Occludin mRNA Research Articles

Effects of dietary β-1,3-glucan addition on the growth performance, mRNA expression in jejunal barrier, and cecal microflora of broilers challenged with Clostridium perfringens

This experiment aimed to explore the interaction of β-1,3-glucan and Clostridium perfringens on the growth performance, intestinal health and cecal microflora of broilers. A total of 384 one-day-old Arbor Acre broilers were sorted into 4 treatments with 6 replications. There were 2 factors in this trial: dietary β-1,3-glucan addition including 0 and 250 mg/kg, intestinal enteritis challenged with Clostridium perfringens attack or not. Results showed that Clostridium perfringens infection disrupted the integrity of the intestinal mucosa by reducing the jejunal Occludin and Claudin-1 mRNA expression of broiler chickens at 21 d of age (P < 0.05). Meanwhile, when considering Clostridium perfringens as the main effect, it also decreased the mRNA expression of the glucose transporter recombinant sodium/glucose cotransporter 1 (SGLT1) at d 21 and the fatty acid transporter liver fatty acid-binding protein (L-FABP) at d 42 (P < 0.05) as well as affect cecum microbial diversity, especially in relative abundance of Firmicutes and Bacteroidetes. In addition, Clostridium perfringens infection reduced body weight, daily weight gain, and feed-gain ratio (FCR) in broilers at d 42 (P < 0.05). The dietary β-1,3-glucan could alleviate intestinal mucosal damage caused by the Clostridium perfringens to some extent. When considering β-1,3-glucan as the main effect, it increased the SGLT1 at 42 d of age (P < 0.05), and stabilized gut microbiota disorder caused by Clostridium perfringens. More over dietary β-1,3-glucan addition increased body weight at 42-day-old (P < 0.05), and improved daily weight gain and FCR during 1 to 42 d (P < 0.05). In conclusion, dietary β-1,3-glucan could improve growth performance and intestinal health in broilers infected with Clostridium perfringens.

Maternal obesity exacerbates the responsiveness of offspring BALB/c mice to cow’s milk protein-induced food allergy

Food allergy has become a significant public health problem affecting a large number of people worldwide. Maternal obesity causes inflammation and alters the immune system of offspring, which may exacerbate their food allergy. The aim of this study was to determine whether offspring mice born to obese mothers would have more serve reactions to cow’s milk protein-induced food allergy, and further investigate the underlying mechanisms. Female offspring BALB/c mice of mothers with normal and high-fat diets were sensitized with β-lactoglobulin (BLG), respectively. Maternal obesity increased the serum immunoglobulin E and mouse mast cell protease levels, though did not have significant influence on anaphylactic symptom score, core temperature and diarrhea rate of offspring mice after BLG sensitization. Furthermore, maternal obesity led to a lower level of occludin mRNA expression in BLG -sensitized mice. The mice born to obese mothers exhibited increased mRNA expression levels of GATA-3, interleukin (IL)-4 and IL-10 in jejunum after BLG sensitization, indicating maternal obesity intensified Th2-type biased immune responses. In conclusion, maternal obesity exerted exacerbating effects on the responsiveness of their offspring to cow’s milk protein sensitization.

In vivo evidence of the prevents DSS-induced colitis of Lactiplantibacillus plantarum L15.

Ulcerative colitis (UC) is challenging to treat and severely impacts patients and families. A previous study reported immunomodulatory and reduction of pro-inflammatory properties for the Lactiplantibacillus plantarum L15. This study aimed to analyze the preventive properties and mechanistic actions in an in vivo colitis model. The histopathological alteration, inflammation cytokines, and intestinal barrier function were analyzed. Subsequently, the cecal gut microbiota contents and products from different groups were detected. Finally, gene expressions related to the NF-κB signaling process were evaluated. L. plantarum L15 significantly decreased disease activity index (DAI), myeloperoxidase activity (MPO), pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) level, and increased weight change, colon length, and production of inflammation-suppressing cytokines. Furthermore, this strain supplementation substantially increased ZO-1, Occludin, and Claudin-1, and MUC2 mRNA expression levels with a corresponding decrease in serum lipopolysaccharide and D-lactic acid contents. In addition, L. plantarum L15 improved gut microbiota composition and increased short-chain fatty acid (SCFAs) in the colon content, which significantly reduced the transfer of NF-κB p65 to the nucleus. Our findings provide a theoretical basis for L. plantarum L15 as a preventive candidate for UC.

