Different Suppressive Effects of Fucoidan and Lentinan on IL-8 mRNA Expression in in Vitro Gut Inflammation

A system for assessing the anti-inflammatory effects of food factors was developed by establishing a co-culture system with intestinal epithelial Caco-2 cells and macrophage RAW264.7 cells. The results indicate that fucoidan and lentinan exhibited different suppressive effects on interleukin-8 (IL-8) mRNA expression in Caco-2 through tumor necrosis factor-α (TNF-α) production from RAW264.7 stimulated with lipopolysaccharide (LPS).     

Lineage-Specific Expression of Bestrophin-2 and Bestrophin-4 in Human Intestinal Epithelial Cells

Intestinal epithelial cells (IECs) regulate the absorption and secretion of anions, such as HCO3^- or Cl^-. Bestrophin genes represent a newly identified group of calcium-activated Cl^- channels (CaCCs). Studies have suggested that, among the four human bestrophin-family genes, bestrophin-2 (BEST2) and bestrophin-4 (BEST4) might be expressed within the intestinal tissue. Consistently, a study showed that BEST2 is expressed by human colonic goblet cells. However, their precise expression pattern along the gastrointestinal tract, or the lineage specificity of the cells expressing these genes, remains largely unknown. Here, we show that BEST2 and BEST4 are expressed in vivo, each in a distinct, lineage-specific manner, in human IECs. While BEST2 was expressed exclusively in colonic goblet cells, BEST4 was expressed in the absorptive cells of both the small intestine and the colon. In addition, we found that BEST2 expression is significantly down-regulated in the active lesions of ulcerative colitis, where goblet cells were depleted, suggesting that BEST2 expression is restricted to goblet cells under both normal and pathologic conditions. Consistently, the induction of goblet cell differentiation by a Notch inhibitor, LY411575, significantly up-regulated the expression of not BEST4 but BEST2 in MUC2-positive HT-29 cells. Conversely, the induction of absorptive cell differentiation up-regulated the expression of BEST4 in villin-positive Caco-2 cells. In addition, we found that the up- or down-regulation of Notch activity leads to the preferential expression of either BEST4 or BEST2, respectively, in LS174T cells. These results collectively confirmed that BEST2 and BEST4 could be added to the lineage-specific genes of humans IECs due to their abilities to clearly identify goblet cells of colonic origin and a distinct subset of absorptive cells, respectively.     

高表达trip-1蛋白对大鼠肾小管上皮细胞转分化的影响

目的:研究上调大鼠肾小管上皮细胞中trip-1蛋白的表达量对TGF-β1诱导的上皮细胞转分化的影响.方法:包装人TRIP-1基因重组腺病毒,用其感染NRK52E细胞36h上调内源性trip-1蛋白表达量,之后用10 ng/ml的TGF-β1对细胞进行刺激诱导,72h后做Western Blot检测细胞中E-cadherin蛋白和α-SMA蛋白表达量.结果:①包装的人TRIP-1基因重组腺病毒感染细胞能够有效上调细胞中trip-1蛋白的表达量.②上调细胞中trip-1蛋白表达量,对TGF-β1引起的NRK52E细胞中E-cadherin蛋白表达水平降低有所抑制,但对TGF-β1引起的NRK52E细胞中α-SMA蛋白表达水平升高没有明显调节作用.结论:上调大鼠肾小管上皮细胞中trip-1蛋白的表达量在一定程度上抑制了TGF-β1诱导的NRK52E细胞转分化.     

IL-8 Promote Carcinogenesis of Primary Epithelial Cells From Familial Adenomatous Polyposis

Accumulated evidences supported IL-8 play an important role during colorectal carcinogenesis. However, few direct-related evidences are available. In this paper, we found that high level of IL-8 was constitutively present both in familial adenomatous polyposis (FAP) tissue and carcinoma tissue. Using primary epithelial cells from FAP samples, we study IL-8 effect on their growth, migration, and colonies formation. The results showed that IL-8 stimulated cells proliferation, migration, and colonies formation. Furthermore, High level of CEA, CK20, and EGFR was detected after exposure to IL-8 in primary epithelial cells. In conclusion, our findings showed that IL-8 promotes the adenoma–carcinoma transition in primary epithelial cells from FAP. Targeting IL-8 at adenoma stage may prevent or reduce carcinogenesis.     

