expression level of liver peroxisome proliferator-activated receptor α (PPARα) (p < 0.05). In terms of intestinal morphology and mucosal barrier function, 150 mg/kg of BAs significantly increased villus height (VH) and VH/crypt depth (CD) in the jejunum (p < 0.05). The addition of 150 and 300 mg/kg of BAs significantly reduced the CD in the ileum, while increasing VH and VH/CD (p<0.05). Additionally, the addition of 150 and 300 mg/kg of BAs significantly increased the expression levels of zonula occludens-1 (ZO-1) and occludin in the jejunum. Simultaneously 150mg/kg and 300mg/kg BAs increased the total short-chain fatty acids (SCFA) concentrations in the jejunum and cecum(p < 0.05).Supplementation with BAs resulted in a significant increase in the ɑ-diversity of cecal microbiota and a decrease in the abundance of Proteobacteria in the cecum. The addition of 150 mg/kg of BAs significantly reduced the abundance of Bacteroidetes and increased the abundance of Firmicutes. Moreover,Linear discriminant analysis Effect Size analysis (LEfSe) showed that the abundances of bacteria producing SCFA and bile salt hydrolases (BSH) were increased in the BAs-treated group. Furthermore, Spearman's analysis showed that the genus Balutia, which is negatively correlated with visceral fat area, was positively correlated with serum high-density lipoprotein cholesterol (HDL-C), while Clostridium was positively correlated with intestinal VH and VH/CD. In conclusion, BAs can be considered an effective feed additive for geese, as they increased SCFA concentration, improve lipid metabolism and intestinal health by enhancing the intestinal mucosal barrier, improving intestinal morphology, and altering the cecal microbiota structure.

The bio-derived material acacetin ameliorated hyperlipidemia and intestinal barrier damage in mice by modulating gut microbiota

The increasing incidence of hyperlipidemia has been linked to disruptions in gut flora. The bio-derived material, acacetin, is a naturally flavonoid compound extracted from various plants that has been shown to protect the liver and lowers blood lipid levels, its potential to mitigate gut barrier damage caused by a high-fat diet (HFD) has not been fully explored. This work aimed to investigate the effects of acacetin on HFD-induced intestinal barrier disruption and its potential interaction with gut microbiota regulation. Forty-eight female ICR mice were divided into three groups: control group (standard diet containing 6% daily energy from fat), HFD group (45% of daily energy from fat), and the acacetin (AC) group (HFD with acacetin 30 mg/kg body weight). The test period lasted for eight weeks. In addition to lipid metabolism parameters, serum lactate dehydrogenase (LDH) activity, diamine oxidase (DAO) activity, and lipid metabolism, we used the real-time fluorescence quantitative method to measure the expression of zonula occludens 1 (ZO-1) and occludin genes. The microorganisms were analyzed by 16 s RNA and functional gene prediction analysis. The results indicated that acacetin treatment could alter serum biochemical parameters and reduce body weight, liver weight gain, and abdominal fat accumulation. Furthermore, acacetin increased the expression levels of ZO-1 and occludin in HFD mice. In addition, acacetin altered the structure, diversity, and function of intestinal flora, characterized by the restoration of the Firmicutes/Bacteroidetes ratio. Additionally, the species abundance were significant correlation with lipid factors, DAO, and LDH. Alistipes and Acetatifactor were the prevalent genus in the AC group. Acacetin downregulated HFD-induced Facultative_anaerobic phenotypes related to Clostridium according to the BugBase analysis. The KEGG study revealed that acacetin altered the functional composition of microorganisms, as evidenced primarily by variations in the abundance of metabolic pathways involved in lipid metabolism and intestinal epithelial injury. The COG category showed acacetin increased the abundance of Cytoskeleton associated with the intestinal barrier. Overall, acacetin ameliorated HFD-induced hyperlipidemia and intestinal barrier damage in mice by modulating intestinal bacteria, exhibited a good clinical application prospect.

