Effect and mechanism of Prunella vulgaris L. extract on alleviating lipopolysaccharide-induced acute mastitis in protecting the blood-milk barrier and reducing inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: According to ancient literature, Prunella vulgaris L. (P vulgaris) alleviates mastitis and has been used in China for many years; however, there are no relevant reports that confirm this or the mechanism of its efficacy. AIM OF THE STUDY: To explore the anti-acute mastitis effect and potential mechanism of P vulgaris extract. MATERIALS AND METHODS: First, the active ingredients and targets of P vulgaris against mastitis were predicted using network pharmacology. Next, the relevant active ingredients were enriched using macroporous resins and verified using UV and UPLC-Q-TOF-MS/MS. Lastly, a mouse model of acute mastitis was established by injecting lipopolysaccharides into the mammary gland and administering P vulgaris extract by oral gavage. The pathological changes in mammary tissue were observed by HE staining. Serum and tissue inflammatory factors were measured by ELISA method. MPO activity in mammary tissue was measured using colorimetry and MPO expression was detected by immunohistochemistry. The expression of tight junction proteins (ZO-1, claudin-3, and occludin) in mammary tissue was detected by immunofluorescence and Western blot. iNOS and COX-2 in mammary tissue were detected by Western blot. MAPK pathway and NF-κB pathway related proteins were also detected by Western blot. RESULTS: Network pharmacology predicted that phenolic acids and flavonoids in P vulgaris had anti-mastitis effects. The contents of total flavonoids and total phenolic acids in P vulgaris extract were 64.5% and 29.4%, respectively. UPLC-Q-TOF-MS/MS confirmed that P vulgaris extract contained phenolic acids and flavonoids. The results of animal experiments showed that P vulgaris extract reduced lipopolysaccharide-induced inflammatory edema, inflammatory cell infiltration, and interstitial congestion of mammary tissue. It also reduced the levels of serum and tissue inflammatory factors TNF-α, IL-6, and IL-1ß, and inhibited the activation of MPO. Furthermore, it downregulated the expression of MAPK and NF-κB pathway-related proteins. The expressions of ZO-1, occludin, and claudin-3 in mammary gland tissues were upregulated. CONCLUSIONS: P vulgaris extract can maintain the integrity of mammary connective tissue and reduce its inflammatory response to prevent acute mastitis. Its mechanism probably involves regulating NF-κB and MAPK pathways.

Ginger Root Extract Improves GI Health in Diabetic Rats by Improving Intestinal Integrity and Mitochondrial Function.

Background Emerging research suggests hyperglycemia can increase intestinal permeability. Ginger and its bioactive compounds have been reported to benefit diabetic animals due to their anti-inflammatory and antioxidant properties. In this study, we revealed the beneficial effect of gingerol-enriched ginger (GEG) on intestinal health (i.e., barrier function, mitochondrial function, and anti-inflammation) in diabetic rats. Methods Thirty-three male Sprague Dawley rats were assigned to three groups: low-fat diet (control group), high-fat-diet (HFD) + streptozotocin (single low dose 35 mg/kg body weight (BW) after 2 weeks of HFD feeding) (DM group), and HFD + streptozotocin + 0.75% GEG in diet (GEG group) for 42 days. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were conducted at baseline and prior to sample collection. Total pancreatic insulin content was determined by ELISA. Total RNA of intestinal tissues was extracted for mRNA expression using qRT-PCR. Results Compared to the DM group, the GEG group had improved glucose tolerance and increased pancreatic insulin content. Compared to those without GEG (DM group), GEG supplementation (GEG group) increased the gene expression of tight junction (Claudin-3) and antioxidant capacity (SOD1), while it decreased the gene expression for mitochondrial fusion (MFN1), fission (FIS1), biogenesis (PGC-1α, TFAM), mitophagy (LC3B, P62, PINK1), and inflammation (NF-κB). Conclusions Ginger root extract improved glucose homeostasis in diabetic rats, in part, via improving intestinal integrity and mitochondrial dysfunction of GI health.