Transgenic Expression of Human Lysophosphatidic Acid Receptor LPA2 in Mouse Intestinal Epithelial Cells Induces Intestinal Dysplasia

Lysophosphatidic acid (LPA) acts on LPA₂ receptor to mediate multiple pathological effects that are associated with tumorigenesis. The absence of LPA₂ attenuates tumor progression in rodent models of colorectal cancer, but whether overexpression of LPA₂ alone can lead to malignant transformation in the intestinal tract has not been studied. In this study, we expressed human LPA₂ in intestinal epithelial cells (IECs) under control of the villin promoter. Less than 4% of F1-generation mice had germline transmission of transgenic (TG) human LPA₂; as such only 3 F1 mice out of 72 genotyped had TG expression. These TG mice appeared anemic with hematochezia and died shortly after birth. TG mice were smaller in size compared with the wild type mouse of the same age and sex. Morphological analysis showed that TG LPA₂ colon had hyper-proliferation of IECs resulting in increased colonic crypt depth. Surprisingly, TG small intestine had villus blunting and decreased IEC proliferation and dysplasia. In both intestine and colon, TG expression of LPA₂ compromised the terminal epithelial differentiation, consistent with epithelial dysplasia. Furthermore, we showed that epithelial dysplasia was observed in founder mouse intestine, correlating LPA₂ overexpression with epithelial dysplasia. The current study demonstrates that overexpression of LPA₂ alone can lead to intestinal dysplasia.     

CCN1 Secretion Induced by Cigarette Smoking Extracts Augments IL-8 Release from Bronchial Epithelial Cells

Inflammation involves in many cigarette smoke (CS) related diseases including the chronic obstructive pulmonary disease (COPD). Lung epithelial cell released IL-8 plays a crucial role in CS induced lung inflammation. CS and cigarette smoke extracts (CSE) both induce IL-8 secretion and subsequently, IL-8 recruits inflammatory cells into the lung parenchyma. However, the molecular and cellular mechanisms by which CSE triggers IL-8 release remain not completely understood. In this study, we identified a novel extracellular matrix (ECM) molecule, CCN1, which mediated CSE induced IL-8 secretion by lung epithelial cells. We first found that CS and CSE up-regulated CCN1 expression and secretion in lung epithelial cells in vivo and in vitro. CSE up-regulated CCN1 via induction of reactive oxygen spices (ROS) and endoplasmic reticulum (ER) stress. p38 MAPK and JNK activation were also found to mediate the signal pathways in CSE induced CCN1. CCN1 was secreted into ECM via Golgi and membrane channel receptor aquaporin4. After CSE exposure, elevated ECM CCN1 functioned via an autocrine or paracrine manner. Importantly, CCN1 activated Wnt pathway receptor LRP6, subsequently stimulated Wnt pathway component Dvl2 and triggered beta-catenin translocation from cell membrane to cytosol and nucleus. Treatment of Wnt pathway inhibitor suppressed CCN1 induced IL-8 secretion from lung epithelial cells. Taken together, CSE increased CCN1 expression and secretion in lung epithelial cells via induction of ROS and ER stress. Increased ECM CCN1 resulted in augmented IL-8 release through the activation of Wnt pathway.     

IL-17A Induces Pendrin Expression and Chloride-Bicarbonate Exchange in Human Bronchial Epithelial Cells

The epithelium plays an active role in the response to inhaled pathogens in part by responding to signals from the immune system. Epithelial responses may include changes in chemokine expression, increased mucin production and antimicrobial peptide secretion, and changes in ion transport. We previously demonstrated that interleukin-17A (IL-17A), which is critical for lung host defense against extracellular bacteria, significantly raised airway surface pH in vitro, a finding that is common to a number of inflammatory diseases. Using microarray analysis of normal human bronchial epithelial (HBE) cells treated with IL-17A, we identified the electroneutral chloride-bicarbonate exchanger Pendrin (SLC26A4) as a potential mediator of this effect. These data were verified by real-time, quantitative PCR that demonstrated a time-dependent increase in Pendrin mRNA expression in HBE cells treated with IL-17A up to 48 h. Using immunoblotting and immunofluorescence, we confirmed that Pendrin protein expression is increased in IL-17 treated HBE cells and that it is primarily localized to the mucosal surface of the cells. Functional studies using live-cell fluorescence to measure intracellular pH demonstrated that IL-17A induced chloride-bicarbonate exchange in HBE cells that was not present in the absence of IL-17A. Furthermore, HBE cells treated with short interfering RNA against Pendrin showed substantially reduced chloride-bicarbonate exchange. These data suggest that Pendrin is part of IL-17A-dependent epithelial changes and that Pendrin may therefore be a therapeutic target in IL-17A-dependent lung disease.     