Pulsatilla decoction improves DSS-induced colitis via modulation of fecal-bacteria-related short-chain fatty acids and intestinal barrier integrity

Ethnopharmacological relevancePulsatilla decoction (PD), is an herbal formula commonly used for the treatment of ulcerative colitis (UC) in clinical practice, but the mechanism of PD alters the colitis remains elusive. Aim of the studyTo evaluate the intervention effect of PD on Dextran Sodium Sulfate (DSS)-induced UC based on gut microbiota and intestinal short-chain fatty acid (SCFAs) metabolism, and to investigate the mechanism of action of PD in treating UC. Materials and methodsA 3% (wt/vol) DSS-induced ulcerative colitis model in C57BL/6 male mice was used to evaluate the effect of oral PD in treating UC. The changes in gut microbiota in mice were analyzed by 16SrDNA gene sequencing, and the content of SCFAs in the intestinal contents of mice was determined by gas chromatography-mass spectrometry (GC-MS). Enzyme-linked immunosorbent assay (ELISA) was applied to analyze the expression of inflammatory cytokines in serum and colonic tissues, and western blotting (WB) was applied to analyze the expression of tight junction proteins in colonic tissues. ResultsPD can alleviate the symptoms of UC mice, Pulsatilla Decoction high dose treatment group (PDHT) shows the best effect. Compared with the DSS group, the PDHT had significantly lower body mass, disease activity index (DAI) score, colonic macroscopic damage index (CMDI) score, and pathological damage score, at the phylum level, the relative abundance of Bacteroidetes increased while that of Firmicutes and Proteobacteria decreased, at the Genus level, the abundance of Bacteroides and Lachnospiraceae.NK4A136.group increased while that of Clostridium. sensu.stricto。, Escherichia. shigella and Turicibacter decreased. Compared with the DSS group, acetate, propionate, and total SCFAs in the PDHT with significantly higher levels. The concentrations of interleukin-1β (L-1β), tumor necrosis factor-alpha (TNF-α), and interleukin-17 (IL-17) decreased whereby the concentration of interleukin-10 (IL-10) increased in the PDHT group. The expression levels of Occludin, zonula occludens-1 (ZO-1), Claudin1, Claudin5, G protein-coupled receptor43 (GPR43) protein, and the relative expression of ZO-1 and Occludin mRNA were significantly increased PDHT group. ConclusionsPD has a good therapeutic effect on UC mice. The pharmacological mechanism is probably maintaining the homeostasis and diversity of gut microbiota, increasing the content of SCFAs, and repairing the colonic mucosal barrier.

Pathological mechanism of intestinal mucosal barrier injury of large intestine dampness-heat syndrome rats and the protective effect of Yujin powder

Large intestine dampness-heat syndrome (LIDHS) is frequently-occurring in the inflammatory intestinal disease of animals and human. Yujin powder (YJP) is a classical prescription for treating LIDHS. To explore the pathological mechanism of intestinal mucosal barrier injury of LIDHS and the protection of YJP, the LIDHS rat model was established through imitating the inducing conditions of LIDHS and treated with YJP. The integrity of ileal and colonic mucosa was detected through histopathological examination. The serum DAO, D-LA and ET levels were detected by ELISA. The mRNA and protein expression levels of Occludin, ZO-1 and MUC2 in ileum and colon were detected using RT-PCR and immunohistochemistry methods, respectively. The results showed that the ileal and colonic epithelium of LIDHS rats were destroyed; the serum DAO, D-LA and ET levels were significantly increased; the mRNA and protein expression levels of Occludin, ZO-1 and MUC2 in ileum and colon were all abnormally expressed. After treatment with YJP, the mucosal integrity was restored; the levels of serum DAO, D-LA and ET, mRNA and protein levels of Occludin and ZO-1 in ileum and colon and MUC2 in ileum were back-regulated; however, MUC2 level in colon was further increased. The results demonstrated that the intestinal mucosal barrier was damaged in LIDHS rats and Occludin, ZO-1 and MUC2 were abnormally expressed, and YJP could repair the intestinal mucosal barrier through up-regulating the expression of Occludin and ZO-1 in ileum and colon as well as MUC2 in colon and down-regulating MUC2 in ileum.

Activation of cannabinoid receptor 2 alleviates acute lung injury in rats with lipopolysaccharide-induced sepsis

To investigate the protective effect of cannabinoid receptor 2 (CB2) activation against acute lung injury in rats with lipopolysaccharide (LPS)-induced sepsis and explore the underlying mechanism. Forty-eight SD rats were randomly assigned into control group, model group, CB2 agonist group and P38 MAPK inhibitor group (n=12). In the latter 3 groups, the rats received intraperitoneal injection of LPS to induce sepsis, and the control rats were given saline injection. In CB2 agonist group, JWH133 (3 mg/kg) was injected intraperitoneally 30 min before LPS injection; in P38 MAPK inhibitor group, the rats received intraperitoneal injection of SB203580 (5 mg/kg) 30 min prior to JWH133 injection. The changes in lung histopathology, water content, fluid clearance rate, inflammatory factors, pulmonary expressions of CB2 and tight junctionrelated genes, and phosphorylation of P38 MAPK in the lung tissues were examined. The rat models of sepsis showed severe damage of alveolar structures with significantly decreased fluid clearance rate, lowered pulmonary expressions of CB2, occludin and ZO-1 mRNA and proteins, increased water content in the lung tissue, and increased phosphorylation level of P38 MAPK and TNF-α and IL-1β levels in lung lavage fluid (all P < 0.05). Treatment with JWH133 improved alveolar pathology in the septic rats, but there was still inflammatory infiltration; lung tissue water content, phosphorylation of P38 MAPK, and TNF-α and IL-1β levels in lung lavage fluid were all significantly decreased, and the fluid clearance rate, pulmonary expressions of CB2, occludin and ZO-1 were significantly increased (all P < 0.05). Additional treatment with SB203580 resulted in further improvements of alveolar pathologies, lowered phosphorylation levels of P38 MAPK in the lung tissue and TNF-α and IL-1β levels in lung lavage fluid, and increased the protein expressions of occludin and ZO-1 (P < 0.05) without causing significant changes in mRNA and protein expression of CB2 (P > 0.05). In rats with LPS-induced sepsis, activation of CB2 can inhibit the p38 MAPK signaling pathway, reduce the release of inflammatory factors in the lung tissues, promote tight junction protein expressions, and thus offer protection against acute lung injury.