Impact of dandelion polysaccharides on growth and immunity response in common carp Cyprinus carpio.

Natural products have gained considerable attention for improving fish growth performance and immunity to enhance disease resistance. This study evaluated the effect of dandelion polysaccharides (DP) on skin mucosal immune parameters, immune-related gene expression, and susceptibility to pathogen challenge in the Common carp Cyprinus carpio. Diets containing four different concentrations of DP (g Kg-1):0 g [basal diet], 0.5 g [D1], 1.5 g [D2], 2.5 g [D3], and 4.0 g [D4] were fed to the carp (average weight: 13.92 ± 0.83 g) for eight weeks. Growth parameters were analyzed four and eight weeks after feeding. Immunological, hematological, and antioxidant parameters were examined eight weeks post-feeding. Growth performance was significantly higher on D3, with a final weight gain of 71.48 ± 1.57 g and a specific growth rate of 3.06 ± 0.12. Among hematological parameters examined, erythrocyte, hematocrit, and mean corpuscular volume (MCV) levels were significantly higher in D3. Skin mucosal immune parameters, such as lysozyme (31.04 ± 1.02 Unit mL-1), alkaline phosphatase (122.6 ± 3.8 IU L-1), and protein level (10.6 ± 0.74 mg mL-1) were significantly higher in D3, while peroxidase activity was higher in D4. Furthermore, SOD activity was higher in D2-D3, whereas catalase activity was higher in D2-D4 (P < 0.05) than in the control. Malondialdehyde level decreased significantly in D3 (5.43 ± 0.36 nmol mL-1); whereas, serum ALT and AST levels were significantly lower on D2-D4. Intestinal tight-junction-related genes ZO-1 and Claudin 7 were significantly higher in the DP-fed groups; however, DP had no significant effect on claudin 3. Occludin expression was higher (p < 0.05) on D3 only. Pro-inflammatory cytokines (IL-1ß and TNF-α) and IFN-γ strongly upregulated in the head kidney at D3. Conversely, the expression of the anti-inflammatory cytokine interleukin-10, HSP70, and TOR were considerably downregulated in D3. Fish from D3 exhibited markedly higher relative post-challenge survival (66.67%) against Aeromonas hydrophila challenge. The results of the present study suggest that dietary supplements of DP at 2.5 g kg-1 can significantly improve the growth performance, skin mucosal, and serum antioxidant parameters, and strengthen the immunity of C. carpio. Therefore, DP is a promising food additive for carp aquaculture.

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-ß mRNA transcription levels, while the opposite was true for plasma AST activity, hepatic MDA contents, intestinal Claudin-15, NF-κB, IL-1ß, 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-ß 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.

Dietary Alpha-Ketoglutarate Partially Abolishes Adverse Changes in the Small Intestine after Gastric Bypass Surgery in a Rat Model.

Alpha-ketoglutarate (AKG) is one of the key metabolites that play a crucial role in cellular energy metabolism. Bariatric surgery is a life-saving procedure, but it carries many gastrointestinal side effects. The present study investigated the beneficial effects of dietary AKG on the structure, integrity, and absorption surface of the small intestine after bariatric surgery. Male 7-week-old Sprague Dowley rats underwent gastric bypass surgery, after which they received AKG, 0.2 g/kg body weight/day, administered in drinking water for 6 weeks. Changes in small intestinal morphology, including histomorphometric parameters of enteric plexuses, immunolocalization of claudin 3, MarvelD3, occludin and zonula ocludens 1 in the intestinal mucosa, and selected hormones, were evaluated. Proliferation, mucosal and submucosal thickness, number of intestinal villi and Paneth cells, and depth of crypts were increased; however, crypt activity, the absorption surface, the expression of claudin 3, MarvelD3, occludin and zonula ocludens 1 in the intestinal epithelium were decreased after gastric bypass surgery. Alpha-ketoglutarate supplementation partially improved intestinal structural parameters and epithelial integrity in rats undergoing this surgical procedure. Dietary AKG can abolish adverse functional changes in the intestinal mucosa, enteric nervous system, hormonal response, and maintenance of the intestinal barrier that occurred after gastric bypass surgery.