Wine-processed Chuanxiong Rhizoma enhances efficacy of aumolertinib against EGFRmutant non-small cell lung cancer xenografts in nude mouse brain

To investigate the effect of wine-processed Chuanxiong Rhizoma (WCR) for enhancing the efficacy of aumolertinib against xenografts of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) in the brain of nude mice. In a co-culture system of hCMEC/D3 and PC9 NSCLC cells, the effect of aqueous extract of WCR (2 mg/mL) combined with aumolertinib (10 and 20 μmol/L) on apoptosis of PC9 cells was investigated using flow cytometry. The effects of WCR extract (0.5, 1, and 2 mg/mL) on transmembrane transport of 8 μmol/L aumolertinib was examined in ABCB1-MDCK monolayer cells. Western blotting was used to detect the expressions of the tight junction proteins related with blood- brain barrier integrity. A nude mouse model bearing NSCLC xenograft in the brain was established to observe the inhibitory effect of WCR (1 mg/g) combined with aumolertinib (10 mg/kg) on tumor growth. Compared with aumolertinib (20 μmol/L) alone, WCR extract (2 mg/mL) combined with aumolertinib significantly increased the apoptosis rate of PC9 cells by 21% (P < 0.01). The combined treatment with WCR (0.5, 1, 2 mg/mL) obviously increased apical-basolateral transport of aumolertinib in ABCB1-MDCK monolayer cells (P < 0.05) and significantly lowered the expression levels of zonula occludens-1, claudin-5 and P-glycoprotein (P < 0.05). In the tumor-bearing mice, compared with aumolertinib alone, the combined treatment with WCR and aumolertinib produced stronger inhibitory effect on tumor growth, improved weight loss, and prolonged the survival time of the nude mice (P < 0.05). Pathological examination showed that the combined treatment obviously increased the apoptosis rate of the tumor cells and alleviated neural injuries in the brain. Immunohistochemistry revealed that WCR treatment significantly reduced the expressions of ZO-1 and claudin-5 in the brain of the mice. WCR combined with aumolertinib shows stronger inhibitory effects against tumor xenografts of EGFR-mutant NSCLC possibly due to the effect of WCR in facilitating the transmembrane transport of aumolertinib by downregulating ZO-1, claudin-5 and P-glycoprotein expression.

High concentrate diet induced inflammatory response and tight junction disruption in the mammary gland of dairy cows.

This study aimed to investigate the effect and mechanism of a high concentrate (HC) diet on the inflammatory response and cellular tight junctions (TJs) in the mammary gland of dairy cows. Twelve lactating Holstein dairy cows were randomly assigned into low concentrate (LC) and HC groups (n=6), which were fed with LC diet and HC diet respectively for 3 weeks. The HC diet lead to subacute ruminant acidosis with a rumen pH<5.6 more than 3h daily. The HC diet triggered an inflammatory response with increased levels of inflammatory cytokines in the lacteal vein, upregulated expression of inflammation-related genes, elevated activity of myeloperoxidase, and inflammatory cells infiltration in the mammary gland. Furthermore, the HC diet induced the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways with enhanced phosphorylation ratios of NF-κB P65, inhibitor of NF-κB (IκB), P38 and extracellular signal-regulated kinase 1/2 (ERK1/2) as well as decreased ratios of DNA methylation and chromatin compaction of genes coding for proinflammatory cytokines, which contributed to the upregulation of proinflammatory cytokine expression. The HC diet also destroyed the integrity of TJ with discontinuous and decreased expression levels of zonula occludens-1, Occludin, Claudin-4 and increased expression level of Claudin-1 in the mammary epithelial cells compared with LC group. Conclusively, the HC diet induced the activation of NF-κB and MAPK signaling pathways and epigenetic modifications, promoted the transcription of proinflammatory cytokines, and finally caused inflammatory response and TJ disruption in the mammary gland of dairy cows.

Restorative effects of Acetobacter ghanensis on the pathogenicity of gliadin-induced modulation of tight junction-associated gene expression in intestinal epithelial cells

Background/Aim: At present, a gluten-free diet is the only efficient way to treat celiac disease (CD). The development of novel approaches to lessen or counteract the pathogenic effects of gluten remains crucial for the treatment of CD. The aim in this investigation was to examine the restorative effects of Acetobacter ghanensis as a novel probiotic against gliadin-induced modulation in the barrier integrity of an intestinal epithelial cell (IEC) model (Caco-2). Methods: Fully differentiated Caco-2 cell monolayers were subjected to enzymatically digested gliadin with a pepsin and trypsin (PT) in the presence or absence of A. ghanensis for 90 min. The relative amounts of zonulin, zonula occludens-1 (ZO-1), claudin-1, and occludin mRNA expression were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Transepithelial electrical resistance (TEER) was evaluated to monitor the barrier integrity of cell monolayers. Statistical analyses were carried out using one- or two-way ANOVA followed by Tukey’s post-hoc analysis for multiple pairwise comparisons. Results: A significant upregulation (4.7-fold) of zonulin was noted in the PT-gliadin treated Caco-2 cells in comparison with the untreated controls (P&lt;0.001). Conversely, gliadin-induced zonulin expression was markedly downregulated in the Caco-2 cells following exposure to A. ghanensis in the presence of PT-gliadin (P&lt;0.001). Furthermore, prominent decreases in the mRNA expression levels of ZO-1 (45%) and occludin (40%) were seen in the PT-gliadin exposed Caco-2 cells compared to the untreated control cells (P&lt;0.001). PT-gliadin in the Caco-2 cells did not significantly alter the mRNA levels of claudin-1 (P=0.172). Similarly to zonulin expression, the decreasing effect of PT-gliadin on ZO-1 was completely attenuated in the PT-gliadin-administrated Caco-2 cells following exposure to A. ghanensis (P&lt;0.001). Conclusion: A. ghanensis restored the pathogenicity of PT-gliadin on intestinal barrier integrity.

