Regulation of intestinal permeability: The role of proteases

The gastrointestinal barrier is-with approximately 400 m^2-the human body's largest surface separating the external environment from the internal milieu. This barrier serves a dual function: permitting the absorption of nutrients, water and electrolytes on the one hand, while limiting host contact with noxious luminal antigens on the other hand. To maintain this selective barrier, junction protein complexes seal the intercellular space between adjacent epithelial cells and regulate the paracellular transport. Increased intestinal permeability is associated with and suggested as a player in the pathophysiology of various gastrointestinal and extraintestinal diseases such as inflammatory bowel disease, celiac disease and type 1 diabetes. The gastrointestinal tract is exposed to high levels of endogenous and exogenous proteases, both in the lumen and in the mucosa. There is increasing evidence to suggest that a dysregulation of the protease/antiprotease balance in the gut contributes to epithelial damage and increased permeability. Excessive proteolysis leads to direct cleavage of intercellular junction proteins, or to opening of the junction proteins via activation of protease activated receptors. In addition, proteases regulate the activity and availability of cytokines and growth factors, which are also known modulators of intestinal permeability. This review aims at outlining the mechanisms by which proteases alter the intestinal permeability. More knowledge on the role of proteases in mucosal homeostasis and gastrointestinal barrier function will definitely contribute to the identification of new therapeutic targets for permeability-related diseases.

Higher doses of ascorbic acid may have the potential to promote nutrient delivery via intestinal paracellular absorption

The significance of plasma ascorbic acid(AA)is underscored by its enzymatic and antioxidant properties as well as involvement in many aspects of health including the synthesis of biomolecules during acute illness,trauma and chronic health conditions.Dietary intake supports maintenance of optimal levels with supplementation at higher doses more likely pursued.Transient increased intestinal paracellular permeability following high dose AA may be utilised to enhance delivery of other micronutrients across the intestinal lumen.The potential mechanism following dietary intake however needs further study but may provide an avenue to increase small intestinal nutrient co transport and absorption,including in acute and chronic illness.

Emodin promoted pancreatic claudin-5 and occludin expression in experimental acute pancreatitis rats

AIM:To investigate the effect of emodin on pancreatic claudin-5 and occludin expression,and pancreatic paracellular permeability in acute pancreatitis(AP).METHODS:Experimental pancreatitis was induced by retrograde injection of 5% sodium taurocholate into the biliopancreatic duct.Emodin was injected via the external jugular vein 0 or 6 h after induction of AP.Rats from sham operation and AP groups were injected with normal saline at the same time.Samples of pancreas were obtained 6 or 12 h after drug administration.Pancreatic morphology was examined with hematoxylin and eosin staining.Pancreatic edema was estimated by measuring tissue water content.Tumor necrosis factor(TNF)-α and interleukin(IL)-6 level were measured by enzyme-linked immunosorbent assay.Pancreatic paracellular permeability was assessed by tissue dye extravasation.Expression of pancreatic claudin-5 and occludin was examined by immunohistology,quantitative real-time reverse transcriptase polymerase chain reaction and western blotting.RESULTS:Pancreatic TNF-α and IL-6 levels,wet/dry ratio,dye extravasation,and histological score were significantly elevated at 3,6 and 12 h following sodium taurocholate infusion;treatment with emodin prevented these changes at all time points.Immunostaining of claudin-5 and occludin was detected in rat pancreas,which was distributed in pancreatic acinar cells,ductal cells and vascular endothelial cells,respectively.Sodium taurocholate infusion significantly decreased pancreatic claudin-5 and occludin mRNA and protein levels at 3,6 and 12 h,and that could be promoted by intravenous administration of emodin at all time points.CONCLUSION:These results demonstrate that emodin could promote pancreatic claudin-5 and occludin expression,and reduce pancreatic paracellular permeability.

Claudin-1 Leads to Strong Formation of Tight Junction in Cultured Mouse Lung Microvascular Endothelial Cells

We aimed to examine paracellular barrier function in cultured mouse lung microvascular endothelial cells (LMECs). The transcellular resistance of LMEC monolayers yielded an electrical resistance of approximately 19 Ω × cm2 at days 6 - 7 in culture when the cells reached confluence, and paracellular permeable clearance of sodium fluorescein was the lowest on day 6 in culture, suggesting the formation of tight junctions (TJs) in cultured LMECs. Moreover, the expression of TJ-associated proteins, occludin, claudin-1 and -4 and zonula occludents 1 (ZO-1) was detected in LMECs at day 6 in culture. However, mRNAs of occludin, claudin-1 and -4 and ZO-1 were already expressed on day 1 after culture, and large variations were absent in the mRNA levels of occludin, claudin-4 and ZO-1 between days 1 and 7 in culture, when the level of each mRNA on day 1 in culture was used as a basal level. However, the claudin-1 mRNA level gradually increased up to approximately 7-fold on day 7 in culture over the basal level. These results indicate that the drastic increase in the mRNA expression level of claudin-1 leads to the strong formation of TJs.