Dietary supplementation of ß-conglycinin, with or without sodium butyrate on the growth, immune response and intestinal health of hybrid grouper.

We investigated the effects of low and high doses of ß-conglycinin and the ameliorative effects of sodium butyrate (based on high-dose ß-conglycinin) on the growth performance, serum immunity, distal intestinal histopathology, and gene, protein expression related to intestinal health in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂). The results revealed that the instantaneous growth rate (IGR) of grouper significantly increased, decreased, and increased in the low-dose ß-conglycinin (bL), high-level ß-conglycinin (bH) and high-level ß-conglycinin plus sodium butyrate (bH-NaB), respectively. The feed coefficient ratio (FCR) was significantly increased in the bH and bH-NaB, serum levels of IFN-γ, IL-1ß, and TNF-α were upregulated in the bH. The intestinal diameter/fold height ratio was significantly increased in the bH. Furthermore, there were increases in nitric oxide (NO), total nitric oxide synthase (total NOS), and peroxynitrite anion (ONOO-) in the bH, and decreases in total NOS and ONOO- in the bH-NaB. In the distal intestine, IL-1ß and TGF-ß1 mRNA levels were downregulated and upregulated, respective in the bL. The mRNA levels of TNF-α and IL-6 were upregulated in the bH, and downregulated in the bH-NaB, respectively. Occludin, claudin3 and ZO-3 mRNA levels were upregulated in the bL, downregulated in the bH and then upregulated in the bH-NaB. No significant differences were observed in the mRNA levels of IFN-γ and jam4. And the p-PI3K p85Tyr458/total PI3K p85 value was significantly increased in the bH and then decreased in the bH-NaB, and the total Akt value was significantly increased in the bH. These indicate ß-conglycinin has a regulatory effect on serum immunity and affect distal intestinal development by modulating distal intestinal injury-related parameters. Within the distal intestinal tract, low- and high-dose ß-conglycinin differentially affect immune responses and tight junctions in the distal intestine, which eventually manifests as a reduction in growth performance. Supplementing feed with sodium butyrate might represent an effective approach for enhancing serum immunity, and protects the intestines from damage caused by high-dose ß-conglycinin.

Combined effects of xylo-oligosaccharides and coated sodium butyrate on growth performance, immune function, and intestinal physical barrier function of broilers.

This study was conducted to investigate the effects of dietary supplementation xylo-oligosaccharides (XOS), coated sodium butyrate (CSB), and their combination on growth performance, immune parameters, and intestinal barrier of broilers. A total of 192 1-day-old chicks were assigned to a 2 × 2 factorial design including two dietary additives (0 and 150 mg/kg XOS and 0 and 400 mg/kg CSB). This trial lasted for 42 days. CSB supplementation increased the thymus and bursa index, blood myeloperoxidase (MPO) activity, and IgG and IgM concentrations, whereas adding XOS only improved IgM concentration (p < .05). A significant interaction was observed for MPO activity. Furthermore, broilers fed CSB and their interaction exhibited increased ileal villus height/crypt depth (VH/CD) and goblet cells numbers in the ileum, as well as decreased ileal CD (p < .05). Broilers fed XOS and CSB individually showed higher ileal VH, the number of goblet cells in the duodenum and jejunum (p < .05). Moreover, XOS and CSB individual supplementation upregulated the expression of claudin3 in the ileum (p < .05). Simultaneously, a significant interaction was found for the ileal expression of claudin3. Overall, XOS and CSB supplementation could improve the development of immune organs, the small intestine morphology, and the intestinal physical barrier of broilers. Although no clear synergy of XOS and CSB was detected, the combination had positively affect broilers intestinal barrier and immune parameters.

Localization of Claudin-3 and Claudin-4 within the Small Intestine of newborn piglets.

