Tight Junctions In Intestinal Epithelial Cells Research Articles

Effect of STING signaling on intestinal barrier damage in severe acute pancreatitis

BackgroundPatients with severe acute pancreatitis (SAP) have a compromised intestinal barrier with decreased barrier function and increased cell death. Intestinal epithelial cells (IECs) create a physicochemical barrier that anchors bacteria in the intestine. Recent studies have shown that the stimulator of interferons genes (STING) signaling pathway plays an important function in a number of inflammatory conditions. MethodsThe rat SAP model was established by retrograde injection of freshly prepared sodium taurocholate into the biliopancreatic duct. Serum amylase (AMY), lipase (LIPA), interleukin (IL)-6, interferon (IFN)-β, tumor necrosis factor (TNF)-α, intestinal-type fatty acid binding protein (FABP2), diamine oxidase (DAO) and endotoxin (ET) levels were measured in rats. H&E staining was used to assess histological changes in the intestine and pancreas. The expression of intestinal epithelial cell tight junction (TJ) proteins and STING signaling pathway proteins and genes were measured by RT- PCR, Western blot and immunofluorescence staining were used to analyze. The expression of STING signaling pathway proteins in pancreas were measured by Western blot were used to analyze. TUNEL was used to detect IECs death. ResultsUpregulation of STING pathway-related proteins and genes occurred after sap-induced IECs. In addition, C-176 reduced serum AMY, LIPA, TNF-α, IL-6, INF-β, FABP2, DAO and endotoxin levels and decreased pancreatic and intestinal histopathological injury in SAP rats; DMXAA aggravated serum AMY, LIPA, TNF-α, IL-6, INF-β, FABP2, DAO and endotoxin levels and increased pancreatic and intestinal histopathological injury in SAP rats. ConlusionsThe results suggest that inhibition of STING signaling can alleviate IECs after SAP, and activation of STING signaling can aggravate IECs after SAP.

Dietary Soybean Lecithin Improves Growth, Immunity, Antioxidant Capability and Intestinal Barrier Functions in Largemouth Bass Micropterus salmoides Juveniles.

This study evaluated the effects of dietary soybean lecithin (SBL) on the growth, haematological indices, immunities, antioxidant capabilities, and inflammatory and intestinal barrier functions because little information of dietary SBL could be obtained in juvenile largemouth bass (Micropterus salmoides). The fish were fed identical diets except for SBL added at 0, 2, 4 and 8%. It was found that 4 and 8% SBL significantly increased fish weight gain and daily growth rate (p < 0.05), while 4% SBL was optimal for enhancing RBC, HGB, PLT, MCV, MCH, WBC and MON in blood, and ALB and ALP in serum (p < 0.05). SBL (4%) also significantly elevated the antioxidant enzymes activities of T-SOD, CAT, GR, GPx, GST and T-AOC and GSH contents; increased mRNA transcription levels of Nrf2, Cu/Zn-SOD, CAT, GR, GST3 and GPx3; and decreased MDA contents. Keap1a and Keap1b levels were markedly down-regulated (p < 0.05). SBL (4%) significantly enhanced levels of the immune factors (ACP, LZM and C3) and the mRNA expression levels of innate immune-related genes (C3, C4, CFD, HEPC and MHC-I) compared with the control groups (0%) (p < 0.05). SBL (4%) significantly increased IgM and T-NOS in the intestine (p < 0.05) and significantly decreased levels of TNF-α, IL-8, IL-1β and IFN-γ and increased TGF-β1 at both transcription and protein levels in the liver and intestine (p < 0.05). The mRNA expression levels of MAPK13, MAPK14 and NF-κB P65 were significantly decreased in the intestine in the 4% SBL groups (p < 0.05). Histological sections also demonstrated that 4% SBL protected intestinal morphological structures compared with controls. This included increased intestinal villus height and muscular thickness (p < 0.05). Furthermore, the mRNA expression levels of the intestinal epithelial cell tight junction proteins (TJs) (ZO-1, claudin-3, claudin-4, claudin-5, claudin-23 and claudin-34) and mucin-5AC were significantly up-regulated in the 4% SBL groups compared with the controls (p < 0.05). In conclusion, these results suggested that 4% dietary SBL could not only improve growth, haematological indices, antioxidant capabilities, immune responses and intestinal functions, but also alleviate inflammatory responses, thereby providing reference information for the feed formulations in cultured largemouth bass.