Claudin-4 is required for AMPK-modulated paraceliular permeability in submandibular gland ceils

Tight junction plays an important rote in mediating paraceUular permeability in epithelia. We previously found that activation of AMP- activated protein kinase （AMPK） increased saliva secretion by modulating paraceUular permeability in submandibular glands. However, the molecular mechanisms underlying AMPK-modulated paraceUular permeability are unknown. In this study, we found that AICAR, an AMPK agonist, increased saliva secretion in the isolated rat submandibular glands, decreased transepithelial electrical resistance （TER）, and increased 4 kDa FITC-dextran flux in cultured SMG-C6 cells. AICAR also induced redistribution of tight junction protein claudin-4, but not claudin-1, claudin-3, occtudin, or ZO-1, from the cytoplasm to the membrane. Moreover, knockdown of claudin-4 by shRNA suppressed while claudin-4 re-expression restored the TER and 4 kDa FITC-dextran flux responses to AICAR. Additionally, AICAR increased ERK1/2 phosphorylation, and inhibition of ERK1/2 by U0126, an ERK1/2 kinase inhibitor, or by siRNA decreased AICAR-induced TER responses. AICAR induced the serine S199 phosphorylation of claudin-4 and enhanced the inter- action of claudin-4 and occludin. Furthermore, pretreatment with U0126 significantly suppressed AMPK-modulated phosphorytation, redistribution, and interaction with occludin of claudin-4. Taken together, these results indicated that claudin-4 played a crucial role in AMPK-rnodutated paraceUular permeability and ERK1/2 was required in AMPK-modulated tight junction barrier function in subman- dibular gland.

Traditional and emerging Fusarium mycotoxins disrupt homeostasis of bovine mammary cells by altering cell permeability and innate immune function

High incidence of traditional and emerging Fusarium mycotoxins in cereal grains and silages can be a potential threat to feed safety and ruminants.Inadequate biodegradation of Fusarium mycotoxins by rumen microflora following ingestion of mycotoxin-contaminated feeds can lead to their circulatory transport to target tissues such as mammary gland.The bovine udder plays a pivotal role in maintaining milk yield and composition,thus,human health.However,toxic effects of Fusarium mycotoxins on bovine mammary gland are rarely studied.In this study,the bovine mammary epithelial cell line was used as an in-vitro model of bovine mammary epithelium to investigate effects of deoxynivalenol(DON),enniatin B(ENB)and beauvericin(BEA)on bovine mammary gland homeostasis.Results indicated that exposure to DON,ENB and BEA for 48 h significantly decreased cell viability in a concentration-dependent manner(P<0.001).Exposure to DON at 0.39μmol/L and BEA at 2.5μmol/L for 48 h also decreased paracellular flux of FITC-40 kDa dextran(P<0.05),whereas none of the mycotoxins affected transepithelial electrical resistance after 48 h exposure.The qPCR was performed for assessment of expression of gene coding tight junction(TJ)proteins,toll-like receptor 4(TLR4)and cytokines after 4,24 and 48 h of exposure.DON,ENB and BEA significantly upregulated the TJ protein zonula occludens-1,whereas markedly downregulated claudin 3(P<0.05).Exposure to DON at 1.35μmol/L for 4 h significantly increased expression of occludin(P<0.01).DON,ENB and BEA significant downregulated TLR4(P<0.05).In contrast,ENB markedly increased expression of cytokines interleukin-6(IL-6)(P<0.001),tumor necrosis factorα(TNF-a)(P<0.05)and transforming growth factor-β(TGF-β)(P<0.01).BEA significantly upregulated IL-6(P<0.001)and TGF-β(P=0.01),but downregulated TNF-α(P<0.001).These results suggest that DON,ENB and BEA can disrupt mammary gland homeostasis by inducing cell death as well as altering its paracellular permeability and expression of genes involved in innate immune function.