Piglets must acquire passive immunity through colostrum within hours after birth to survive. How colostral macromolecules traverse the small intestinal epithelium may include nonselective pinocytosis and paracellular transport through tight junction proteins located between epithelial cells. Claudin proteins-3 and -4 contribute to the epithelial tight junctions (TJs) on the apical aspect of lateral surfaces of intestinal epithelial cells (IECs) where they help regulate ion and macromolecule movement across the intestinal epithelium. Throughout the small intestine of newborn piglets, Claudin-3 was localized to the lateral and basolateral surface of intestinal epithelial cells as well as the membrane of large vacuoles. In the duodenum and jejunum, Claudin-4 was localized to the apical surface independent of tight junction regions. In the ileum, Claudin-4 was localized to the lateral and basolateral surfaces indicating region-specific differences and noncanonical patterns of Claudin-4 localization independent of tight junction regions. Understanding the timing of changes in surface localization of Claudin-3 and Claudin-4 and how they may coincide with changes in small intestinal permeability may help develop new protective strategies against infectious diseases within newborn piglets.

Ginseng Extract Ameliorates the Negative Physiological Effects of Heat Stress by Supporting Heat Shock Response and Improving Intestinal Barrier Integrity: Evidence from Studies with Heat-Stressed Caco-2 Cells, C. elegans and Growing Broilers.

Climatic changes and heat stress have become a great challenge in the livestock industry, negatively affecting, in particular, poultry feed intake and intestinal barrier malfunction. Recently, phytogenic feed additives were applied to reduce heat stress effects on animal farming. Here, we investigated the effects of ginseng extract using various in vitro and in vivo experiments. Quantitative real-time PCR, transepithelial electrical resistance measurements and survival assays under heat stress conditions were carried out in various model systems, including Caco-2 cells, Caenorhabditis elegans and jejunum samples of broilers. Under heat stress conditions, ginseng treatment lowered the expression of HSPA1A (Caco-2) and the heat shock protein genes hsp-1 and hsp-16.2 (both in C. elegans), while all three of the tested genes encoding tight junction proteins, CLDN3, OCLN and CLDN1 (Caco-2), were upregulated. In addition, we observed prolonged survival under heat stress in Caenorhabditis elegans, and a better performance of growing ginseng-fed broilers by the increased gene expression of selected heat shock and tight junction proteins. The presence of ginseng extract resulted in a reduced decrease in transepithelial resistance under heat shock conditions. Finally, LC-MS analysis was performed to quantitate the most prominent ginsenosides in the extract used for this study, being Re, Rg1, Rc, Rb2 and Rd. In conclusion, ginseng extract was found to be a suitable feed additive in animal nutrition to reduce the negative physiological effects caused by heat stress.

Uridine/UMP metabolism and their function on the gut in segregated early weaned piglets.

Uridine monophosphate (UMP) is a major nucleotide analogue in mammalian milk and uridine (UR) is its gastro-intestinal metabolite in vivo. This study aims to investigate the functional effects of UMP and UR on the gut in vitro and in vivo. Twenty-one piglets were randomly allotted into three groups, the control group, UMP group and UR group, and orally administered UMP or UR for 10 days. Results showed that UMP and UR supplements improved the ADG of piglets, and decreased the diarrhea rate. UR increased the jejunum villus length/crypt depth ratio, Claudin-3 and E-cadherin expression, and the pyrimidine nucleotide metabolic enzymes including CMPK1, RRM2, UPRT, CTPS1 and CTPS2 in the duodenal mucosa. Both the UMP and UR decreased the expression of CAD and RRM2 at the jejunal mucosa. Moreover, UMP and UR increased the apoptosis ratio of intestinal epithelial cells in in vivo and in vitro experiments. Taken together, oral administration of UR and UMP could improve the small intestinal morphology, promote epithelial cell apoptosis and renewal of intestinal villus tips, and benefit intestinal development and health thus improving the growth performance and reducing the risk of diarrhea in early-weaned piglets.

Naringenin promotes recovery from colonic damage through suppression of epithelial tumor necrosis factor-α production and induction of M2-type macrophages in colitic mice.