Angelica oil restores the intestinal barrier function by suppressing S100A8/A9 signalling in mice with ulcerative colitis

BackgroundUlcerative colitis (UC) progression is driven by the activation of immune cells that release pro-inflammatory mediators to disrupt intestinal epithelial barrier integrity. This study aimed to investigate the potential protective effects of Angelica oil (AO) on the intestinal epithelial barrier in mice with UC and the underlying mechanisms. MethodsImprovement of the disease state and protective effect of AO on the intestinal epithelial barrier were observed in mice with dextran sulphate sodium salt (DSS)-induced UC. Protein microarrays were used to screen AO-affected cytokine pools and their recruited immune cells for accumulation in the tissues. Furthermore, quantitative proteomics was applied to search for AO-acting molecules and to verify in vitro the functions of key molecules between inflammation and the intestinal mucosal barrier. ResultsAO significantly alleviated intestinal inflammation, reduced intestinal permeability, and retained barrier function in mice with UC. Furthermore, cytokines inhibited by AO mainly promoted monocyte and neutrophil activation or chemotaxis. Moreover, proteomic screening revealed that S100A8/A9 was a key molecule significantly regulated by AO, and its mediated TLR4/NF-κB pathway was also inhibited. Finally, we verified that AO inhibited the activation of the S100A8/A9/TLR4 signalling pathway and enhanced the expression of tight junctions (TJs) proteins using a cellular model of intestinal barrier damage induced by S100A8/A9 or macrophage-derived medium. And the enhancement of TJs in intestinal epithelial cells and the inhibition of inflammatory signalling by AO were significantly attenuated due to the application of S100A8/A9 monoclonal antibody. ConclusionThese results demonstrated that AO improves intestinal mucosal barrier damage in the inflammatory environment of mice with UC by inhibiting the expression of S100A8/A9 and the activation of its downstream TLR4/NF-κB signalling pathway.

The Role of Epigenomic Regulatory Pathways in the Gut-Brain Axis and Visceral Hyperalgesia.

The gut-brain axis (GBA) is broadly accepted to describe the bidirectional circuit that links the gastrointestinal tract with the central nervous system (CNS). Interest in the GBA has grown dramatically over past two decades along with advances in our understanding of the importance of the axis in the pathophysiology of numerous common clinical disorders including mood disorders, neurodegenerative disease, diabetes mellitus, non-alcohol fatty liver disease (NAFLD) and enhanced abdominal pain (visceral hyperalgesia). Paralleling the growing interest in the GBA, there have been seminal developments in our understanding of how environmental factors such as psychological stress and other extrinsic factors alter gene expression, primarily via epigenomic regulatory mechanisms. This process has been driven by advances in next-generation multi-omics methods and bioinformatics. Recent reviews address various components of GBA, but the role of epigenomic regulatory pathways in chronic stress-associated visceral hyperalgesia in relevant regions of the GBA including the amygdala, spinal cord, primary afferent (nociceptive) neurons, and the intestinal barrier has not been addressed. Rapidly developing evidence suggests that intestinal epithelial barrier dysfunction and microbial dysbiosis play a potentially significant role in chronic stress-associated visceral hyperalgesia in nociceptive neurons innervating the lower intestine via downregulation in intestinal epithelial cell tight junction protein expression and increase in paracellular permeability. These observations support an important role for the regulatory epigenome in the development of future diagnostics and therapeutic interventions in clinical disorders affecting the GBA.

Gut Microbiota Modulation by Polyphenols from Aronia melanocarpa of LPS-Induced Liver Diseases in Rats.

Aronia melanocarpa polyphenols (AMPs) can alleviate the degree of liver diseases in rats. However, the mechanism by which this is achieved through gut microbiota modulation remains unclear. Here, a rich-polyphenol extract of A. melanocarpa (AMPs) was used to treat lipopolysaccharide (LPS)-induced liver diseases in rats. To gain insights into the anti-LPS-induced liver disease, liver function index, expression of apoptosis proteins, inflammatory factors, and activation of inflammatory signaling pathways were determined with western blot analysis, immunohistochemistry, and 16S rRNA sequencing or quantitative real-time polymerase chain reaction (qRT-PCR). After AMPs treatment, the gut microbiota composition was modulated, promoting the intestinal barrier function by increasing the expression of intestinal epithelial cell tight junction proteins to reduce the LPS content in serum. The expression levels of inflammatory factors interleukin 6 (IL-6), interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and related mRNAs were reduced. These results showed that AMPs, as a bioactive substance, could enhance the intestinal barrier function and modulate the gut microbiota of LPS-induced liver diseases.