Expression and Localization of GEN1 in Mouse Mammary Epithelial Cells

GEN1, a Holliday junction resolvase, is involved in homologous repair of DNA double strand break and in maintaining centrosome integrity. Although GEN1 mutants have been reported in breast cancer patients and cell lines, little is currently known about the functions of GEN1 in the development and oncogenic transformation of mammary gland. In the present study, we demonstrate that GEN1 expression is correlated with mammary epithelial cell proliferation, differentiation in various physiological stages as well as casein. By immunofluorescence analysis, the centrosomal association of GEN1 is confirmed in mammary epithelial cells. Additionally, GEN1 is likely involved in DNA damage response of breast cancer cell lines. These results suggest that GEN1 may play an important role in the development of mammary gland; its response upon DNA damage indicates that GEN1 gene alteration may contribute to breast cancer formation.     

IL-22 Negatively Regulates Helicobacter pylori-Induced CCL20 Expression in Gastric Epithelial Cells

Helicobacter pylori is a Gram-negative bacterium that infects the human gastric mucosa and causes various gastric diseases. H. pylori infection induces the production of inflammatory chemokine CCL20 in gastric mucosa and leads to gastric inflammation. Given that the IL-22/IL-22R axis plays a critical role in the regulation of homeostasis and inflammation of epithelial cells at barrier surfaces, we investigated the effect of IL-22 on CCL20 expression induced by H. pylori. We demonstrated that H. pylori infection of the gastric epithelia-derived AGS cells significantly induced CCL20 expression and the induction was inhibited by IL-22. Functional analysis of the CCL20 promoter revealed that the H. pylori-induced CCL20 expression required the activation of NF-κB, and that IL-22 inhibited the induction by attenuating NF-κB activation. Knockdown of endogenous STAT3 by either short interfering RNAs or a short hairpin RNA significantly reduced the inhibitory effect of IL-22. Furthermore, STAT3 phosphorylation elicited by IL-22 was crucial for the inhibition of H. pylori-induced CCL20 expression. Consistent with the in vitro data showing that IL-22 negatively regulated H. pylori-induced CCL20 expression in gastric epithelial cells, studies on the tissue sections from patients with H. pylori infection also revealed an inverse association of IL-22 expression and CCL20 expression in vivo. Together, our findings suggest that IL-22 plays a role in the control of overproduction of the inflammatory chemokine and thus may protect the gastric mucosa from inflammation-mediated damage.     

兔机械性角膜上皮损伤对结膜杯状细胞及结膜上皮细胞的作用研究

目的探讨机械性角膜上皮损伤对结膜杯状细胞及结膜上皮细胞的作用。方法选取雄性新西兰大白兔12只,建立机械性角膜上皮损伤模型（角膜中央直径8 mm上皮刮除）,建模后使用盐酸林可霉素滴眼,用法为3次/日,1滴/次,观察时间为7 d。在模型建立后第1、4、7天共3个时间点进行结膜印迹细胞学检查、结膜组织透射电镜检查,对结膜上皮细胞及杯状细胞数量及形态进行分析。结果成功建立机械性角膜上皮损伤模型。结膜印迹细胞学检查显示,造模前结膜杯状细胞数量平均值为66.367±2.466（个/每200μm×150μm面积）,Nelson 0级;造模后第1天,结膜杯状细胞数量明显下降,平均值为2.933±0.242（个/每200μm×150μm面积）,Nelson 3级;造模后第4天,结膜杯状细胞数量开始恢复,平均值为17.350±0.991（个/每200μm×150μm面积）,Nelson 2级;造模后第7天,结膜杯状细胞数量已明显恢复,平均值为32.467±2.244（个/每200μm×150μm面积）,Nelson 1级。结膜组织透射电镜检查可见到造模后结膜杯状细胞大量减少,分泌颗粒排空,细胞凋亡,结膜上皮细胞脱落坏死,胞核固缩,胞质中可见溶酶体,上皮下及上皮细胞间炎症细胞浸润;随时间推移,结膜杯状细胞数量及形态逐渐恢复,初期细胞形态欠规则,结膜上皮细胞胞间隙大,连接松散;后期杯状细胞数量明显恢复,形态饱满,分泌功能开始恢复。结膜上皮细胞分化好,细胞连接较为紧密。结论机械性角膜上皮损伤可造成结膜杯状细胞的数量下降及分泌增加,同时可造成结膜上皮细胞凋亡增加,炎症细胞浸润。结膜杯状细胞的数量、功能以及结膜上皮细胞正常结构可在一定时间内自行修复。     