Our previous study demonstrated that supplemental naringenin reduced the development of colitis induced by dextran sodium sulfate (DSS) in mice, however, the effect of naringenin on the recovery from colonic damage was totally unknown. The primary purpose was to investigate if naringenin promoted recovery from colonic damage in DSS-administered mice and colonic tissues. When mice were fed diets lacking or containing naringenin (0.3%, w/w) for 11 days after colitis induction through DSS administration, the supplemental naringenin was found to promote a reversal of body weight loss and suppress tumor necrosis factor (TNF)-α mRNA expression in the DSS-administered mice. Moreover, protein expression of two tight junction proteins, claudin-3 and junctional adhesion molecule-A, was higher in DSS-administered mice that were fed naringenin than in the mice that did not receive naringenin. To examine the early mechanisms underlying the naringenin-mediated reduction of colonic damage, the inflamed colonic tissues of DSS-administered mice were incubated with or without naringenin for 24 hours; in tissues incubated with naringenin, TNF-α production was lower and interleukin (IL)-10 and CD206 mRNA expression was higher than in tissues incubated without naringenin, but naringenin did not affect the expression of the tight junction proteins. Flow cytometry results further demonstrated that naringenin reduced TNF-α-positive epithelial cells, but not macrophages, and promoted the polarization of M2-type macrophages in the colonic tissues. Thus, supplemental naringenin promoted recovery from colonic damage in mice with colitis, and suppression of epithelial TNF-α production and induction of M2-type macrophages might represent the early mechanisms underlying this naringenin effect.

Claudin-3 regulates bile canalicular paracellular barrier and cholesterol gallstone core formation in mice.

BACKGROUND & AIMS: Most cholesterol gallstones have a core consisting of inorganic and/or organic calcium salts, although the mechanisms of core formation are poorly understood. We examined whether the paracellular permeability of ions at hepatic tight junctions is involved in the core formation of cholesterol gallstones, with particular interest in the role of phosphate ion, a common food additive and preservative. METHODS: We focused on claudin-3 (Cldn3), a paracellular barrier-forming tight junction protein whose expression in mouse liver decreases with age. Since Cldn3-knockout mice exhibited gallstone diseases, we used them to assess the causal relationship between paracellular phosphate ion permeability and the core formation of cholesterol gallstones. RESULTS: In the liver of Cldn3-knockout mice, the paracellular phosphate ion permeability through hepatic tight junctions was significantly increased, resulting in calcium phosphate core formation. Cholesterol overdose caused cholesterol gallstone disease in these mice. CONCLUSION: We revealed that in the hepatobiliary system, Cldn3 functions as a paracellular barrier for phosphate ions, to help maintain biliary ion homeostasis. We provide in vivo evidence that elevated phosphate ion concentrations play a major role in the lifestyle- and age-related risks of developing cholesterol gallstone disease under cholesterol overdose. LAY SUMMARY: Herein, we reveal a new mechanism for cholesterol gallstone formation, in which increased paracellular phosphate ion permeability across hepatobiliary epithelia causes calcium phosphate core formation and cholesterol gallstones. Thus, altered phosphate ion metabolism under cholesterol overdose plays a major role in the lifestyle- and age-related risks of developing cholesterol gallstone disease.

Cytotoxicity of Clostridium perfringens enterotoxin depends on the conditions of claudin-4 in ovarian carcinoma cells.

Currently, Clostridium perfringens enterotoxin (CPE) is being investigated as an anti-cancer drug for tumors expressing the tight junction (TJ) transmembrane proteins claudin-3 and/or claudin-4. However, the optimal conditions for CPE cytotoxicity are still unclear. Our objectives were to determine the optimal conditions for CPE as an anti-cancer drug for treating ovarian cancer in vitro and in vivo. In our experiments, cells at low culture density showed higher sensitivity to CPE, suggesting that claudins at TJs were poorly accessible to CPE compared with those at the edge of cell colonies. Ovarian cancer cells cultured under calcium-depleted pretreatment conditions to disrupt TJs and to knock-down TJ proteins and E-cadherin production altered CPE cytotoxicity, which was mainly dependent on claudin-4 expression. These results suggest that the condition of claudin-4 at the cell surface is important for CPE cytotoxicity. Our in vivo experiments showed that a high dose of CPE is required for the effective treatment of peritoneal dissemination of ovarian cancer cells. Here, we suggest that the accessibility of CPE to claudins is important for its cytotoxicity and depends on the conditions of claudin-4 in vitro. In addition, E-cadherin expression in ovarian cancer cells affects the efficiency of CPE in vivo.

