Expression of Claudin-2 in human esophageal squamous cell carcinoma tissues and its effect on the malignant biological behaviors of KYSE-450 cells / 中国肿瘤生物治疗杂志

@#目的：分析Claudin-2蛋白在食管鳞癌（esophageal squamous cell carcinomas，ESCC）组织中的表达及其与患者临床病理特征、5年生存率的关系，探索其对ESCC细胞KYSE450的增殖、迁移和侵袭的影响。方法：选取河南省肿瘤医院2010至2013年间初治的ESCC患者手术切除肿瘤组织52例及其中20例对应的癌旁组织标本，采用免疫组化和WB法检测Claudin-2的表达并分析其与患者临床病理特征和5年生存率的关系。WB法检测ESCC细胞（KYSE450、KYSE150、KYSE510、KYSE140）和人永生化食管上皮细胞SHEE中Claudin-2的表达，构建Claudin-2 shRNA慢病毒载体并转染KYSE450细胞构建敲低Claudin-2表达的细胞系，进一步通过克隆形成实验、细胞划痕实验及Transwell实验检测敲低Claudin-2对KYSE450细胞增殖、迁移和侵袭的影响。结果：ESCC组织中Claudin-2阳性率显著高于癌旁组织（P<0.05），ESCC组织中Claudin-2的表达与淋巴结转移有关（P<0.05）。Claudin-2表达阳性患者5年生存率显著低于阴性者（P<0.05）。成功构建敲低Claudin-2表达的KYSE450细胞系。sh-Claudin-2组细胞的克隆形成数、伤口愈合率和侵袭细胞数均显著低于sh-NT组和对照组（P<0.05）。结论：ESCC组织中Claudin-2的表达高于癌旁组织，且与患者5年生存率呈负相关，Claudin-2能够增强KYSE450细胞的增殖、迁移和侵袭能力。

Curcuma wenyujin Y. H. Chen et C. Ling n-Butyl Alcohol Extract Inhibits AGS Cell Helicobacter pylori Promoted Invasiveness by Down-Regulating Caudal Type Homeobox Transcription Factor and Claudin-2 Expression / 中国结合医学杂志

OBJECTIVE@#To investigate the effects and possible mechanisms of action of Curcuma wenyujin Y. H. Chen et C. Ling n-Butyl alcohol extract (CWNAE) on repression of human gastric cancer (GC) AGS cell invasion induced by co-culturing with Helicobacter pylori (HP).@*METHODS@#AGS cells were cultured with HP of positive or negative cytotoxin-associated gene A (CagA) and vacuolating cytotoxin gene A (VacA) expression (CagA+/- or VacA+/-) and divided into 5 group. Group A was cultured without HP as a control, Group B with HP, Group C with HP, Group D with HP and CWNAE, and Group E with HP and CWNAE. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and tumor invasion assays, examinations of morphology and ultramicroscopic structures, quantitative real-time polymerase chain reaction and Western blots were performed to measure the effects and uncover the mechanisms behind these effects of HP and CWNAE on the epithelial-mesenchymal transition (EMT) of AGS cells.@*RESULTS@#The 10% inhibitory concentration of CWNAE against AGS cells after a 48 h incubation was 19.73±1.30 μg/mL. More AGS cells were elongated after co-culturing with HP than after culturing with HP. In tumor invasion assays, HP significantly enhanced the invasiveness of AGS cells compared to the other experimental groups (all P value <0.05), and this effect was inhibited by CWNAE. Treatment with CWNAE normalized tight junctions and reduced the number of pseudopodia of AGS cells co-cultured with HP. HP up-regulated zincfinger ebox binding homeobox 1 (ZEB1) in AGS cells after co-culturing for 24 h. Expression of caudal type homeobox transcription factor (CDX-2) and claudin-2 was significantly increased by HP (P<0.05), but not by HP.@*CONCLUSION@#HP promoted the invasiveness of AGS cells through up-regulation of ZEB1 transcription and claudin-2 and CDX-2 expression. CWNAE inhibited these effects of HP on AGS cells by down-regulating ZEB1 transcription, and CDX-2 and claudin-2 expression.

Alanyl-glutamine protects the intestinal barrier function in trained rats against the impact of acute exhaustive exercise