Effect of Dietary Lactose Supplementation on Growth Performance and Intestinal Epithelium Functions in Weaned Pigs Challenged by Rotavirus.

Simple SummaryLactose is a kind of carbohydrate that exists in mammal milk. It has some physiological functions, such as providing energy, regulating gut microbiota, and affecting immunity. Rotavirus (RV) is the main pathogen that induces severe diarrhea in piglets, which impairs their growth and development. In this study, we investigated whether different levels (4% and 6%) of dietary lactose supplementation alleviates RV-induced diarrhea in weaned piglets. The results showed that lactose administration relieved the negative effect of RV on growth, which was derived from the improvement of nutrient utilization, gut barrier function, and immunity. Moreover, supplementing 6% lactose in the diets had a tendency to alleviate diarrhea in RV-infected piglets. Thus, we suggest that the diet of weaned piglets should be supplemented with more than 4% lactose (especially in the early period of weaning) if the cost of feed can be afforded.The purpose of this study was to investigate whether dietary lactose supplementation relieves rotavirus (RV)-induced diarrhea and gut dysfunction. Thirty-six crossbred weaned piglets were randomly allocated into three groups and fed diets containing 0, 4%, and 6% lactose for 20 days. On Day 15, half of the piglets in each group were orally infused with RV. RV infection impaired growth performance; induced severe diarrhea; decreased serum D-xylose concentration and morphology and sIgA level of jejunal mucosa; downregulated MUC1, MUC2, occludin, Bcl-2, IL-4, pBD3, pBD2, and pBD1 mRNA expression of jejunal mucosa and/or mesenteric lymph nodes; upregulated Bax, caspase-3, IL-2, IFN-γ, and IFN-β mRNA expression of jejunal mucosa and/or mesenteric lymph nodes; and damaged microbiota and metabolites of cecal digesta in weaned piglets (p < 0.05). Dietary lactose supplementation improved nutrient digestibility and growth performance and relieved the negative influence of RV challenge on intestinal barrier function, mRNA expression of cytokines, and host defense peptides of jejunal mucosa and/or mesenteric lymph nodes in weaned piglets (p < 0.05). Dietary administration of 6% lactose tended to relieve diarrhea (p = 0.07). These results suggest that lactose in feed increases growth performance and has a tendency to alleviate RV-induced diarrhea, derived from the improvement of nutrient utilization, gut barrier function, and immunity.

Rhizoma curcumae Longae ameliorates high dietary carbohydrate-induced hepatic oxidative stress, inflammation in golden pompano Trachinotus ovatus

In general, starch, as a complex carbohydrate, is the most economical energy source in aquaculture for its relatively low cost. However, excessive dietary levels of carbohydrate result in pathological conditions. An 8-week feeding trial with CT (control diet, containing 21% carbohydrate), HC (a high-carbohydrate diet, containing 50% carbohydrate) and HCR (a HC diet supplemented with 0.015% Rhizoma curcumae Longae) was performed to investigate the protective effect of curcumin on high-carbohydrate-induced hepatic oxidative stress and intestine lesion in juvenile Trachinotus ovatus. In the current study, HC group significantly decreased WGR, SGR, plasma CAT activity, intestinal C4 levels, hepatic Nrf2, Keap1, Bach1, HO1, CAT, and GPX mRNA expression as well as ZO-1, Occludin, and Claudin-3, TGF-β mRNA transcription levels, while the opposite was true for plasma AST activity, hepatic MDA contents, intestinal Claudin-15, NF-κB, IL-1β, IL-6, and TNF-α mRNA expression. In contrast with the HC group, the HCR group significantly increased the activities of hepatic CAT, SOD, intestinal C3, C4, IgG and LZM levels, hepatic Nrf2, Bach1, CAT, and GPX mRNA expression as well as intestinal ZO-1, Occludin, Claudin-3, TGF-β and IL-10 mRNA expression levels, but the opposite trend was found in plasma triglyceride content, hepatic lipid deposition, hepatic Keap1 mRNA level as well as intestinal NF-κB, IL-6. In conclusion, high-carbohydrate diet can cause detrimental effect on physiological health status in Trachinotus ovatus, while adding Rhizoma curcumae Longae can improve hepatic and intestinal health status via attenuating the oxidative stress, inflammation, and reducing lipid deposition.

