ZO-1 mRNA Research Articles

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.

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.

The replacement of bacitracin methylene disalicylate with Bacillus subtilis PB6 in the diet of male Cherry Valley Ducks reduces the feed conversion ratio by improving intestinal health and modulating gut microbiota

In this study, we compared the impacts of Bacillus subtilis PB6 (BS) and bacitracin methylene disalicylate (BMD) on the growth performance, intestinal morphology, expression of tight connection protein, and cecal microbiota community of male ducks through a 42-d trial. Three-hundred and sixty male Cherry Valley meat-type ducklings (1-day-old) were distributed into 3 groups of 6 replicates: CON group (control, basal diet), BMD group (basal diet + 45 mg/kg BMD, active ingredient dose in the feed), and BS group (basal diet + 2 × 107 CFU/kg BS in the feed). Results showed that supplementing the diet with BS reduced the average daily feed intake (ADFI) during d 15 to 42 and d 1 to 42 compared with the CON group (P = 0.032). It also reduced feed conversion ratio (FCR) during d 15 to 42 and d 1 to 42 (P < 0.05) relative to the other groups. The ileal villus height (VH) and villus height /crypt depth ratio (V/C) were increased (P < 0.05) in both the BS and BMD groups, and the jejunal VH and V/C ratio were increased in the BS group (P < 0.05). Relative to the CON, BS supplementation was associated with numerical augmentation of goblet cells in the jejunal mucosa and upregulation of jejunal zonula occludens (ZO-1) and ileal mucin2 (P < 0.05) mRNA levels. Analysis showed a negative correlation between FCR (d 0–42) and VH, V/C, and the number of goblet cells in the jejunum (P < 0.05). Additionally, BMD or BS supplementation altered the alpha diversity of colonic microbiota (P < 0.05). Correlation analysis revealed that Butyricimonas, Enterobacteriaceae, Clostridiaceae, and Tannerellaceae were positively associated with the acetic acid and butyrate concentrations (P < 0.05). Taken together, the supplementation of BS in the diet of male ducks was conducive to reducing FCR by meliorating intestinal morphology, upregulating ZO-1 and mucin2 mRNA levels, regulating the abundance of microbiota, and metabolites, and having a greater effect than BMD supplementation.

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.

Niacin Improves Intestinal Health through Up-Regulation of AQPs Expression Induced by GPR109A.

(1) Background: Changes in the expression of aquaporins (AQPs) in the intestine are proved to be associated with the attenuation of diarrhea. Diarrhea is a severe problem for postweaning piglets. Therefore, this study aimed to investigate whether niacin could alleviate diarrhea in weaned piglets by regulating AQPs expression and the underlying mechanisms; (2) Methods: 72 weaned piglets (Duroc × (Landrace × Yorkshire), 21 d old, 6.60 ± 0.05 kg) were randomly allotted into 3 groups for a 14-day feeding trial. Each treatment group included 6 replicate pens and each pen included 4 barrows (n = 24/treatment). Piglets were fed a basal diet (CON), a basal diet supplemented with 20.4 mg niacin/kg diet (NA) or the basal diet administered an antagonist for the GPR109A receptor (MPN). Additionally, an established porcine intestinal epithelial cell line (IPEC-J2) was used to investigate the protective effects and underlying mechanism of niacin on AQPs expression after Escherichia coli K88 (ETEC K88) treatment; (3) Results: Piglets fed niacin-supplemented diet had significantly decreased diarrhea rate, and increased mRNA and protein level of ZO-1, AQP 1 and AQP 3 in the colon compared with those administered a fed diet supplemented with an antagonist (p < 0.05). In addition, ETEC K88 treatment significantly reduced the cell viability, cell migration, and mRNA and protein expression of AQP1, AQP3, AQP7, AQP9, AQP11, and GPR109A in IPEC-J2 cells (p < 0.05). However, supplementation with niacin significantly prevented the ETEC K88-induced decline in the cell viability, cell migration, and the expression level of AQPs mRNA and protein in IPEC-J2 cells (p < 0.05). Furthermore, siRNA GPR109A knockdown significantly abrogated the protective effect of niacin on ETEC K88-induced cell damage (p < 0.05); (4) Conclusions: Niacin supplementation increased AQPs and ZO-1 expression to reduce diarrhea and intestinal damage through GPR109A pathway in weaned piglets.

Role of Short Chain Fatty Acids to Counteract Inflammatory Stress and Mucus Production in Human Intestinal HT29-MTX-E12 Cells.