Probiotic Bacillus enhance the intestinal epithelial cell barrier and immune function of piglets.

Bacillus is widely used in the livestock industry. This study was designed to evaluate the effects of probiotic Bacillus amyloliquefaciens SC06 (Ba), originally isolated from soil, in piglets diet as an alternative to antibiotics (aureomycin), mainly on intestinal epithelial barrier and immune function. Ninety piglets were divided into three groups: G1 (containing 150 mg/kg aureomycin in the diet); G2 (containing 75 mg/kg aureomycin and 1×108 cfu/kg Ba in the diet); G3 (containing 2×108 cfu/kg Ba in the diet without any antibiotics). The results showed that, compared with the antibiotic group (G1), villus length, crypt depth and villus length/crypt depth ratio of intestine significantly increased in the G2 and G3 groups. In addition, intestinal villi morphology, goblet-cell number, mitochondria structure and tight junction proteins of intestinal epithelial cells in G2 and G3 were better than in G1. The relative gene expression of intestinal mucosal defensin-1, claudin3, claudin4, and human mucin-1 in G3 was significantly lower, while the expression of villin was significantly higher than in the antibiotic group. Probiotic Ba could significantly decrease serum interferon (IFN)-α, IFN-γ, interleukin (IL)-1ß, and IL-4 levels, whereas increase tumour necrosis factor (TNF)-α and IL-6 secretion. Ba could also significantly decrease cytokines TNF-α, IFN-γ, IL-1ß, and IL-4 level in liver, whereas it significantly increased IFN-α. Furthermore, replacing antibiotics with Ba also significantly down-regulated gene expression of TNF and IL-1α in intestinal mucosa, but up-regulated IL-6 and IL-8 transcription. Dietary addition of Ba could significantly reduce the gene expression of nuclear factor kappa beta (NFκB)-p50 and Toll-like receptor (TLR)6, while there was no significant difference for that of myeloid differentiation primary response 88, TNF receptor-associated factor-6, nucleotide-binding oligomerisation domain-containing protein 1, TLR2, TLR4, and TLR9. Taken together, our findings demonstrated that probiotic Ba could increase the intestinal epithelial cell barrier and immune function by improving intestinal mucosa structure, tight junctions and by activating the TLRs signalling pathway.

Perioperative oral supplementation with fish oil promotes liver regeneration following partial hepatectomy in mice via AMPK activation.

The present study aimed to observe the effects of perioperative oral supplementation with fish oil (FO) on liver regeneration in mice and examine the potential mechanism. A total of 120 male ICR mice were randomly divided into 5 groups: Sham, Control, fish oil (FO), Compound C [the AMP­activated protein kinase (AMPK) inhibitor dorsomorphin], and Compound C + FO. Changes in liver function, alterations in hepatocyte proliferation and in the expression of polarization markers, and activation of AMPK signaling were examined following partial hepatectomy (PH). The results demonstrated that restoration of serum alanine aminotransferase (ALT) and total bilirubin (TBIL) levels were significantly faster in FO­treated mice compared with Control mice, and this effect was suppressed by treatment with Compound C. FO­treated mice exhibited increased numbers of Ki­67 positive hepatocytes and their postoperative liver­to­body weight ratio was significantly increased compared with the Control mice, which was also suppressed by co­treatment with the AMPK inhibitor. Furthermore, protein expression of Occludin, Claudin­3, tight junction protein 1 and bile salt export pump was gradually increased in FO­treated mice compared with Control, whereas Compound C treatment reversed this effect. Therefore, the present study revealed that perioperative oral supplementation with FO may promote liver regeneration and improved liver function in mice following PH through AMPK activation.