Oryzanol Ameliorates DSS-Stimulated Gut Barrier Damage via Targeting the Gut Microbiota Accompanied by the TLR4/NF-κB/NLRP3 Cascade Response In Vivo.

Inflammatory bowel disease (IBD) is a global chronic disease with a long duration and repeated relapse. Currently, there is still a lack of effective approaches to prevent IBD. Food-derived oryzanol (ORY) possesses extensive biological activities, such as ameliorating bowel diseases, antioxidation, and antiobesity. However, the mechanism of ORY in preventing colitis remains unclear. The present research aims to explore the potential mechanism of ORY in dextran sulfate sodium (DSS)-stimulated colitis in a rat model. The results showed that the symptoms of colitis were significantly improved with the administration of ORY. Mechanismly, the expression levels of Zonula occludens-1 (ZO-1), Claudin-1, Occludin, MUC2, and TFF3 were elevated through ORY treatment, suggesting that oral ORY relieved the degree of gut barrier damage of colitis rats. Meanwhile, 16S sequencing results found that ORY supplementation increased the abundances of Alloprevotella, Roseburia, Treponema, Muribaculaceae, and Ruminococcus, which are associated with the synthesis of short-chain fatty acids (SCFAs). Moreover, GC-MS results confirmed that ORY supplementation reversed the DSS-induced reduction of acetic acid, butyric acid, and total acid. Further research indicated that ORY intervention downregulated the TLR4/NF-κB/NLRP3 pathway, which is closely linked to the expression of proinflammatory cytokines and colon injury. Taken together, ORY ameliorates DSS-stimulated gut barrier damage and inflammatory responses via the gut microbiota-TLR4/NF-κB/NLRP3 signaling axis.

MFE40—the active fraction of Mume Fructus alcohol extract—alleviates Crohn's disease and its complications

Ethnopharmacological relevanceMume Fructus (MF) is a well-known traditional Chinese medicine used to treat chronic cough, prolonged diarrhea, and other inflammation-related diseases. We previously confirmed the anti-colitis effect of its ethanol extract on a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced Crohn's disease (CD) rat model. However, the active ingredients and underlying mechanisms of MF remain unknown. Aim of the studyTo clarify the material basis and potential mechanism of the ethanol extract of MF (MFE) in alleviating CD and its complications, such as lung injury and intestinal obstruction. Materials and methodsMF was extracted with 80% ethanol aqueous solution and separated with 0, 40, and 100% ethanol aqueous solutions. MFE and its fractions were screened in a TNBS-induced CD rat model. For the bioactive fraction, the chemical composition was identified and quantified using ultrahigh-performance liquid chromatography coupled with diode-array detection and quadrupole time-of-flight tandem mass spectrometry. Interleukin (IL)-1β, IL-6, IL-17, transforming growth factor (TGF)-β, and lipopolysaccharide (LPS) levels in the colon, lungs, and/or plasma were detected using enzyme-linked immunosorbent assays. The expression levels of zonula occludens-1 (ZO-1) and occludin in the colon were measured using immunohistochemical staining, and the intestinal microbiota and short-chain fatty acid (SCFA) levels were analyzed using 16S rRNA gene sequencing and gas chromatography/mass spectrometry. ResultsThe 40% ethanol fraction of MF (MFE40), which mainly contained methyl citrate, ethyl citrate, and caffeoylquinic acid ethyl esters, was identified as the active fraction that could alleviate CD in rats. MFE40 could ameliorate inflammation and fibrosis in the colon and lung tissues by inhibiting the secretion of cytokines, such as IL-1β, IL-6, IL-17, and TGF-β, along with intestinal obstruction and lung injury in CD rats. The possible mechanisms of MFE40 were related to increased expression of ZO-1 and occludin in the colon, reduction in plasma LPS levels, and restoration of SCFAs via reduction in the relative abundance of Adlercreutzia, Clostridium_sensu_stricto_1, Erysipelatoclostridium, Faecalibaculum, norank_f_Erysipelotrichaceae, Phascolarctobacterium Coriobacteriaceae_UGG_002, and Allobaculum and increase in the relative abundance of Escherichia shigella, Christensenella, Acetivibrio_ethanolgignens, and Butyricicoccus. MFE40 had no significant influence on the inflammatory factors in healthy rats. ConclusionsCitrate esters and hydroxycinnamate esters are the main active constituents of MFE40. MFE40 exhibited a remission effect on CD rats and inhibited intestinal obstruction and lung injury via anti-inflammatory effects and regulation of the intestinal microbiota–gut–lung homeostasis.