Mechanism of gasdermin D on intestinal injury in severe acute pancreatitis by mediating pyroptosis

To investigate the function of gasdermin D (GSDMD) in intestinal damage of mice with severe acute pancreatitis (SAP). The healthy C57BL/6 mice were divided into four groups randomly, including normal saline (NS) group, small interfering RNA (siRNA)-NS group, SAP model group and siRNA-SAP group, with 6 mice in each group. The SAP mouse model was reproduced by intraperitoneal injection of caerulein 50 μg/kg combined with lipopolysaccharide (LPS) 10 mg/kg; the NS group was given the same amount of NS; in the siRNA-SAP group and siRNA-NS group, siRNA 50 mg/kg was injected through the tail vein three times before modeling or injection of NS. The blood of mice eyeball in each group was taken 12 hours after modeling, and serum interleukins (IL-1β, IL-18) levels were detected by enzyme linked immunosorbent assay (ELISA). The mice were sacrificed to observe the general changes in abdominal cavity, the pancreas and ileum tissues were taken to observe the pathological changes under a light microscope. The expression of long-chain non-coding RNA uc.173 (lnc uc.173) was detected by reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical method was used to detect the expression of tight junction proteins zonula occluden-1 (ZO-1) and Occludin in intestinal mucosal epithelial cells. Western blotting was used to detect the GSDMD protein expression level in the intestinal tissue. The serum levels of IL-1β and IL-18 in the SAP model group were significantly higher than those in the NS group and the siRNA-NS group [IL-1β (ng/L): 146.66±1.40 vs. 44.48±5.76, 81.49±10.75, IL-18 (ng/L): 950.47±177.09 vs. 115.43±16.40, 84.84±21.90, all P < 0.05]; and the levels of IL-1β and IL-18 in the siRNA-SAP group were significantly lower than those in the SAP model group [IL-1β (ng/L): 116.26±15.54 vs. 146.66±1.40, IL-18 (ng/L): 689.96±126.08 vs. 950.47±177.09, both P < 0.05]. General observation showed that there were no obvious abnormalities in the abdominal cavity of the mice in the NS and siRNA-NS groups; the mice in the SAP model group and the siRNA-SAP group had different degrees of edema and congestion in the intestine; compared with the SAP model group, the abnormalities in the siRNA-SAP group was significantly reduced. Under light microscope, there were no obvious changes in the pancreas and intestinal mucosa in the NS group and the siRNA-NS group; the pancreatic tissue of the SAP model group and the siRNA-SAP group had different degrees of edema, inflammatory cell infiltration, and lobular structure damage, and the intestinal mucosa was damaged to a certain degree, and the villi were broken to varying degrees, but the damage in the siRNA-SAP group was lighter. The results of RT-PCR showed that the expression of lnc uc.173 in the intestinal tissues of the model SAP group was significantly lower than that of the NS group and the siRNA-NS group (2-ΔΔCt: 0.26±0.12 vs. 1.01±0.37, 0.67±0.32, both P < 0.05), while the expression of lnc uc.173 in the siRNA-SAP group was significantly higher than that in the SAP model group (2-ΔΔCt: 0.60±0.39 vs. 0.26±0.12, P < 0.05). Immunohistochemistry showed that ZO-1 and Occludin proteins in the NS group were distributed along the epithelial cells of the intestinal mucosa, showing a strong expression; ZO-1 and Occludin expressions were significantly reduced in the SAP model group and siRNA-SAP group, but the expressions in the siRNA-SAP group was higher than that in the SAP model group. Western blotting showed that the expression level of GSDMD protein in the intestinal tissues of the SAP model group was significantly higher than that of the NS group and the siRNA-NS group [GSDMD protein (GSDMD-N/β-actin): 1.99±0.46 vs. 1, 1.00±0.78, both P < 0.05]. Compared with the SAP model group, the expression of GSDMD protein in the siRNA-SAP group was significantly decreased [GSDMD protein (GSDMD-N/β-actin): 1.42±0.42 vs. 1.99±0.46, P < 0.05]. The systemic inflammatory response and intestinal mucosal barrier damage of SAP mice may be related to the increase of GSDMD expression in intestinal tissues. GSDMD mediates cell pyrolysis to promote the release of inflammatory factors, cause intestinal injury, and down-regulate the expression of intestinal epithelial cell tight junction proteins such as ZO-1 and Occludin, resulting in intestinal mucosal damage.