Claudin-3 and occludin content in the glands of colonic mucosa devoid from fecal stream submitted to topical intervention with oil extract of Curcuma longa.

PURPOSE:: To evaluate the inflammatory intensity and measure the tissue content of the proteins claudin-3 and occludin in the colonic mucosa without fecal stream submit to intervention with curcumin. METHODS:: Thirty-six rats were submitted to a proximal colostomy and a distal mucous fistula and divided into two groups according to sacrifice to be performed two or four weeks. Each group was divided into three subgroups according daily application of enemas containing saline, curcumin at 50 mg/kg/day or 200 mg/kg/day. Colitis was diagnosed by histological analysis. Claudin-3 and occludin were determined by immunohistochemistry. The tissue content of claudin-3 and occludin were quantified by computer-assisted image analysis. Mann-Whitney, Student t and ANOVA tests were used to analyze the results establishing the level of significance of 5% for both (p<0.05). RESULTS:: Curcumin at both concentrations reduces the inflammation and preserves the tissue content of the proteins claudin-3 and occludin, which was related to the concentration used and to the time of the intervention. CONCLUSION:: The application of enemas with curcumin reduces inflammation and preserves the tissue content of the proteins claudin-3 and occludin in the colonic mucosa devoid from the fecal stream.

Dietary Tryptophan Enhanced the Expression of Tight Junction Protein ZO-1 in Intestine.

Dietary amino acids provide various beneficial effects for our health. The aim of the present study was to assess the effects of tryptophan (Trp) supplementation on barrier function. Ninety-six healthy finishing pigs (initial body weight 51.49 ± 1.12 kg) were randomly allocated into 2 treatment groups, control group, and 0.2% Trp group. The control group was fed the basal diet, and 0.2% Trp group was fed basal diet plus 0.2% Trp. The trial period is 60 d. Compared with control group, the mRNA abundance of claudin-3 and zonula occluden-1 (ZO-1) in the jejunum in 0.2% Trp group (P < 0.05) was increased. According to immunohistochemistry and immunoblotting test, the expression of ZO-1 in jejunum in 0.2% Trp group was also significantly increased compared with the control group (P < 0.05). These results revealed that Trp enhanced the expression of tight junction protein ZO-1 in the intestine of pig model. Trp may be potential and beneficial dietary functional factor for regulating the intestinal development and inhibiting intestinal aging.

Claudin-3 and occludin content in the glands of colonic mucosa devoid from fecal stream submitted to topical intervention with oil extract of Curcuma longa

Abstract Purpose: To evaluate the inflammatory intensity and measure the tissue content of the proteins claudin-3 and occludin in the colonic mucosa without fecal stream submit to intervention with curcumin. Methods: Thirty-six rats were submitted to a proximal colostomy and a distal mucous fistula and divided into two groups according to sacrifice to be performed two or four weeks. Each group was divided into three subgroups according daily application of enemas containing saline, curcumin at 50 mg/kg/day or 200 mg/kg/day. Colitis was diagnosed by histological analysis. Claudin-3 and occludin were determined by immunohistochemistry. The tissue content of claudin-3 and occludin were quantified by computer-assisted image analysis. Mann-Whitney, Student t and ANOVA tests were used to analyze the results establishing the level of significance of 5% for both (p<0.05). Results: Curcumin at both concentrations reduces the inflammation and preserves the tissue content of the proteins claudin-3 and occludin, which was related to the concentration used and to the time of the intervention. Conclusion: The application of enemas with curcumin reduces inflammation and preserves the tissue content of the proteins claudin-3 and occludin in the colonic mucosa devoid from the fecal stream.

Galacto-oligosaccharides Protect the Intestinal Barrier by Maintaining the Tight Junction Network and Modulating the Inflammatory Responses after a Challenge with the Mycotoxin Deoxynivalenol in Human Caco-2 Cell Monolayers and B6C3F1 Mice.