Interleukin-13 Inhibits Lipopolysaccharide-Induced BPIFA1 Expression in Nasal Epithelial Cells

Short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is expressed in human nasopharyngeal and respiratory epithelium and has demonstrated antimicrobial activity. SPLUNC1 is now referred to as bactericidal/permeability-increasing fold containing family A, member 1 (BPIFA1). Reduced BPIFA1 expression is associated with bacterial colonization in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Interleukin 13 (IL-13), predominately secreted by T helper 2 (T_H2) cells, has been found to contribute to airway allergies and suppress BPIFA1 expression in nasal epithelial cells. However, the molecular mechanism of IL-13 perturbation of bacterial infection and BPIFA1 expression in host airways remains unclear. In this study, we found that lipopolysaccharide (LPS)-induced BPIFA1 expression in nasal epithelial cells was mediated through the JNK/c-Jun signaling pathway and AP-1 activation. We further demonstrated that IL-13 downregulated the LPS-induced activation of phosphorylated JNK and c-Jun, followed by attenuation of BPIFA1 expression. Moreover, the immunohistochemical analysis showed that IL-13 prominently suppressed BPIFA1 expression in eosinophilic CRSwNP patients with bacterial infection. Taken together, these results suggest that IL-13 plays a critical role in attenuation of bacteria-induced BPIFA1 expression that may result in eosinophilic CRSwNP.     

Effects of Weaning on Intestinal Upper Villus Epithelial Cells of Piglets

The intestinal upper villus epithelial cells represent the differentiated epithelial cells and play key role in digesting and absorbing lumenal nutrients. Weaning stress commonly results in a decrease in villus height and intestinal dysfunction in piglets. However, no study have been conducted to test the effects of weaning on the physiology and functions of upper villus epithelial cells. A total of 40 piglets from 8 litters were weaned at 14 days of age and one piglet from each litter was killed at 0 d (w0d), 1 d (w1d), 3 d (w3d), 5 d (w5d), and 7 d (w7d) after weaning, respectively. The upper villus epithelial cells in mid-jejunum were isolated using the distended intestinal sac method. The expression of proteins in upper villus epithelial cells was analyzed using the isobaric tags for relative and absolute quantification or Western blotting. The expression of proteins involved in energy metabolism, Golgi vesicle transport, protein amino acid glycosylation, secretion by cell, transmembrane transport, ion transport, nucleotide catabolic process, translational initiation, and epithelial cell differentiation and apoptosis, was mainly reduced during the post-weaning period, and these processes may be regulated by mTOR signaling pathway. These results indicated that weaning inhibited various cellular processes in jejunal upper villus epithelial cells, and provided potential new directions for exploring the effects of weaning on the functions of intestine and improving intestinal functions in weaning piglets.     

Up-Regulation of Intestinal Epithelial Cell Derived IL-7 Expression by Keratinocyte Growth Factor through STAT1/IRF-1, IRF-2 Pathway

Background

Epithelial cells(EC)-derived interleukin-7 (IL-7) plays a crucial role in control of development and homeostasis of neighboring intraepithelial lymphocytes (IEL), and keratinocyte growth factor (KGF) exerts protective effects on intestinal epithelial cells and up-regulates EC-derived IL-7 expression through KGFR pathway. This study was to further investigate the molecular mechanism involved in the regulation of IL-7 expression by KGF in the intestine.