Effect of herb-partitioned moxibustion in improving tight junctions of intestinal epithelium in Crohn disease mediated by TNF-α-NF-κB-MLCK pathway

To explore the effect of herb-partitioned moxibustion (HPM) on tight junctions (TJs) of intestinal epithelial cells in Crohn disease (CD) mediated by tumor necrosis factor-α (TNF-α)-nuclear factor kappa B (NF-κB)-myosin-light-chain kinase (MLCK) pathway. Forty-eight male Sprague-Dawley rats were randomly divided into a normal control (NC) group, a model control (MC) group, an HPM group and a mesalazine (MESA) group, with 12 rats in each group. Trinitrobenzene sulfonic acid (TNBS) was administered to establish CD models. When the model was confirmed a success, the HPM group rats were treated with HPM at Tianshu (ST 25) and Qihai (CV 6), while the MESA group rats were given MESA solution by lavage. When the intervention finished, the colonic epithelial tissues were separated, purified and cultured in each group to establish the intestinal epithelial barrier model in vitro, and TNF-α was added (100 ng/mL) in the culture medium and maintained for 24 h to establish an increased epithelial permeability model. Transepithelial electrical resistance (TEER) was used to examine the permeability of the barrier; Western blot was used to observe the expressions of the proteins related to TJs of intestinal epithelial cells mediated by TNF-α-NF-κB-MLCK pathway; immunofluorescence staining was used to observe the expressions and distributions of tight junction proteins in the intestinal epithelium. After TNF-α induction, compared with the MC+TNF-α group, the TEER value increased significantly in the HPM+TNF-α and MESA+TNF-α groups (both P<0.001); the expressions of nuclear factor kappa B (NF-κB) p65, MLCK, myosin light chain (MLC), tumor necrosis factor receptor-associated factor 6 (TRAF6) and receptor interaction protein-1 (RIP1) decreased significantly (P<0.01 or P<0.05), and the expression of zinc finger protein A20 (A20) increased significantly (P<0.01); the expressions of occludin, claudin-1, zonula occludens protein 1 (ZO-1) and F-actin also increased significantly (all P<0.01). Compared with the MESA+TNF-α group, the expressions of MLC, occludin, claudin-1, ZO-1 and F-actin increased significantly in the HPM+TNF-α group (P<0.01 or P<0.05). HPM can protect or repair the damage of intestinal epithelial barrier in CD rats, which may be achieved through modulating the abnormal TJs in intestinal epithelium mediated by TNF-α-NF-κB-MLCK pathway.

Histone H3K9 methylation regulates chronic stress and IL-6-induced colon epithelial permeability and visceral pain.

Chronic stress is associated with activation of the HPA axis, elevation in pro-inflammatory cytokines, decrease in intestinal epithelial cell tight junction (TJ) proteins, and enhanced visceral pain. It is unknown whether epigenetic regulatory pathways play a role in chronic stress-induced intestinal barrier dysfunction and visceral hyperalgesia. Young adult male rats were subjected to water avoidance stress±H3K9 methylation inhibitors or siRNAs. Visceral pain response was assessed. Differentiated Caco-2/BBE cells and human colonoids were treated with cortisol or IL-6±antagonists. Expression of TJ, IL-6, and H3K9 methylation status at gene promoters was measured. Transepithelial electrical resistance and FITC-dextran permeability were evaluated. Chronic stress induced IL-6 up-regulation prior to a decrease in TJ proteins in the rat colon. The IL-6 level inversely correlated with occludin expression. Treatment with IL-6 decreased occludin and induced visceral hyperalgesia. Chronic stress and IL-6 increased H3K9 methylation and decreased transcriptional GR binding to the occludin gene promoter, leading to down-regulation of protein expression and increase in paracellular permeability. Intrarectal administration of a H3K9 methylation antagonist prevented chronic stress-induced visceral hyperalgesia in the rat. In a human colonoid model, cortisol decreased occludin expression, which was prevented by the GR antagonist RU486, and IL-6 increased H3K9 methylation and decreased TJ protein levels, which were prevented by inhibitors of H3K9 methylation. Our findings support a novel role for methylation of the repressive histone H3K9 to regulate chronic stress, pro-inflammatory cytokine-mediated reduction in colon TJ protein levels, and increase in paracellular permeability and visceral hyperalgesia.