BACKGROUND: The integrity of the epithelial layer in the gastrointestinal tract protects organisms from exposure to luminal antigens, which are considered the primary cause of chronic intestinal inflammation and allergic responses. The common wheat-associated fungal toxin deoxynivalenol acts as a specific disruptor of the intestinal tight junction network and hence might contribute to the pathogenesis of inflammatory bowel diseases. OBJECTIVE: The aim of the current study was to assess whether defined galacto-oligosaccharides (GOSs) can prevent deoxynivalenol-induced epithelial dysfunction. METHODS: Human epithelial intestinal Caco-2 cells, pretreated with different concentrations of GOSs (0.5%, 1%, and 2%) for 24 h, were stimulated with 4.2-µM deoxynivalenol (24 h), and 6/7-wk-old male B6C3F1 mice were fed a diet supplemented with 1% GOSs for 2 wk before being orally exposed to deoxynivalenol (25 mg/kg body weight, 6 h). Barrier integrity was determined by measuring transepithelial electrical resistance (TEER) and intestinal permeability to marker molecules. A calcium switch assay was conducted to study the assembly of epithelial tight junction proteins. Alterations in tight junction and cytokine expression were assessed by quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, or ELISA, and their localization was visualized by immunofluorescence microscopy. Sections of the proximal and distal small intestine were stained with hematoxylin/eosin for histomorphometric analysis. RESULTS: The in vitro data showed that medium supplemented with 2% GOSs improved tight junction assembly reaching an acceleration of 85% after 6 h (P < 0.05). In turn, GOSs prevented the deoxynivalenol-induced loss of epithelial barrier function as measured by TEER (114% of control), and paracellular flux of Lucifer yellow (82.7% of prechallenge values, P < 0.05). Moreover, GOSs stabilized the expression and cellular distribution of claudin3 and suppressed by >50% the deoxynivalenol-induced synthesis and release of interleukin-8 [IL8/chemokine CXC motif ligand (CXCL8)] (P < 0.05). In mice, GOSs prevented the deoxynivalenol-induced mRNA overexpression of claudin3 (P = 0.022) and CXCL8 homolog keratinocyte hemoattractant (Kc) (Cxcl1) (P = 0.06) as well as the deoxynivalenol-induced morphologic defects. CONCLUSIONS: The results demonstrate that GOSs stimulate the tight junction assembly and in turn mitigate the deleterious effects of deoxynivalenol on the intestinal barrier of Caco-2 cells and on villus architecture of B6C3F1 mice.

Simvastatin attenuates sepsis-induced blood-brain barrier integrity loss.

BACKGROUND: Systemic inflammation and oxidative stress are crucial in mediating blood-brain barrier (BBB) integrity loss during sepsis. Simvastatin possess potent anti-inflammation and antioxidation capacity. We sought to elucidate whether an acute bolus of simvastatin could mitigate BBB integrity loss in a rodent model of polymicrobial sepsis. METHODS: A total of 96 adult male rats (200-250 g) were randomized to receive cecal ligation and puncture (CLP), CLP plus simvastatin, sham operation, or sham operation plus simvastatin (n = 24 in each group). After maintaining for 24 h, BBB integrity in the surviving rats was determined. RESULTS: CLP significantly induced BBB integrity loss, as grading of Evans blue staining of the brains, BBB permeability to Evans blue dye, and brain edema levels in rats receiving CLP were significantly higher than those receiving sham operation. In contrast, grading of Evans blue staining (P = 0.020), BBB permeability to Evans blue dye (P = 0.031), and brain edema levels (P = 0.009) in rats receiving CLP plus simvastatin were significantly lower than those receiving CLP alone. Tight junction proteins claudin-3 and claudin-5 in endothelial cells are major structural components of BBB. Our data revealed that concentrations of claudin-3 and claudin-5 in rats receiving CLP were significantly lower than those receiving CLP plus simvastatin (P = 0.010 and 0.007). Immunohistochemistry further revealed significant fragmentation of claudin-3 and claudin-5 in rats receiving CLP. Moreover, levels of claudin-3 and claudin-5 fragmentation in rats receiving CLP plus simvastatin were significantly lower than those receiving CLP. CONCLUSIONS: Simvastatin mitigates BBB integrity loss in a rodent model of polymicrobial sepsis.