Methods

Intestinal epithelial cells (LoVo cells) and adult C57BL/6J mice were treated with KGF. Epithelial cell proliferation was studied by flow cytometry for BrdU-incorporation and by immunohistochemistry for PCNA staining. Western blot was used to detect the changes of expression of P-Tyr-STAT1, STAT1, and IL-7 by inhibiting STAT1. Alterations of nuclear extracts and total proteins of IRF-1, IRF-2 and IL-7 following IRF-1 and IRF-2 RNA interference with KGF treatment were also measured with western blot. Moreover, IL-7 mRNA expressions were also detected by Real-time PCR and IL-7 protein level in culture supernatants was measured by enzyme linked immunosorbent assay(ELISA).

Results

KGF administration significantly increased LoVo cell proliferation and also increased intestinal wet weight, villus height, crypt depth and crypt cell proliferation in mice. KGF treatment led to increased levels of P-Tyr-STAT1, RAPA and AG490 both blocked P-Tyr-STAT1 and IL-7 expression in LoVo cells. IRF-1 and IRF-2 expression in vivo and in vitro were also up-regulated by KGF, and IL-7 expression was decreased after IRF-1 and IRF-2 expression was silenced by interfering RNA, respectively.

Conclusion

KGF could up-regulate IL-7 expression through the STAT1/IRF-1, IRF-2 signaling pathway, which is a new insight in potential effects of KGF on the intestinal mucosal immune system.     

The P2Y6 Receptor Mediates Clostridium difficile Toxin-Induced CXCL8/IL-8 Production and Intestinal Epithelial Barrier Dysfunction

C. difficile is a Gram-positive spore-forming anaerobic bacterium that is the leading cause of nosocomial diarrhea in the developed world. The pathogenesis of C. difficile infections (CDI) is driven by toxin A (TcdA) and toxin B (TcdB), secreted factors that trigger the release of inflammatory mediators and contribute to disruption of the intestinal epithelial barrier. Neutrophils play a key role in the inflammatory response and the induction of pseudomembranous colitis in CDI. TcdA and TcdB alter cytoskeletal signaling and trigger the release of CXCL8/IL-8, a potent neutrophil chemoattractant, from intestinal epithelial cells; however, little is known about the surface receptor(s) that mediate these events. In the current study, we sought to assess whether toxin-induced CXCL8/IL-8 release and barrier dysfunction are driven by the activation of the P2Y₆ receptor following the release of UDP, a danger signal, from intoxicated Caco-2 cells. Caco-2 cells express a functional P2Y₆ receptor and release measurable amounts of UDP upon exposure to TcdA/B. Toxin-induced CXCL8/IL-8 production and release were attenuated in the presence of a selective P2Y₆ inhibitor (MRS2578). This was associated with inhibition of TcdA/B-induced activation of NFκB. Blockade of the P2Y₆ receptor also attenuated toxin-induced barrier dysfunction in polarized Caco-2 cells. Lastly, pretreating mice with the P2Y₆ receptor antagonists (MSR2578) attenuated TcdA/B-induced inflammation and intestinal permeability in an intrarectal toxin exposure model. Taken together these data outline a novel role for the P2Y₆ receptor in the induction of CXCL8/IL-8 production and barrier dysfunction in response to C. difficile toxin exposure and may provide a new therapeutic target for the treatment of CDI.     

可溶性人TNFR75的表达、纯化及活性鉴定

将编码可溶性人TNFR75 (shTNFR75 )的cDNA与酵母整合载体pPICZαA重组 ,构建的重组质粒线性化后转染酵母细胞GS 115 ,获得了shTNFR75在酵母细胞中遗传性稳定表达酵母工程细胞 .甲醇诱导的pPICZαA shTNFR75 GS115重组酵母工程细胞培养上清 ,经CNBr活化的TNF Sepharose4B亲和层析柱纯化 ,纯化产物纯度为 92 % ;经Western印迹分析 ,可被TNFR75单克隆抗体特异性识别 ,分子量约为 31kD ;受体配体结合试验 ,shTNFR75纯化产物与rhTNFα和rhTNFβ的结合能力与其阳性对照基本相同 ;中和试验显示 ,该shTNFR75可完全阻断TNF对L92 9细胞的细胞毒活性 .表明酵母系统分泌表达的shTNFR75产物具有良好的结合TNF的能力 .