Bovine lactoferricin ameliorates intestinal inflammation and mucosal barrier lesions in colitis through NF-κB/NLRP3 signaling pathways

• LfcinB ameliorates intestinal tissue morphology and distribution disorder of tight junction proteins in an established model of acute colitis. • LfcinB improves the production and distribution of mucin in colon of rats. • LfcinB alters the immune status of acute UC rats and inhibits the activities of inflammatory mediators. • NF-κB/NLRP3 signaling pathway involved the protection role of LfcinB on intestinal inflammation and mucosal barrier. Bovine lactoferricin (LfcinB) has biological properties such as anti-inflammatory and immunological activity. This study aimed to investigate the protective effects of LfcinB on mucosal injury during the pathological process of ulcerative colitis in rats. The thirty-two rats were randomly assigned to four groups: control (C group), colitis model (DSS group; oral intake of 5.0% dextran sodium sulfate), colitis model with LfcinB (LBD group; 5 mg/kg for 7 days, starting while DSS treatment) and LfcinB (LfcinB group; 5 mg/kg for 7 days) group. Treatment with LfcinB reduced the disease activity index and diamine oxidase concentration in rats with colitis. Histology analysis revealed a better preservation of the epithelium as well as attenuated inflammatory infiltration of the colon epithelium in LfcinB-treated rats. LfcinB treatment also downregulated the colonic expression of IL-1β and restored the expression levels of zonula occludens-1, occludin and mucin 2, by suppressing DSS-induced increases in inflammation and myeloperoxidase concentration in the rats of LBD group. During this process, the decreased expression levels of ASC, Caspase-1, NLRP3, and NF-κB in the rats of LBD group were characterized by quantitative RT-PCR. These results suggest LfcinB ameliorates DSS-induced changes in intestinal inflammation and mucosal barrier in colitis rats, possibly through NF-κB/NLRP3 signaling pathway.

Effects of magnolol and honokiol blend on performance, egg quality, hepatic lipid metabolism, and intestinal morphology of hens at late laying cycle

Magnolol and its isomer honokiol are polyphenols with anti-oxidative and anti-inflammatory activities. We evaluated the effects of magnolol and honokiol supplementation alone or in combination with hen diets during the late laying cycle. A total of 540 Jingfen pink-shell laying hens (50 weeks old) were randomly assigned to six treatments: a control diet and diets supplemented with 300 mg/kg magnolol (M300), honokiol (H300), or 300 mg/kg total phenols with a magnolol/honokiol ratio of 2:1 (M200H100), 1:2 (M100H200), and 1:1 (M150H150). Compared with that of the control, all supplementation groups had higher laying rates and the M300, M100H200, and M150H150 groups showed comparatively lower feed conversion ratios. Magnolol and honokiol supplementation increased the Haugh units of fresh eggs at week 62 and alleviated the decline of the Haugh units of eggs stored for 14 days. Compared with that of the control group, the serum total antioxidant capacity of the M100H200 and M150H150 groups significantly increased, and all supplementation groups had higher total antioxidant capacity and lower malondialdehyde content in the liver. With respect to lipid metabolism, the M200H100 and M150H150 groups had lower total and relative liver weights compared with those of the control and H300 groups. The mRNA expression levels of CCAAT enhancer binding protein alpha, sterol regulatory element binding protein-1, fatty acid synthase and stearyl coenzyme A desaturase 1 involved in lipogenesis; microsomal triglyceride transfer protein and apolipoprotein B involved in fatty acid transport; and the proinflammatory cytokine interleukin-1 beta were lower in all supplementation groups compared with those in the control. With respect to gut health, the heights of the jejunum and ileum villi significantly increased in all supplementation groups compared with those of the control, and the jejunum villus heights of the M300 and M150H150 groups were higher than those of the H300 and M100H200 groups. The H300 and M150H150 groups had higher mRNA expression levels of zonula occludens-1 in the ileum compared with those in the control and M300 groups, whereas all supplementation groups had higher mRNA levels of claudin-1 than that of the control group. In conclusion, magnolol and honokiol improved hen performance and the albumen quality of fresh and stored eggs by improving the antioxidant capacity, liver lipid metabolism, and intestinal health of laying hens. The combination of magnolol and honokiol at a 1:1 ratio may be an optimal choice for hen diet supplementation.