Regulation of the intestinal barrier by nutrients: The role of tight junctions.

Tight junctions (TJs) play an important role in intestinal barrier function. TJs in intestinal epithelial cells are composed of different junctional molecules, such as claudin and occludin, and regulate the paracellular permeability of water, ions, and macromolecules in adjacent cells. One of the most important roles of the TJ structure is to provide a physical barrier to luminal inflammatory molecules. Impaired integrity and structure of the TJ barrier result in a forcible activation of immune cells and chronic inflammation in different tissues. According to recent studies, the intestinal TJ barrier could be regulated, as a potential target, by dietary factors to prevent and reduce different inflammatory disorders, although the precise mechanisms underlying the dietary regulation remain unclear. This review summarizes currently available information on the regulation of the intestinal TJ barrier by food components.

The Mechanism of Mica on Protecting NSAIDs Induced Small Intestinal Mucosal Barrier Injury via ncRuPAR-PAR2 Pathway

Objective: The study was to detect the role of ncRuPAR and Protease activated receptor (PAR)2 in the mechanism of Non-steroidal anti-inflammatory drugs(NSAIDs) induced small intestinal mucosal barrier injury. And to clarify the possible mechanism of mica in treatment NSAIDs induced small intestinal disease. Methods: Caco-2 cells were cultured and divided into 6 groups: control group, diclofenac sodium group, control RNAa group, ncRuPAR RNA activation (RNAa) group, diclofenac sodium + mica group, diclofenac sodium + FSLLRY-NH2 group. 40 μmol/L diclofenac sodium treatment for 24 hours to establish NSAIDs induced small intestinal epithelial cell injury model, 10% mica-containing serum treatment for 24h, 100uM PAR2 blocker FSLLRY-NH2 treatment for 24h. Blank vector or ncRuPAR overexpression vector were transfected into cells for 72h. Then Transwell assay were performed to detect cell permeability, qPCR were used to detect RNA expression of PAR2 and ncRuPAR, and western blot were performed to detect PAR2, occludin and claudin-1 expression. Result: NSAIDs-induced small intestinal epithelial cell injury model was successfully built. Compared with the control group, ncRuPAR and PAR2 were up-regulated, occludin and claudin-1 were down-regulated, cell permeability were increased in diclofenac sodium group (P 0.05). Compared with the control RNAa group, overexpression of ncRuPAR significantly up-regulated the expression of ncRuPAR and PAR2, down-regulated the expression of occludin and claudin-1, and increased cell permeability (P<0.01). Conclusion: When NSAIDs induced small intestinal injury occurs, ncRuPAR activation could up-regulate PAR2, causing the destruction of intestinal epithelial cell tight junction and the increase of intestinal epithelial cells permeability. Mica-containing serum has a similar effect as PAR2 blocker, which could antagonize the above-mentioned damage process and play a role in protecting the mechanical barrier function of the intestinal mucosa. Funding Statement: This research was supported by funding from Zhejiang Provincial Natural Science Foundation of China under Grant No.LY18H030001； the Medicine and Health Science and Technology Plan Projects in Zhejiang province(2017KY413), Traditional Chinese Medicine Science and Technology Plan of Zhejiang Province(2017ZA089, 2016ZB071, 2015ZZ012, 2014ZA030); National Natural Science Foundation of China (81573760);Medical Health Platform Plan Projects of Zhejiang Province (2015RCA020); Zhejiang Provincial Natural Science Foundation of China (LY16H030010). Declaration of Interests: The authors state: There is no conflict of interest. Ethics Approval Statement: Not required.