Effects of early post-hatch feeding on the growth performance, hormone secretion, intestinal morphology, and intestinal microbiota structure in broilers

This study aimed to investigate the effects of time access to post-hatch feeding on the growth performance, hormone secretion, intestinal morphology, and intestinal microbiota structure of broilers. A total of 900 broilers were randomly allocated to 3 treatment groups, with 6 replicates of 50 broilers each. The 3 treatments were: immediate feeding (Group 2 h), delayed access to feed for 24 h (Group 24 h), and delayed access to feed for 48 h (Group 48 h). The experiment lasted for 50 d. Results revealed that Group 2 h had a higher average daily gain (ADG) and average daily feed intake (ADFI) as well as a lower feed-to-gain ratio (F/G) than Group 48 h during the starter period (P < 0.05). Compared with Group 48 h, broilers in Group 2 h exhibited significantly elevated villus height (VH) and villus height to crypt depth ratio (VH: CD) in the duodenum, increased Occludin, and Claudin-1 mRNA expression in the jejunum but decreased crypt depth (CD) in the duodenum at 50 d (P < 0.05). Meanwhile, broilers in Groups 2 h and 24 h had increased glycogen (Gn) and protein (Pro) levels in breast muscle and TG levels in the liver, as well as a higher concentration of serum T3, T4, and IGF-1 compared with Group 48 h at 21 d (P < 0.05). Besides, intestinal microbiota consisted primarily of Firmicutes, Bacteroidetes, and Proteobacteria at the phylum level at 21 d and 50 d; at the genus level, broilers in Group 2 h displayed significantly reduced abundance of Escherichia at 21 d and Bacteroides at 50 d compared with Group 48 h (P < 0.05). Collectively, these findings signal that early post-hatch feeding measures, especially at 21 d, improve hormone secretion, intestinal morphology, and the growth performance of broilers by enhancing intestinal health and modulating the intestinal microbiota.

Dietary Glucose Ameliorates Impaired Intestinal Development and Immune Homeostasis Disorders Induced by Chronic Cold Stress in Pig Model.

Endotherms are easily challenged by chronic cold stress. In this study, the development and injury of the small intestine in the Min pig model and Yorkshire pig model under chronic cold stress, and the molecular mechanisms by which glucose supplementation reduces small intestinal mucosal damage were investigated. The results showed that morphological structure lesions of the jejunal mucosa and ileal mucosa were visible in Yorkshire pigs under chronic cold stress. Meanwhile, the Occludin mRNA and protein expression in jejunal mucosa of Yorkshire pigs was decreased. Chronic cold stress enhanced the expression of Toll-like receptor 4 (TLR4), the myeloid differentiation main response 88 (MyD88), nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3), cleaved caspase-1, mature-IL-1β, and high-mobility group box 1 (HMGB 1) mRNA and protein expression in jejunal mucosa of Yorkshire pigs, whereas the mRNA and protein of Bax was triggered in ileal mucosa. In Min pigs, no such deleterious consequences were observed. Dietary glucose supplementation ameliorates small intestinal mucosal injury, declined TLR4 and MyD88 expression in jejunal mucosa. In conclusion, chronic cold stress induced the small intestinal mucosa damage in Yorkshire pigs, whereas glucose supplementation mitigated the deleterious effects of chronic cold stress on the small intestine.

Tryptophan Supplementation Enhances Intestinal Health by Improving Gut Barrier Function, Alleviating Inflammation, and Modulating Intestinal Microbiome in Lipopolysaccharide-Challenged Piglets.

Tryptophan (Trp) can modify the gut microbiota. However, there is no information about the effect of Trp on intestinal microbiota after lipopolysaccharide (LPS) challenge. This study aimed to investigate the effect of Trp on intestinal barrier function, inflammation, antioxidant status, and microbiota in LPS-challenged piglets. A total of 18 weaned castrated piglets were randomly divided into three treatments with 6 replicate per treatment, namely, (i) non-challenged control (CON); (ii) LPS-challenged control (LPS-CON); and (iii) LPS + 0.2% Trp (LPS-Trp). After feeding with control or 0.2% tryptophan-supplemented diets for 35 days, pigs were intraperitoneally injected with LPS (100 μg/kg body weight) or saline. At 4 h post-challenge, all pigs were slaughtered, and colonic samples were collected. The samples were analyzed for gut microbiota, fatty acids, antioxidant parameters, and the expression of mRNA and protein. The community bar chart showed that Trp supplementation to LPS-challenged pigs increased the relative abundance of Anaerostipes (P < 0.05) and tended to increase the relative abundance of V9D2013_group (P = 0.09), while decreased the relative abundance of Corynebacterium (P < 0.05) and unclassified_c__Bacteroidia (P < 0.01). Gas chromatography showed that Trp increased the concentrations of acetate, propionate, butyrate, and isovalerate in the colonic digesta (P < 0.05). Trp reduced the mRNA level of pro-inflammatory cytokines (P < 0.01), and increased mRNA level of aryl hydrocarbon receptor, cytochrome P450 (CYP) 1A1 and CYP1B1 (P < 0.05). Correlation analysis results showed that acetate, propionate, and butyrate concentrations were positively correlated with mRNA level of occludin and CYP1B1 (P < 0.05), and were negatively correlated with pro-inflammatory cytokines gene expression (P < 0.05). Isovalerate concentration was positively correlated with catalase activity (P < 0.05), and was negatively correlated with pro-inflammatory cytokines gene expression (P < 0.05). Furthermore, Trp enhanced the antioxidant activities (P < 0.01), and increased mRNA and protein expressions of claudin-1, occludin, and zonula occludens-1 (P < 0.01) after LPS challenge. These results suggest that Trp enhanced intestinal health by a modulated intestinal microbiota composition, improved the short chain fatty acids synthesis, reduced inflammation, increased antioxidant capacity, and improved intestinal barrier function.