Short chain fatty acids (SCFAs), especially butyrate (BUT), are known to promote intestinal health, but their role in the protection of intestinal barrier integrity is poorly characterized. The aim of the study was to set up an in vitro model of human colon epithelium using HT29-MTX-E12 cells to delineate the potential role of SCFAs under stress conditions. Accordingly, the HT29-MTX-E12 cells were differentiated for 42 days and subsequently exposed to dextran sulphate sodium (DSS). Further, the effects of BUT or its mixture with acetate and propionate (SCFAs-MIX) were tested to study proliferation, epithelial integrity and mucus production. The results showed that the concentration of 10% DSS for 24 h decreased the TEER about 50% compared to the control in HT29-MTX-E12 cells. The pre-treatment on HT29-MTX-E12 cells with BUT or SCFAs-MIX at specific concentrations significantly (p < 0.05) reduced the DSS-induced damage on epithelial cell integrity and permeability. Further, the treatment with specific concentrations of BUT and SCFAs-MIX for 24 h significantly promoted ZO-1, MUC2 and MUC5AC mRNA expression (p < 0.005). The present study demonstrated the suitability of HT29-MTX-E12 cells treated with DSS as an in vitro stress model of inflammatory bowel disease, which enabled us to understand the effect of bioactive SCFAs on the intestinal barrier.

Exosomally Targeting microRNA23a Ameliorates Microvascular Endothelial Barrier Dysfunction Following Rickettsial Infection.

Spotted fever group rickettsioses caused by Rickettsia (R) are devastating human infections, which mainly target microvascular endothelial cells (ECs) and can induce lethal EC barrier dysfunction in the brain and lungs. Our previous evidence reveals that exosomes (Exos) derived from rickettsial-infected ECs, namely R-ECExos, can induce disruption of the tight junctional (TJ) protein ZO-1 and barrier dysfunction of human normal recipient brain microvascular endothelial cells (BMECs). However, the underlying mechanism remains elusive. Given that we have observed that microRNA23a (miR23a), a negative regulator of endothelial ZO-1 mRNA, is selectively sorted into R-ECExos, the aim of the present study was to characterize the potential functional role of exosomal miR23a delivered by R-ECExos in normal recipient BMECs. We demonstrated that EC-derived Exos (ECExos) have the capacity to deliver oligonucleotide RNAs to normal recipient BMECs in an RNase-abundant environment. miR23a in ECExos impairs normal recipient BMEC barrier function, directly targeting TJ protein ZO-1 mRNAs. In separate studies using a traditional in vitro model and a novel single living-cell biomechanical assay, our group demonstrated that miR23a anti-sense oligonucleotide-enriched ECExos ameliorate R-ECExo-provoked recipient BMEC dysfunction in association with stabilization of ZO-1 in a dose-dependent manner. These results suggest that Exo-based therapy could potentially prove to be a promising strategy to improve vascular barrier function during bacterial infection and concomitant inflammation.

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.

Effects of Taraxacum mongolicum Hand.-Mazz. (dandelion) on growth performance, expression of genes coding for tight junction protein and mucin, microbiota composition and short chain fatty acids in ileum of broiler chickens