Effect of Aging on Experimental Colitis in Mice and Its Associated Mechanism

Objective To explore whether aging increases severity of colitis in mice and its mechanism.Methods Young (6-8 weeks)and aged (56 weeks) C57Bl/6 mice were divided into the control and experimental group (n=5,each). Dextran sodium sulfate(DSS) was used to induce acute colitis mouse model in the experimental group.The mRNA expressions of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in colon were measured by RT-PCR. Tight junctions (TJs) of intestinal epithelial cells was examined by transmission electron microscopy (TEM). Protein expressions of E-cadherin and occludin were detected by Western blotting and immunohistochemistry in colon.Results Compared with the young DSS-induced mice,the aged DSS-induced mice had more weight loss(t=3.679,P=0.006),higher disease indexes (t=2.496,P=0.037),higher histologic scores(U=0.000,P=0.008) and higher colonic IL-6 level (U=4.000,P=0.191). The TJs of intestinal epithelial cells were discontinuous in old healthy rats,and the TJs were destroyed significantly in both young and aged DSS-induced mice. Compared with the young DSS-induced mice,the aged DSS-induced mice had decreased protein expressions of E-cadherin (t=0.184,P=0.863)and occludin (t=0.399,P=0.710).Conclusions Aging leads to more severe disease following DSS challenge. Age-related deterioration in the functions of the gastrointestinal barrier and integrity may be one of the possible mechanisms.

Modulation of the xenobiotic transformation system and inflammatory response by ochratoxin A exposure using a co-culture system of Caco-2 and HepG2 cells

Cytotoxicity of ochratoxin A (OTA) was evaluated using the MTS assay, and membrane integrity was measured using transepithelial electrical resistance (TEER). A transwell system was used to investigate the effect of OTA on the expression of the CYP450 (1A1, 2A6, 2B6, 3A4 and 3A5), NAT2, COX-2, LOX-5, and MRP2 genes in Caco-2 and HepG2 cells. TEER decreased by a mean of 63.2% after 24 h in Caco-2 differentiated cells without inducing cell detachment; revealing damage to the intestinal epithelial cell tight junction proteins and an increase in cell permeability. Gene expression analysis showed that modulation of gene expression by OTA was higher in Caco-2 cells than in HepG2 cells, and generally, the duration of exposure to OTA had a more significant effect than the OTA dose. A general OTA down-regulation effect was observed in Caco-2 cells, in contrast with the down- and up-regulation observed in HepG2 cells. In Caco-2 cells, CYP1A1 was the gene with the highest regulation, followed by CYP3A4 and CYP3A5. Conversely, in HepG2 cells, CYP2B6 was highly regulated at 3 and 12 h compared to the other cytochromes; CYP1A1 was slightly modulated during the first 12 h, but an overexpression was observed at 24 h.Our data support the involvement of the COX-2 and 5-LOX genes in liver metabolism of OTA. On the basis of the gene expression analysis, the results suggest a possible impairment in OTA secretion at the intestinal and hepatic level due to MRP2 repression. In addition, we provide evidence of the effect of OTA on NAT2 gene expression, which had not been reported before.

What could probiotic do for us?

Gastrointestinal microbes play important roles in the health and disease of the host. There are many documented evidences which demonstrated that disturbance of intestinal microbiota is linked to the risk of developing infectious, inflammatory and allergic diseases. Human intestine is home for a complex consortium of 1013–1014 microbial cells. Interactions between the intestinal microbes, pathogens and the host lead to exclusion of toxins (mycotoxins) and pathogens (colonization resistance), interference in disease progression as demonstrated in the prevention of oral infection, dental caries, diarrhoeas (Antibiotic Associated Diarrhoea, Travellers’ Diarrhoea and Rotavirus Diarrhoea), postoperative infection, respiratory infection and certain cancers. The group of beneficial intestinal microbes termed probiotics alter intestinal epithelial cell tight junction and immunological functions. Lately, laboratory and clinical studies demonstrated gut-brain axis communication and intestinal microbial (both pathogens and probiotics) modulation of host psycho-neuroimmunological functions, in relation to depression, anxiety and memory dysfunction. These open up many possibilities of probiotics supplementation for moderating intestinal microbiota as an approach in disease prevention and treatment.