Evaluation of LncRNA uc.173 and Occludin in Iranian Patients with Inflammatory Bowel Disease Compared with Healthy Individuals

Background &amp; Objective: Inflammatory bowel disease (IBD) is characterized by the chronic gastrointestinal inflammation. The two common forms of IBD are ulcerative colitis (UC) and Crohn’s disease (CD) that are distinguished by their location and depth of involvement in the diffuse inflammation of the colonic mucosa and affects the rectum (proctitis). A novel class of LncRNAs transcribed from ultra-conserved regions (UCRs) is a recently identified ultra-conserved region (T-UCR) transcript that is involved in the cellular function in a variety of pathways. However, the regulation of LncRNA uc.173 in IBD remains to be fully elucidated. In this study, we aimed to examine the expression of LncRNA uc.173 and Occludin genes in an Iranian population with inflammatory bowel disease.&#x0D; Materials &amp; Methods: This case-control study was performed on 33 inflammatory bowel disease patients including 13 Crohn’s disease, 20 ulcerative colitis and 20 healthy controls. The mRNA levels of LncRNA uc.173 and Occludin genes were assessed using the quantitative Real-time polymerase chain reaction. The B2M was used as an internal control. The 2 -ΔΔCq method was used to determine the expression fold changes.&#x0D; Results: Statistically, the level of the LncRNA uc.173 gene expression between the UC and normal tissues increased significantly (P=0.0024). Also, the expression analysis revealed no significant difference between the samples of CD patients compared to the controls (P&gt;0.05). In order to further evaluate the role of LncRNA uc.173 in IBD, the associations between the transcript levels of the LncRNA uc.173 and Occludin mRNA demonstrated significant difference in the CD tissue (R=0.59; P=0.002). In our study, the mRNA expression of Occludin gene did not show any changes in the IBD patients compared to the healthy controls.&#x0D; Conclusion: The increased expression of LncRNA uc.173 in the tissues of UC patients may be considered as a diagnostic or prognostic biomarker. Also, there was no correlation found between Occludin and LncRNA uc.173 expressions in the IBD patients’ tissues.

Danhong injection combined with tPA protects the BBB through Notch-VEGF signaling pathway on long-term outcomes of thrombolytic therapy

Current therapy for ischemic stroke primarily relies on tissue plasminogen activator (tPA), but it is limited by narrow treatment time window, bleeding complications and neurotoxicity. The preliminary study of tPA plus Danhong injection (DHI) shows that it can significantly reduce the side effects of tPA and improve its thrombolytic effect, but the mechanism of this action has not been further studied. In this study, the rats were randomly divided into sham group, vehicle group, DHI group (4 mL/kg), tPA group (5 mg/kg) and DHI+tPA group (4 mL/kg+ 2.5 mg/kg), administered intravenously 4.5 h since focal embolic stroke modeling. After 3 days and 7 days of cerebral ischemia, the neurological function of each treatment group was significantly improved compared with the vehicle group. The combination of DHI and tPA significantly reduced Evans blue (EB) penetration as well as the expressions of the proteins MMP-9, PAI-1 and P-selectin, while upregulating the expressions of claudin-5, occludin, and ZO-1 mRNA. Furthermore, the effect of continuous 7-day treatment was more conspicuous than 3-day treatment. Then, it significantly reduced the expressions of the proteins DLL-4 and VEGFR-2, increased the expressions of Notch-1, HIF-1α and HES-1 mRNA, and promoted the expressions of VEGF/HIF-1α-positive cells at 14 days following stroke. Hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM) also showed that it improved pathological changes of ischemic brain tissue and the cerebral cortex micro-structure. These indicate that DHI combined with tPA may significantly ameliorate blood-brain barrier (BBB) disruption by activating Notch-VEGF signaling pathway to promote angiogenesis for long-term outcomes.