BackgroundDandelion is becoming an exploitable alternative to the widely prohibited antibiotics in the poultry production. This research aimed to investigate the effects of dandelion on the growth performance and intestinal barrier function of broiler chickens maintained under standard condition of management. One-hundred and sixty 1-day-old Arbor Acres (AA) male broiler chickens were randomly divided into four groups, with five replicates of eight birds each. The birds were fed a basal diet supplemented without (control group, [CON]) or with 500 (low dose [LD]) or 1000 (high dose [HD]) mg/kg dandelion or with 250 mg/kg chlortetracycline 20% premix (CTC) for 42 days, including the starter phase (d 1 to 21) and the grower phase (d 22 to 42). Body weight (BW) of each bird and feed consumption of each replicate were measured at d 21 and d 42. The ileal tissues were collected on day 21 and 42 to determine expression of genes coding for tight junction protein and mucin as well as ELISA analysis for immune factor. The ileal digesta was collected for microbiota and short chain fatty acids analysis.ResultsCompared with CON group, during day 1–21, the average daily feed intake (ADFI) and feed/gain ratio (F/G) of LD group were lower (P < 0.05); during day 22–42, the F/G of LD and CTC group tended to be lower (P = 0.07); during the overall phase, the ADFI of HD and CTC groups were decreased (P < 0.05), and the F/G of dandelion and CTC groups tended to be decreased (P = 0.07). On day 21, the relative mRNA expression of claudin, occludin-1 and mucin1 in dandelion groups were up-regulated (P < 0.05), and the ZO-1 mRNA expression in CTC group was increased (P < 0.05); on day 42, the claudin and mucin1 transcripts in LD group and ZO-1 transcripts in HD and CTC group were up-regulated (P < 0.05), while the occludin-1 and mucin1 transcripts in CTC group was significantly down-regulated (P < 0.05). In addition, the contents of TNF-α in dandelion groups were lower than that in CTC group (P < 0.05). In the analysis of ileal microbiota, on day 21, decreased α-diversity was observed in HD and CTC groups (P < 0.05). Meanwhile, on day 21, the relative abundance of Firmicutes in dandelion groups tended to be higher (P = 0.09), the relative abundance of Lactobacillus in LD and CTC group were increased (P < 0.05), while Bacteroidete, Bacteroides, and Alistipes relative abundance in dandelion and CTC groups were decreased (P < 0.05). On day 42, the Actinobacteriota relative abundance in CTC group tended to be higher (P = 0.05), and Lysinibacillus relative abundance of CTC group was higher (P = 0.02). Compared with CON group, on day 21, the propionic acid and butyric acid content in CTC group were higher, the butyric acid content in HD group was lower (P < 0.10).ConclusionIn summary, dietary dandelion supplementation at 500 mg/kg of diet enhanced growth performance of broilers by improving the intestinal barrier function. Dandelion can be supplemented in the diet as an antibiotics alternative to enhance production in poultry industry.

Bifidobacterium longum subsp. longum K5 alleviates inflammatory response and prevents intestinal barrier injury induced by LPS in vitro based on comparative genomics

• K5 inhibited TNF-α, IL-1β, IL-6, ROS and NO level better than K15 in the LPS-induced RAW 264.7 cell model. • K5 increased TEER value and decreased paracellular permeability in LPS-induced Caco-2 cell monolayers model. • Comparative genomics showed K5 had a specific gene encoding fatty acid synthase associated with the immune system. • Comparative genomics showed K5 and K15 had four genes with only an amino acid mutation. Excessive immune responses and intestinal barrier injury are leading causes of inflammatory bowel diseases (IBD). Previously, we demonstrated that B. longum subsp . longum K5 alleviated DSS-stimulated colitis in mice, while K15 could not. Here, we investigated both strains' underlying mechanisms regarding their anti-inflammatory and intestinal barrier protective capacities. We assessed the potential characters of two strains, anti-inflammation activities in the LPS-induced RAW 264.7 cell model, and intestinal epithelial barrier functions in the LPS-induced Caco-2 cell monolayers model. Results showed that B. longum subsp . longum K5 exhibited high inhibition against the adhesion of E. coli ATCC 25922 to HT-29 and acetic acid production. B. longum subsp. longum K5 (MOI 1:100) significantly decreased the pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and inflammatory mediator ROS, NO level in LPS (1 μg/mL) -induced RAW 264.7 cells. It also significantly increased TEER value and decreased paracellular permeability of Caco-2 cells stimulated by LPS. B. longum subsp. longum K5 (MOI 1:100) up-regulated ZO-1 , occlaudin and claudin-1 mRNA expression, down-regulated the mRNA expression level of TLR4 and pro-inflammatory cytokines ( TNF-α, IL-1β, IL-6 ) in LPS (100 μg/mL) -stimulated Caco-2 cells. While B. longum subsp. longum K15 did not do as well as B. longum subsp. longum K5 on these assays. Furthermore, the whole-genome sequencing results revealed a gene encoding fatty acid synthase associated with the immune system in K5 specific genes, LacI family related with carbohydrate metabolism, and serine/threonine-protein kinase involved in signal transduction with an amino acid mutation. Our findings provided the theoretical basis for B. longum subsp . longum K5 to be used as a candidate for IBD therapy.