Preliminary study on time-dependent changes of intestinal tract and brain-gut axis in mice model of Parkinson's disease induced by paraquat

Objective: To observe the intestinal time-dependent changes in Parkinson's disease (PD) mouse model constructed by intraperitoneal injection of paraquat (PQ) and to establish the brain-gut axis connection initially. Methods: In October 2019, 48 mice were randomly divided into treated group and control groups: treated 4-week (P-4) group, treated 6-week (P-6) group, treated 8-week (P-8) group, control 4-week (C-4) group, control 6-week (C-6) group, and control 8-week (C-8) group. The treated group was injected with 15 mg/kg PQ solution and the control group was injected with 0.9% saline (0.2 ml/20 g) by intraperitoneal injection twice a week. After the initial state (0 weeks) and the treatment at the end of 4, 6 and 8 weeks, the mood changes and motor functions of mice were assessed by neurobehavioral tests (open field test, pole climbing test, tail suspension test and elevated plus maze test) . And the number of fecal pellets for 1 h and water content were calculated to assess the functional status of the gastrointestinal tract. Western blotting experiments were performed to detect the expression levels of α-synuclein (α-syn) and tyrosine hydroxylase (TH) in the nigrostriatal region of the mouse brain, the tight junction markers zonula occludens-1 (ZO-1) and Occludin, the inflammatory markers of integrin αM subunit (CD11b) , inducible nitric oxide synthase (iNOS) , high mobility group box 1 (HMGB1) , interleukin-1β (IL-1β) , and the neuronal markers βⅢ-tubulin and α-syn protein in the colon.Immunohistochemical staining was performed to detect the expression levels of colonic tight junction proteins ZO-1 and Occludin. Immunofluorescence staining was performed to detect the expression levels of TH in the substantia nigra region of the midbrain, and the co-localization of colonic intestine neuronal marker (βⅢ-tubulin) and Ser129 α-syn in the colonic. Results: Compared with the initial state (0 weeks) and C-8 group, mice in the P-8 group had significantly higher pole climbing test scores and resting time, and significantly lower total active distance, mean active speed, percentage of open arm entry and 1 h fecal instances (P<0.05) . After poisoning, the 1 h fecal water content of model mice first increased and then decreased, the P-4 and P-6 groups were significantly higher than the simultaneous point control group, and the P-8 groups were significantly lower than the initial state (P<0.05) . Compared with control, P-4 and P-6 groups, the expression levels of ZO-1 and Occludin in the P-8 group were significantly decreased (P<0.05) . Compared with control group, the expression levels of CD11b and IL-1β in the P-4 group were significantly increased (P<0.05) . Compared with control and P-4 group, the expression levels of CD11b, iNOS, HMGB1 and IL-1β in the P-6 and P-8 groups were significantly increased (P<0.05) . Compared with the control and P-4 groups, the expression levels of βⅢ-tubulin in the colon of mice in the P-8 group were significantly decreased, and the expression levels of α-syn and Ser129 α-syn were significantly increased (P<0.05) . The expression level of Ser129 α-syn in the colon of model mice was negatively correlated with the expression level of βⅢ-tubulin (r(s)=-0.9149, 95%CI: -0.9771--0.7085, P<0.001) . Ser129 α-syn and βⅢ-tubulin co-localization in the colonic intermuscular plexus region increased gradually with the time of exposure. Compared with the control, P-4 and P-6 groups, the expression level of TH in the nigrostriatal region of the brain was significantly decreased, and the expression levels of α-syn and Ser129 α-syn were significantly increased in the P-8 group (P<0.05) . Correlation analysis showed that the relative expression level of Ser129 α-syn in the nigrostriatal region of the brain was negatively correlated with the expression level of TH in the model mice (r(s)=-0.9716, 95% CI: -0.9925--0.8953, P<0.001) . Conclusion: The PD mouse model is successfully established by PQ, and the intestinal function of the model mice is reduced in a time-dependent manner. And on this basis, it is preliminary determined that the abnormal aggregation of α-syn may be an important substance connecting the brain-gut axis.