Lactobacillus acidophilus Supplementation Restores Gut Epithelial Integrities and Barrier Functions in Non-specific Diarrhea

Objectives: The study was aimed to evaluate the roles of Lactobacillus acidophilus supplementation in maintaining intestinal epithelial integrities, tight junction proteins, and adhesion molecules in non-specific diarrhea.&#x0D; Methods: In this study, we used the pre-weaned BL/6 pups (3 weeks of age, same litters) as a model animal. We supplied the non-sterilized and poor-quality water to experimental pups (n=7) to develop non-specific diarrheic symptoms. Then diarrheic pups were supplemented with Lactobacillus acidophilus (LA) for three consecutive weeks. The control group (n=5) was supplied with sterilized water and no LA. The sampling and analysis were performed on day 0, day 7, day 14, and day 21. The expressions of pro-inflammatory cytokines (IL-6, TNF-a) and tight junction proteins (TJPs) of gut mucosa were determined using qRT-PCR. And the serum cytokines level was screened through sandwich ELISA.&#x0D; Results: The intestinal cytoskeletal integrity becomes disrupted and characterized by lower ZO-1, Occludin, Claudin-1, Claudin-5, and JAM mRNA expressions upon real-time qRT-PCR. However, Claudin-4 was found to be not affected and illustrated with a higher expression like control pups. Interestingly, supplementing Lactobacillus acidophilus was found to maintain gut integrity and effectively reduce diarrheic symptoms. Like the control pups, the Lactobacillus acidophilus supplemented pups exhibited a higher expression of gut epithelial TJPs and adhesion molecules. Moreover, the diseased pups produced significantly increased IL-6, and TNF-α production in blood serum, compared to control BL/6 pups. &#x0D; Conclusion: We concluded that L. acidophilus supplementation might orchestrate the equilibrium of gut health and immunity against non-specific diarrhea.

Curcumin Improved Intestinal Epithelial Barrier Integrity by Up-Regulating ZO-1/Occludin/Claudin-1 in Septic Rats.

Objective To investigate the protective effect and mechanism of curcumin on intestinal barrier function in rats with enterogenic sepsis. Methods Rats were divided into Sham group (Sham), Model group (Model), low-dose curcumin group (100 mg/kg), and high-dose curcumin group (200 mg/kg), with 10 rats in each group. Sepsis model was established in model group, low-dose curcumin group, and high-dose curcumin group. After drug intervention, hematoxylin-eosin (HE) staining was used to observe the histopathological changes of small intestine in each group. The levels of TNF-α, IL-1β, and IL-6 in serum and intestinal tissues of rats were determined by ELISA. The expression of ZO-1, occludin, and claudin-1 in ileum was detected by QRT-PCR and Western blot. Western blotting was used to detect the expression of ERK/JNK signaling pathway, NF-κB p65, apoptosis-related proteins Caspase-3, and TNF-α in rat intestinal tissues. Results HE staining showed that curcumin treatment reduced epithelial cell shedding, interstitial edema, and apoptosis. Compared with model group, DAO activity, serum intestinal fatty acid binding protein (I-FABP), TNF-α, IL-6, and IL-1β expression in curcumin group were decreased in a dose-dependent manner. Curcumin can upregulate the mRNA and protein expression levels of ZO-1, occludin, and claudin-1 in ileum of CLP-induced rats. In addition, curcumin inhibits NF-κB p65 activation and apoptosis by regulating ERK/JNK signaling pathway. Conclusion Curcumin can reduce inflammatory response and upregulate the expression of intestinal tight junction proteins ZO-1, occludin, and claudin-1 in rats with enterogenic sepsis, and protect intestinal barrier function.

Protective Effect of Lemon Essential Oil and Its Major Active Component, D-Limonene, on Intestinal Injury and Inflammation of E. coli-Challenged Mice.

Inflammatory diseases are a major threat to public health. Natural plant essential oils (EOs) possess anti-inflammatory and anti-oxidative activities. The objective of this study was to investigate the anti-inflammatory effect and mode of action of lemon essential oil (LEO), and its main active component, d-limonene, with different doses on intestinal inflammation of mice. Sixty-four 5-week-old male balb/c mice weighing 22.0 ± 1.5 g were randomly assigned into one of 8 treatments (n = 8/treatment), including normal saline group (NS), Escherichia coli (E. coli) group, and either LEO and d-limonene essential oil (DEO) group supplemented at 300, 600, and 1,200 mg/kg of BW, respectively. After the pre-feeding period, the mice were fasted for 12 h, the mice in the NS group and the E. coli group were gavaged with normal saline, and the mice in the LEO group and DEO group were gavaged with respective dose of EOs for 1 week. One hour after the end of gavage on the 7th day, except that the mice in the normal saline group were intraperitoneally injected with normal saline, the mice in the other groups were intraperitoneally injected with the same concentration of E. coli (108 cfu/ml, 0.15 ml per mouse). The antioxidant indexes were measured including superoxide dismutase (SOD), malondialdehyde (MDA), and myeloperoxidase (MPO) in plasma obtained by taking blood from mouse eyeballs. The inflammatory indexes were measured including interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor alpha (TNF-α) in plasma. The tight junction protein indicators were tested include zona occludens 1 protein (ZO-1), occludin and claudin in mouse duodenum. We found that all of the above indexes for E. coli group were different (P< 0.05) with the NS group. The interaction of EO and dose (E × D) were significant (P < 0.01) for all of the indexes. In addition, LEO at 300 mg/kg BW and DEO at 600 mg/kg BW had better antioxidant and anti-inflammation activity on the infected mice, which reduced (P < 0.05) the plasma concentrations of MDA, MPO, TNF-α, IL-1β, and IL-6, but increased (P < 0.01) the concentrations of SOD. Hematoxylin-eosin (H&E) staining of duodenum observation showed that LEO and DEO reduced inflammatory cell infiltration and maintain the orderly arrangement of epithelial cells. Moreover, supplementation of LEO at 600 mg/kg and DEO at 300 mg/kg BW alleviated (P < 0.05) intestinal barrier injury for increasing the relative expression of ZO-1, occludin and claudin mRNA in mice duodenum. These results showed that the pre-treatment with LEO and DEO had protection of intestinal tissue and inflammation in E. coli infected mice. Both LEO and DEO exhibited activity of antioxidant, anti-inflammatory and alleviating intestinal injury, whereas, compared with DEO, LEO can be active at a lower dosage. Furthermore, as the main active component of LEO, the d-limonene appeared to play not only the major role, but also the joint action with other active components of LEO.