Effects of Prebiotic Fructooligosaccharides on Cytokine and Tight Junction Protein Expression in Cultured Intestinal Epithelial Cells

Prebiotic molecules such as fructooligosaccharides (FOS) can promote the growth and metabolism of beneficial bacteria in the host intestine. There are several published studies suggesting that prebiotic supplementation can alleviate symptoms of colitis in animal models. In addition, a recent study showed evidence for direct effects of FOS on tight junction protein expression in cultured intestinal epithelial cells. This study aimed to assess effects of FOS on pro‐inflammatory cytokine and tight junction protein expression in CMT‐93 and Caco‐2 colonic epithelial cells. Cells were treated with 0.1 mg/ml, 1 mg/ml or 10 mg/ml FOS and expression of cyclooxygenase‐2 (COX‐2), pro‐inflammatory cytokines interleukin (IL)‐1β and IL‐6, and tight junction proteins claudin and ZO‐1 were evaluated by real‐time qPCR. The number of viable cells was analyzed after overnight exposure to FOS using a cell survival assay. Expression of ZO‐1 and claudin and localization of nuclear factor kappa‐B (NFkB) were evaluated by immunofluorescent staining. Surprisingly, FOS‐treated CMT‐93 cells showed a slight reduction in claudin mRNA expression compared to untreated cells. ZO‐1 and COX‐2 mRNA expression were not altered by FOS treatment compared to untreated controls. Immunofluorescent staining showed no translocation of NFkB to the nucleus after 2 hours of exposure to FOS. In conclusion, prebiotic fructooligosaccharides had minimal to no effects on the mRNA expression of claudin, ZO‐1, and COX‐2. In addition, FOS did not activate the NFkB pathway at the time points studied. These data suggest that FOS may not cause inflammatory responses in intestinal epithelial cells.

Minimally Invasive Surgery for ICH Evacuation Combined With Deferoxamine Treatment Increased Perihematomal Claudin-5 and ZO-1 Expression Levels and Decreased BBB Permeability in Rabbits.

ObjectiveTo investigate the role of minimally invasive surgery (MIS) in intracerebral hemorrhage (ICH) evacuation combined with deferoxamine (DFX) treatment on perihematomal tight junction protein (claudin-5 and ZO-1) expression levels and blood-brain barrier (BBB) permeability in rabbits.MethodsWe randomly assigned 65 male rabbits (weight: 1.9–2.6 kg) to a normal control group (NC group, 13 rabbits), hemorrhage model group (HM group, 13), DFX treatment group (DFX group, 13 rabbits), MIS group (MIS group, 13 rabbits), or MIS combined with DFX treatment group (MIS + DFX group, 13 rabbits). ICH was established in all of the groups except the NC group. MIS was performed to evacuate the hematoma 6 h after the ICH model was created in the MIS and MIS + DFX groups. The DFX and MIS + DFX groups were treated with DFX (100 mg/kg, dissolved in 2 mL of 0.9% saline solution, administered intramuscularly) at 2 h, and then every 12 h for 7 d. The same dose of 0.9% saline solution was administered to the NC, HM, and MIS groups at the same time points. Sixty-five rabbits were divided into 5 groups, and 13 rabbits in each group. Neurological deficit (i.e., Purdy's score) was recorded in all rabbits before euthanasia (N total = 65). In each group, 2 rabbits were used for iron concentration measurement (N total = 10), 2 rabbits were used for brain water content measurement (N total = 10), 3 rabbits were used for BBB permeability measurement (N total = 15), 3 rabbits were used for claudin-5, ZO-1 expression detection by Western Blotting (N total = 15), and 3 rabbits were used for claudin-5, ZO-1 mRNA detection by real-time PCR (N total = 15). On day 7, the rabbits were sacrificed and the perihematomal brain tissue was harvested to test the iron concentration, brain water content (BWC), tight junction proteins (claudin-5 and ZO-1) expression, and BBB permeability.ResultsPurdy's score, iron concentration, and BWC were lower in the MIS and MIS + DFX groups compared to the HM and DFX groups. The MIS + DFX group showed a significant decrease in these indicators. The use of MIS to evacuate the hematoma led to increased expression levels of claudin-5 and ZO-1, as well as decreased BBB permeability. The MIS + DFX group exhibited a remarkable increase in claudin-5 and ZO-1 expression levels and a significant decrease in BBB permeability.ConclusionsMIS combined with DFX treatment could increase the expression levels of perihematomal tight junction proteins (claudin-5 and ZO-1) expression, reduce BBB permeability, and improve the neurological function. MIS combined with DFX treatment may also prevent secondary brain damage following ICH.