Effects of Xylooligosaccharides on Lipid Metabolism, Inflammation, and Gut Microbiota in C57BL/6J Mice Fed a High-Fat Diet.

Xylooligosaccharide (XOS) is a source of prebiotics with multiple biological activities. The present study aimed to investigate the effects of XOS on mice fed a high-fat diet. Mice were fed either a normal diet or a high-fat diet supplemented without or with XOS (250 and 500 mg/kg), respectively, for 12 weeks. The results showed that the XOS inhibited mouse weight gain, decreased the epididymal adipose index, and improved the blood lipid levels, including triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels. Moreover, XOS reduced the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and alleviated the damage to the liver caused by the high-fat diet. XOS also reduced hyperlipidemia-associated inflammatory responses. Additionally, quantitative real-time polymerase chain reaction results showed that XOS intervention activated the AMP-activated protein kinase (AMPK) pathway to regulate the fat synthesis, decomposition, and β oxidation; upregulated the mRNA expression levels of carnitine palmitoyl transferase 1 (CPT-1), peroxisome proliferator–activated receptors α (PPAR-α), and cholesterol 7-alpha hydroxylase (CYP7A1); and downregulated the mRNA expression levels of acetyl-CoA carboxylase (ACC), CCAAT/enhancer-binding protein alpha (C/EBPα), and lipoprotein lipase (LPL). On the other hand, XOS enhanced the mRNA expression levels of zonula occludens-1 (ZO-1), occludin, and claudin-1 in the small intestine; increased the strength of the intestinal barrier; and optimized the composition of the intestinal microbiota. Therefore, it was concluded that XOS regulated the intestinal barrier, changed the intestinal microecology, and played an important role in preventing hyperlipidemia through the unique anatomical advantages of the gut–liver axis.

Effects of Coated Cysteamine on Oxidative Stress and Inflammation in Weaned Pigs.

Simple SummaryOxidative stress and inflammation are common problems in livestock and poultry production, and have a serious impact on animal welfare and profitability. Finding an effective way to deal with oxidative stress and inflammation is an urgent problem for the modern pig industry. Cysteamine has been shown to play a crucial role in the growth performance, digestive metabolism, immune function, and fecundity of animals. In this study, we found that dietary supplementation of coated cysteamine could enhance the level of immunoglobulin and the expression of intestinal immune factors in the weaned pigs induced by diquat. It is proved that dietary supplementation with coated cysteamine can improve the intestine barrier function and immune function.This study aimed to explore the effects of dietary coated cysteamine on oxidative stress and inflammation in diquat-induced weaning pigs. Twenty-four pigs were randomly assigned to three dietary groups with eight replicates: the control (fed base diet), diquat (fed base diet), and coated cysteamine + diquat groups (fed 80 mg/kg cysteamine). The experiment was conducted for 21 d, and consisted of a pre-starter period (14 d) and a starter period (7 d). Coated cysteamine treatment significantly increased (p < 0.05) the final weight and average daily gain (ADG) in pigs. The contents of alkaline phosphatase (ALP), immunoglobulin G (IgG), serine (Ser), and isoleucine (Ile) were elevated (p < 0.05) while the contents of albumin (ALB) and aspartic acid (Asp) were reduced (p < 0.05) in the serum after coated cysteamine supplementation. Coated cysteamine supplementation resulted in greater (p < 0.05) serum superoxide dismutase (SOD) activity, the expression of interleukin-10 (IL-10) mRNA in the colon, and the CuSOD mRNA expression in the jejunum (p < 0.05) and colon (p = 0.073). Coated cysteamine supplementation showed an increasing trend in villus height (p = 0.060), villus height/crypt depth (V/C) (p = 0.056), the expression levels of zonula occludens-1 (ZO-1) mRNA (p = 0.061), and Occludin mRNA (p = 0.074) in the jejunum. In summary, dietary supplementation with coated cysteamine improves the intestinal barrier function of the jejunum by increasing the immunoglobulin content and the relative expression of intestinal immune factor mRNA in pigs while alleviating oxidative stress and inflammatory reactions caused by diquat.