Regulation of Tight Junctions by Sex Hormones in Goat Mammary Epithelial Cells.

Simple SummaryHow ovarian hormones affect goat lactation by regulating cell–cell junctions is still unclear. Through the in vivo and in vitro assays, we found that ovarian hormones could elevate cell–cell junction protein expression, which may affect the intercellular space and molecule transportation between the goat mammary epithelial cells. Our assessment suggests that ovarian hormones may affect goat milk production by regulating the cell–cell junction protein expression between mammary epithelial cells.The sex hormones of estrogen and progesterone (P4) play a vital role in mammary gland development and milk lactation in ruminants. The tight junction (TJ) between adjacent secretory epithelial cells is instrumental in establishing the mammary blood–milk barrier. However, whether estrogen and P4 exert their effect on mammary function via regulating TJ remain unclear. Here, to clarify the role of 17-β estradiol (E2) and P4 in the regulation of TJ in goat mammary gland, we first explored the relationships between the concentrations of E2, P4, and the protein expression of claudin-1, claudin-3, occludin, and ZO-1 during the mammary gland development in goat. Then, we further explored the mRNA and protein expression of claudin-1, claudin-3, occludin, and ZO-1 in the goat mammary epithelial cells (GMECs) in vitro under different concentrations of E2 and P4. The results demonstrated that the protein expression of claudin-1 decreased, but occludin and ZO-1 increased with the decline in E2 and P4 during the transition from pregnancy to lactation. In the in vitro studies, E2 exerted a positive effect on the mRNA expression of claudin-1, and accelerated the proteins’ expression of claudin-1 and ZO-1 in GMECs; P4 upregulated the mRNA expression of claudin-1, claudin-3, occludin, and ZO-1, and also improved the protein expression of claudin-1, claudin-3, and ZO-1 in the GMECs. The results demonstrated that E2 and P4 play an important role in regulating the expression of the mammary TJ components, which may ultimately affect the mammary gland development and milk lactation.

LPS‐induced endotoxemia promotes blood‐brain barrier permeability via TSP1‐dependent TGFβ1 activation

IntroductionDuring disease states, the blood‐brain barrier (BBB) can become permeable due to endothelial injury or dysregulation of tight junction proteins. Endotoxemia, which induces systemic inflammation, causes BBB dysfunction and neuroinflammation. We have shown that transforming growth factor beta 1 (TGFβ1) is upregulated during systemic inflammation and promotes BBB permeability by disrupting claudin‐5 and occludin. Therefore, we hypothesize that TGFβ1 is upregulated during endotoxemia and induces BBB permeability by disrupting tight junction proteins.MethodsC57Bl/6 mice were administered 3 mg/kg of LPS and monitored for neurological decline. In a second set of mice 6 hours after LPS administration, an antagonist to TGFβ1 activation, LSKL, or control, SLLK, were injected (1 mg/kg/ip). Mice were euthanized 24 hours after LPS injection and tissue was collected. In a subset of animals, 3 hours prior to euthanasia, Evans Blue was injected (8%/ip) to assess BBB function. TGFβ1, claudin‐5, occludin, IL‐1β, IL6 and TNFα expression was assessed by RTPCR, immunoblotting and immunofluorescence. In vitro studies using mouse brain endothelial cells (bEnd.3 cells) were treated with LPS (10 μg/ml) or rTSP1 (1 μg/ml) to activate TGFβ1. TGFβ1, TSP1, claudin‐5 and occludin expression was assessed by immunofluorescence or RTPCR. Barrier function was determined in bEnd.3 cell monolayers using TEER or 10‐kDa FITC‐dextran diffusion. Data were analyzed using GraphPad Prism with results presented as mean +/‐ SEM. Differences between two groups were compared using Student’s t‐test and differences between three groups were determined using ANOVA.ResultsTGFβ1 expression was increased in cortex and cerebellum of LPS‐treated mice. This was associated with decreased claudin‐5 and occludin mRNA expression and immunofluorescence staining. Treatment of bEnd.3 cells with LPS increased TGFβ1 and TSP1 expression, decreased TEER, and increased FITC‐dextran diffusion. Supplementation of bEnd.3 cells with rTSP1 led to decreased claudin‐5 and occludin mRNA expression, decreased TEER, and increased FITC‐dextran diffusion across the transwell. LSKL treatment in LPS‐treated mice reduced sickness behavior, reduced Evan’s blue penetrance into the brain, restored tight junction expression and alleviated neuroinflammation when compared to SLLK‐treated mice.ConclusionsStrategies to target TGFβ1 signaling may prove effective at alleviating endotoxin‐induced BBB dysfunction.