N-methyl-D-aspartic acid increases tight junction protein destruction in brain endothelial cell via caveolin-1-associated ERK1/2 signaling

N-methyl-D-aspartic acid (NMDA), a glutamate analog, can activate N-Methyl-D-Aspartate receptor (NMDAR) to induce vascular endothelial cell injury but the mechanisms are not fully understood. The present study intended to evaluate the role of caveolin-1 (Cav-1) in NMDA-induced dysfunction of human brain microvascular endothelial cells (HBEC-5i), and verify that endothelial NMDAR activation mediates the disruption of tight junction barrier integrity via extracellular signal-regulated kinase (ERK)1/2 pathway. The expression of NMDAR NR1 were confirmed firstly in HBEC-5i and compared with that in mouse brain by Western blot. To study the role of Cav-1 in NMDA mediated reduction of tight junction protein zonula occludens- (ZO) 1 expression, HBEC-5i were transduced with Cav-1 shRNA or Control shRNA, and the Cav-1 knockdown rate tested with qRT-PCR and Western blot is 99.98% or 87.5%, respectively. NMDA exposure decreased mRNA and protein levels of tight junction protein ZO-1 and suppressed transendothelial electrical resistance (TEER) values in HBEC-5i but blocked by NMDAR antagonist MK801. In addition, NMDA induced Cav-1 and ERK1/2 sequential phosphorylation，but these effects were attenuated by silencing the Cav-1 gene with shRNA and ERK1/2 inhibitor U0126, respectively. These results show that the functional presence of NMDAR NR1 in HBEC-5i. Endothelial NMDAR NR1 activation regulate the maintenance of HBEC-5i-constructed tight junction barrier integrity via the caveolin-1-associated ERK1/2 signaling pathway.

Orange Pectin with Compact Conformation Effectively Alleviates Acute Colitis in Mice.

A comprehensive understanding of the relationships between the structure and function is critical for the targeted preparation of functional pectins. In this study, we compared the alleviating effects of five orange pectins (200 mg/kg) extracted using acid (P2), alkali (P10), cellulase (C), acid + cellulase (P2 + C), and alkali + cellulase (P10 + C) on dextran sodium sulfate-induced acute colitis. The physiological and histopathological indicators revealed that the alleviating effects were most significant for P10 + C, followed by P10, P2 + C, P2, and C. P10 + C increased the diversity and relative abundance of Akkermansia, leading to increased generation of colonic short-chain fatty acids as well as mRNA and protein expressions of GPR43, GPR109A, claudin-1, ZO-1, and occludin. Therefore, proinflammatory cytokines were decreased, and anti-inflammatory cytokines were increased. A compact conformation of P10 + C contributed to the alleviation effects on acute colitis. Alkali + cellulase-extracted orange pectin with a compact conformation has potential as adjuvant treatment for intestinal inflammation.

L-Isoleucine Administration Alleviates DSS-Induced Colitis by Regulating TLR4/MyD88/NF-κB Pathway in Rats.

Inflammatory bowel disease (namely, colitis) severely impairs human health. Isoleucine is reported to regulate immune function (such as the production of immunoreactive substances). The aim of this study was to investigate whether l-isoleucine administration might alleviate dextran sulfate sodium (DSS)-induced colitis in rats. In the in vitro trial, IEC-18 cells were treated by 4 mmol/L l-isoleucine for 12 h, which relieved the decrease of cell viability that was induced by TNF-α (10 ng/ml) challenge for 24 h (P <0.05). Then, in the in vivo experiment, a total of 44 Wistar rats were allotted into 2 groups that were fed l-isoleucine-supplemented diet and control diet for 35 d. From 15 to 35 d, half of the rats in the 2 groups drank the 4% DSS-adding water. Average daily gain, average daily feed intake and feed conversion of rats were impaired by DSS challenge (P <0.05). Drinking the DSS-supplementing water also increased disease activity index (DAI) and serum urea nitrogen level (P <0.05), shortened colonic length (P <0.05), impaired colonic enterocyte apoptosis, cell cycle, and the ZO-1 mRNA expression (P <0.05), increased the ratio of CD11c-, CD64-, and CD169-positive cells in colon (P <0.05), and induced extensive ulcer, infiltration of inflammatory cells, and collagenous fiber hyperplasia in colon. However, dietary l-isoleucine supplementation attenuated the negative effect of DSS challenge on growth performance (P <0.05), DAI (P <0.05), colonic length and enterocyte apoptosis (P <0.05), and dysfunction of colonic histology, and downregulated the ratio of CD11c-, CD64-, and CD169-positive cells, pro-inflammation cytokines and the mRNA expression of TLR4, MyD88, and NF-κB in the colon of rats (P <0.05). These results suggest that supplementing l-isoleucine in diet improved the DSS-induced growth stunting and colonic damage in rats, which could be associated with the downregulation of inflammation via regulating TLR4/MyD88/NF-κB pathway in colon.