Mixtures of natural antimicrobials can reduce Campylobacter jejuni, Salmonella enterica and Clostridium perfringens infections and cellular inflammatory response in MDCK cells

BackgroundThe classification of natural antimicrobials as potential antibiotic replacements is still hampered by the absence of clear biological mechanisms behind their mode of action. This study investigated the mechanisms underlying the anti-bacterial effect of a mixture of natural antimicrobials (maltodextrin, citric acid, sodium citrate, malic acid, citrus extract and olive extract) against Campylobacter jejuni RC039, Salmonella enterica SE 10/72 and Clostridium perfringens ATCC® 13124 invasion of Madin–Darby Canine Kidney cells (MDCK).ResultsMinimum sub-inhibitory concentrations were determined for Campylobacter jejuni (0.25%), Salmonella enterica (0.50%) and Clostridium perfringens (0.50%) required for the in vitro infection assays with MDCK cells. The antimicrobial mixture significantly reduced the virulence of all three pathogens towards MDCK cells and restored the integrity of cellular tight junctions through increased transepithelial resistance (TEER) and higher expression levels of ZO-1 (zonula occludens 1) and occludin. This study also identified the ERK (external regulated kinase) signalling pathway as a key mechanism in blocking the pro-inflammatory cytokine production (IL-1β, IL-6, IL-8, TNF-α) in infected cells. The reduction in hydrogen peroxide (H2O2) production and release by infected MDCK cells, in the presence of the antimicrobial mixture, was also associated with less tetrathionate formed by oxidation of thiosulphate (p < 0.0001).ConclusionThe present study describes for the first time that mixtures of natural antimicrobials can prevent the formation of substrates used by bacterial pathogens to grow and survive in anaerobic environments (e.g. tetrathionate). Moreover, we provide further insights into pathogen invasion mechanisms through restoration of cellular structures and describe their ability to block the ERK–MAPK kinase pathway responsible for inflammatory cytokine release

Lactobacillus delbrueckii alleviates depression-like behavior through inhibiting toll-like receptor 4 (TLR4) signaling in mice.

BackgroundThe intestinal flora can influence behavior through the microbiota-gut-brain axis and is closely related to the occurrence and development of nervous system diseases such as depression. Probiotics like Lactobacillus may regulate the balance of the intestinal flora and play an active role in preventing and treating depression.MethodsEight-week-old C57BL/6J mice (n=32) were randomly and equally divided into a normal control group, a control + Lac group, a model group, and a model + Lac group. The model and model + Lac groups were intraperitoneally injected with 1.2 mg/kg lipopolysaccharide for 7 days, and the behavior of the mice was assessed 24 hours later. The normal and model groups received intragastric administration of saline daily, while the control + Lac and model + Lac groups were given 109 cfu Lac intragastrically daily for 7 days. The inhibitory effect of Lac and its fermentation products on depression-related bacteria were examined in vitro.ResultsLac effectively inhibited the production of depression-like behaviors in mice. The expression levels of zonula occludens-1 (ZO-1) and E-cadherin in the small intestine in the model group were significantly decreased, but Lac abrogated this effect. Overactivation of microglia and decreased expression of dopamine transporter (DAT) in brain tissues, which are closely related to depression, were also abrogated by Lac treatment. Furthermore, the expression of toll-like receptor 4 (TLR4) and nod-like receptor protein-3 (NLRP3), as well as the level of interleukin-1 beta (IL-1β) in the intestine and brain, were all significantly increased; however, these effects were subsequently abrogated by Lac. Moreover, Lac inhibited dysbiosis through its metabolites.ConclusionsLac has a remarkable antidepressant function, which it performs through the inhibition of dysbiosis (via its metabolites) and pattern recognition receptor TLR4 signaling.

The effects of multispecies probiotic formulations on growth performance, hepatic metabolism, intestinal integrity and fecal microbiota in growing-finishing pigs