Porcine Corneas Incubated at Low Humidity Present Characteristic Features Found in Dry Eye Disease

Dry eye is a multifactorial disease that affects the ocular surface and tear fluid. Current treatment options include lubricant eye drop application several times a day. However, these eye drops often cause local side effects like ocular allergies or blurred vision after the application. To test new treatment options, a robust dry eye model is needed. Here, a porcine ex vivo model was established by means of incubation of porcine corneas in low humidity (LH) and characterized by histological damage evaluation, epithelial thickness and by relevant dry eye markers, such as interleukin 1 beta (IL-1β), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), occludin and galectin-3. In the dry eye model proposed, an increased secretion of IL-1β was observed, as well as an upregulation of NF-κB, occludin and galectin-3 mRNA expression. Moreover, the model presented a higher rate of cell death in comparison to the controls. These effects could be reversed with successful treatment of dexamethasone (dexa) and partially reversed with hyaluronic acid (HA) containing eye drops. Furthermore, medium-molecular-weight HA stimulated an increase in IL-1β in the model proposed. In conclusion, this dry eye model mimics the in vivo condition and hence allows for animal-free testing of novel dry eye treatments.

Effects of dietary pantothenic acid on growth, antioxidant ability and innate immune response in juvenile black carp

An 8-week feeding study was conducted to examine how dietary pantothenic acid (PA) affects growth, antioxidant ability and innate immunity in juvenile black carp (Mylopharyngodon piceus). Juvenile fish (7.35 ± 0.06 g) were fed diets with PA at 0, 5, 10, 20, 40 and 80 mg/kg, respectively. The highest weight gain (WG), specific growth rate (SGR) and protein efficiency ratio (PER) were obtained with 10 mg/kg dietary PA (p < 0.05), as well as the lowest feed conversion ratio (FCR). Adequate dietary PA (10 and 20 mg/kg) markedly upregulated neuropeptide Y (NPY), orexin (ORX) and ghrelin (GRL) and downregulated cholecystokinin (CCK) in the brain of these animals (p < 0.05). Intestinal sodium-dependent multivitamin transporter (SMVT) expression and α amylase, trypsin, chymotrypsin and lipase activities were all improved after supplementation of PA at 10 or 20 mg/kg (p < 0.05). In addition, adequate dietary PA (10 and 20 mg/kg) significantly increased the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR), as well as the amounts of reduced glutathione (GSH) and total antioxidant capacity (T-AOC) in the liver of these animals (p < 0.05). Meanwhile, liver glutathione S-transferase (GST) activity and malondialdehyde (MDA) amounts levels were both markedly decreased with dietary PA at 10 or 20 mg/kg (p < 0.05). Furthermore, adequate dietary PA significantly elevated the mRNA amounts of NFE2-related nuclear factor 2 (Nrf2) and downregulated Kelch-like-ECH-associated protein 1a (Keap1a) and Keap1b (p < 0.05), and remarkably upregulated innate defense molecules, including lysozyme (LYZ), natural resistance-associated macrophage protein (NRAMP), liver-expressed antimicrobial peptide 2 (LEAP-2), hepcidin (HEPC), complement 3 (C3) and complement 9 (C9) in the liver (p < 0.05). Meanwhile, elevated dietary PA resulted in increased LYZ activity and C3 content and decreased aspartate aminotransferase (ALT) and aspartate aminotransferase (AST) activities in serum samples from juvenile black carp compared with the groups fed PA-deficient diets (p < 0.05). Moreover, 10, 20 and 40 mg/kg PA-containing diets starkly downregulated intestinal interleukin 8 (IL-8) and IL-11 at the mRNA level in comparison with the PA-deficient diet (0 mg/kg) (p < 0.05). However, PA supplementation at 10, 20 or 40 mg/kg resulted in increased intestinal zonula occludens-1 (ZO-1), ZO-2, ZO-3, claudin 3, claudin 4, claudin 8, claudin 15 and occludin mRNA levels in M. piceus in comparison with the 0 mg/kg PA group (p < 0.05). Jointly, the above findings suggested adequate dietary PA (10 and 20 mg/kg) enhances feed utilization and growth performance, as well as antioxidant capacities and non-specific immune reactions in juvenile M. piceus.