Age-related changes in small intestinal mucosa epithelium architecture and epithelial tight junction in rat models

Functions of the intestinal mucosal barrier are often impaired in the elderly and are closely associated with many age-related diseases. However, mechanisms by which aging influences intestinal barrier function still remain unclear. The aim of this study was to investigate age-related changes in small intestinal morphology, bacteria contents and expression of epithelial tight junction (TJ) proteins. Thirty Sprague-Dawley rats were divided into groups: young (3months), adult (12months), and old (24months). The small intestinal mucosal architecture and TJ of intestinal epithelial cells were examined by light microscopy and transmission electron microscopy. Jejunum and cecum contents were cultured to identify and measure bacterial species. mRNA expression of Zonula occludens-1 (ZO-1) and occludin were measured by semi-quantitative RT-PCR. Protein expression of ZO-1 and occludin were detected by immunohistochemistry and Western blot. Normal ileum villi, which were thick and regularly arranged, though increasingly scattered and atrophic in character with shorter and narrower dimensions (P<0.01), were observed in old rats, along with an elevated number of Gram-positive and Gram-negative bacteria in the jejunum. The TJs of intestinal epithelial cells, as detected by transmission electron microscopy, were wider and discontinuous in old rats. Age-induced down-regulation of mRNA expression and decreased protein expression of ZO-1 and occludin were observed in the ileum (P<0.01). Our study indicated that age-related intestinal barrier dysfunction may be associated with mucosal atrophy, damages to TJ structure, increased small intestine bacteria counts, and decreased epithelial TJ protein.

Abstract P6-08-07: Claudin-20 Promotes an Aggressive Phenotype in Human Breast Cancer Cells

Abstract Background: Claudin-20 is a member of the claudin family of transmembrane proteins located in the tight junction (TJ) of cells of epithelial origin. Claudin-20 was originally described as being distributed in the TJ of intestinal epithelial cells. Due to the increasing evidence supporting the role of TJ proteins in preventing tumour cell metastatic behaviour, this study sought to evaluate the effect of claudin-20 overexpression in human breast cancer cells. Methods: Breast cancer primary tumours (n= 114) and matched background tissue (n=30) were processed for RNA extraction. RNA was reverse transcribed and quantified before analysis by Q-PCR to obtain survival curves. 42 human cancer cell lines were screened for expression of claudin-20. Invasive and non-invasive breast cancer cell lines, MDA-MB-231 (MDAwt) and MCF-7 (MCF-7wt) respectively, which exhibited negligible expression were chosen for over-expression of the claudin-20 gene (MDAexp &amp; MCF-7exp). The gene, after amplification from normal breast cDNA was cloned into a GFP-Fusion TOPO plasmid before electroporation. Expression of the gene product was confirmed by RT-PCR and fluorescent microscopy. Functional testing was carried out using TER (trans-epithelial resistance), paracellular permeability (PCP), cell invasion, cell adhesion and cell growth assays. In addition, in vivo growth was evaluated using an athymic nu/nu mice model. Results: Q-PCR data showed that high claudin-20 expression was correlated with poor survival of patients with breast cancer (p=0.022). We therefore decided to investigate the effect of claudin-20 over-expression in human breast cancer cell behaviour. Following transformation of the breast cancer cell lines MDA-MB-231 &amp; MCF-7 with a claudin-20 expression construct, the expression of the gene was demonstrated by the presence of mRNA expression and by the protein signal clearly observable at the cell-cell membrane. Claudin-20 transformed cells showed significantly increased invasion compared to WT cells (MDAexp 0.332±0.02 RU, MDAwt 0.304±0.001; p=0.05: MCF-7exp0 0.07±0.002 RU, MCF-7wt 0.05±0.0001; p=0.0014). Both MDAexp and MCF-7exp cells were significantly less adhesive than WT cells, (number of cells adhering to matrix: MDAexp 0.2±0.02 RU versus MDAwt 0.27±0.004, p=0.007; MCF-7exp 0.043±0.0005 RU versus MCF-7wt 0.052±0.0.005, p=0.05). There was no effect on growth (either in vitro or in vivo) for either cell line. Over-expression of Claudin-20 resulted in reduced TER (induced by the motogen HGF at 25 ng/ml: MDAexp change in TER at 2h -74+/−1.5 vs MDAwt at 2h -40+/−4.4, p=0.0007). Interestingly, this was not mirrored by PCP, as over-expression of claudin-20 caused a reduction in permeability (MDAexp change in PCP at 2h 182+/−5 vs MDAwt at 2h 260+/−10). Conclusion: The introduction of claudin-20 into human breast cancer cells resulted in breast cancer cells with an aggressive phenotype and reduced trans-epithelial resistance. There was no corresponding decrease in paracellular permeability, indicating that this claudin has a differential function in epithelial TJ. This provides further insight into the importance of correctly functioning TJ in preventing the progression of human breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-08-07.