Probiotics have received attention as alternatives to antibiotics due to their relative biosafety. Despite the health-promoting effects of multispecies probiotic formulation (MPF) in weaning pigs were well-documented, the health-promoting potential of MPF in growing-finishing pigs remain limited. Therefore, we conducted a six-week experiment to investigate the beneficial effects of MPF supplementation (the formulations containing Lactobacillus plantarum CJLP243, L. fermentum LF21, L. salivarius E4101, Leuconostoc paramesenteroides KJP421, Bacillus subtilis CJMPB957 and B. licheniformis CJMPB283) on intestinal environments and hepatic lipid metabolism in growing-finishing pigs. The results showed that the growth performance of MPF group was improved compared to control group during the experimental period without affecting feed intake. The dietary MPF supplementation also significantly increased the jejunal mRNA expression levels of zonula occludens-1 (ZO-1) and decreased that of interleukin-12 (IL-12). Dietary MPF supplementation was found to not only reduce the hepatic triglyceride concentration but down-regulate hepatic mRNA expression levels of lipid metabolism-related markers, such as as sterol regulatory element-binding transcription factor 1c (SREBP-1c), CCAAT/enhancer binding protein α (CEBPα), acetyl coA carboxylase (ACC) and carnitine palmitoyl transferase 1β (CPT1β). Nonetheless, MPF supplementation also had the capability to modulate gut microbiota populations, including Clostridiaceae, Lachnospiraceae, and Turicibacter. Collectively, our results suggest that dietary MPF inclusion could be a promising approach to promote the growth performance and the overall health of growing-finishing pigs via modulation of gut microbiota.

LPS-induced epithelial barrier disruption via hyperactivation of CACC and ENaC.

Gram-negative bacterial lipopolysaccharide (LPS) increases the susceptibility of cells to pathogenic diseases, including inflammatory diseases and septic syndrome. In our experiments, we examined whether LPS induces epithelial barrier disruption in secretory epithelia and further investigated its underlying mechanism. The activities of Ca2+-activated Cl- channels (CACC) and epithelial Na+ channels (ENaC) were monitored with a short-circuit current using an Ussing chamber. Epithelial membrane integrity was estimated via transepithelial electrical resistance and paracellular permeability assays. We found that the apical application of LPS evoked short-circuit current (Isc) through the activation of CACC and ENaC. Although LPS disrupted epithelial barrier integrity, this was restored with the inhibition of CACC and ENaC, indicating the role of CACC and ENaC in the regulation of paracellular pathways. We confirmed that LPS, CACC, or ENaC activation evoked apical membrane depolarization. The exposure to a high-K+ buffer increased paracellular permeability. LPS induced the rapid redistribution of zonula occludens-1 (ZO-1) and reduced the expression levels of ZO-1 in tight junctions through apical membrane depolarization and tyrosine phosphorylation. However, the LPS-induced epithelial barrier disruption and degradation of ZO-1 were largely recovered by blocking CACC and ENaC. Furthermore, although LPS-impaired epithelial barrier became vulnerable to secondary bacterial infections, this vulnerability was prevented by inhibiting CACC and ENaC. We concluded that LPS induces the disruption of epithelial barrier integrity through the activation of CACC and ENaC, resulting in apical membrane depolarization and the subsequent tyrosine phosphorylation of ZO-1.

Manno-oligosaccharide attenuates inflammation and intestinal epithelium injury in weaned pigs upon enterotoxigenic Escherichia coli K88 challenge.

To explore the effect of manno-oligosaccharide (MOS) on intestinal health in weaned pigs upon enterotoxigenic Escherichia coli K88 (ETEC) challenge, thirty-two male weaned pigs were randomly assigned into four groups. Pigs fed with a basal diet or basal diet containing MOS (0·6 g/kg) were orally infused with ETEC or culture medium. Results showed that MOS significantly elevated the digestibility of crude protein and gross energy in both ETEC-challenged and non-challenged pigs (P < 0·05). MOS also elevated serum concentrations of IgA and IgM (P < 0·05), but decreased serum concentrations of TNF-α, IL-1β and IL-6 (P < 0·05) in ETEC-challenged pigs. Interestingly, MOS increased villus height and the ratio of villus height:crypt depth in duodenum and ileum (P < 0·05). MOS also increased duodenal sucrase and ileal lactase activity in ETEC-challenged pigs (P < 0·05). MOS decreased the abundance of E. coli, but increased the abundance of Lactobacillus, Bifidobacterium and Bacillus in caecum (P < 0·05). Importantly, MOS not only elevated the expression levels of zonula occludens-1 (ZO-1), claudin-1 and GLUT-2 in duodenum (P < 0·05) but also elevated the expression levels of ZO-1, GLUT-2 and L-type amino acid transporter-1 in ileum (P < 0·05) upon ETEC challenge. These results suggested that MOS can alleviate inflammation and intestinal injury in weaned pigs upon ETEC challenge, which was associated with suppressed secretion of inflammatory cytokines and elevated serum Ig, as well as improved intestinal epithelium functions and microbiota.