Strenuous exercise triggers deleterious effects on the intestinal epithelium, but their mechanisms are still uncertain. Here, we investigated whether a prolonged training and an additional exhaustive training protocol alter intestinal permeability and the putative effect of alanyl-glutamine (AG) pretreatment in this condition. Rats were allocated into 5 different groups: 1) sedentary; 2 and 3) trained (50 min per day, 5 days per week for 12 weeks) with or without 6 weeks oral (1.5 g/kg) AG supplementation; 4 and 5) trained and subjected to an additional exhaustive test protocol with or without oral AG supplementation. Venous blood samples were collected to determine gasometrical indices at the end of the 12-week protocol or after exhaustive test. Lactate and glucose levels were determined before, during, and after the exhaustive test. Ileum tissue collected after all experimental procedures was used for gene expression analysis of Zonula occludens 1 (ZO-1), occludin, claudin-2, and oligopeptide transporter 1 (PepT-1). Intestinal permeability was assessed by urinary lactulose/mannitol test collected after the 12-week protocol or the exhaustive test. The exhaustive test decreased pH and base excess and increased pCO2. Training sessions delayed exhaustion time and reduced the changes in blood glucose and lactate levels. Trained rats exhibited upregulation of PEPT-1, ZO-1, and occludin mRNA, which were partially protected by AG. Exhaustive exercise induced intestinal paracellular leakage associated with the upregulation of claudin-2, a phenomenon protected by AG treatment. Thus, AG partially prevented intestinal training adaptations but also blocked paracellular leakage during exhaustive exercise involving claudin-2 and occludin gene expression.

Effect of chronic cerebral hypoperfusion on expression of occludin and claudin-2 in rat ileum mucosa / 中国组织工程研究

BACKGROUND: Previous studies have found that chronic cerebral hypoperfusion can cause changes in the intestinal mucosal barrier in rats, and Occludin and Claudin are important components of the intestinal mucosal barrier.OBJECTIVE: To explore the effect of chronic cerebral hypoperfusion on the ileum mucosal barrier. METHODS: Twenty male Sprague-Dawley rats were randomly divided into sham operation (sham) group and chronic cerebral hypoperfusion model group. Animal model of chronic cerebral hypoperfusion was built by permanent bilateral common carotid artery ligation. The bilateral common carotid arteries of the sham group were isolated without ligation. The animals were killed at 4 weeks after operation, and their ileums were isolated for morphological observation and pathological scoring using hematoxylin-eosin staining. Apoptosis in the ileum cells was detected using TUNEL. Western blot was used to detect the expression of Claudin-2 and Occludin. immunohistochemical staining was used to detect the expression of Occludin. The study protocol was approved by the Animal Experiment Ethics Committee of General Hospital of Western Theater Command. RESULTS AND CONCLUSION: Compared with the sham group, hematoxylin-eosin staining showed that the ileum in the model group did not develop obvious injury with no higher pathological scores (P > 0.05). Western blot results indicated that compared with the sham group, the expression of claudin-2 was increased in the ileum tissue of the rats with chronic cerebral hypoperfusion, while the expression of Occludin was decreased (P < 0.05). TUNEL fluorescence staining showed that the apoptotic rate in the model group was significantly increased compared with the sham group (P < 0.05). Immunohistochemical results showed that the expression of Occludin in the ileum tissue of the chronic cerebral hypoperfusion group was significantly reduced (P < 0.05). These findings indicate that chronic cerebral hypoperfusion may damage the intestinal mucosal barrier by downregulating Occludin expression and upregulating Claudin-2 expression.

Study on Role and Mechanism of Claudin-2 in Formation of Cholesterol Gallstone / 胃肠病学

Background: Claudin protein family is an important component for construction of tight cell junction. Claudin-2 is a forming protein of paracellular channel and is highly expressed in hepatocytes and bile duct cells, which has selective permeability of water molecules and cations. Claudin-2 plays a vital role in the physiological regulation of digestive and urinary systems. Aims: To investigate the role and mechanism of claudin-2 in formation of cholesterol gallstone and bile secretion. Methods: Liver and gallbladder tissues from claudin-2 knockout (Cldn-2-/-) and wild type (Cldn-2+/+) mice were collected. Expressions of claudin family protein were determined by Western blotting. HE staining, immunofluorescence staining, electron microscope were used to analyze the effect of claudin-2 gene knockout on liver and gallbladder tissue structure. Regulatory roles of claudin-2 in bile component and flow were analyzed. Results: Claudin family proteins were highly expressed in liver and gallbladder tissue in Cldn-2+/+ mice. No significant difference in structure of liver and gallbladder tissue was found between Cldn-2-/- mice and Cldn-2+/+ mice. Compared with Cldn-2+/+ mice, bile flow rate was significantly decreased (P<0.05), concentration of bile was significantly increased (P<0.05), and contents of bile acid, cholesterol, bilirubin, phospholipid in liver and gallbladder bile were significantly increased in Cldn-2-/- mice (P<0.05). Conclusions: Claudin-2 regulates paracellular water flow which is required for proper regulation of bile composition and flow. Dysregulation of this process increases the susceptibility to cholesterol gallstone related diseases in mice.

Effects of traditional Chinese medicine colquhounia root tablet on the expression of tight junction protein ;claudin-2 and ZO-1 in bronchial epithelium tissue of rats with acute lung injury induced by oleic acid / 中华危重病急救医学

Objective To investigate the effects of traditional Chinese medicine colquhounia root tablet on the expression of tight junction protein claudin-2 and ZO-1 in bronchial epithelium tissues of rats with acute lung injury (ALI), and to study the mechanism of protective effect of colquhounia root tablet on ALI. Methods Twenty-four healthy male Sprague-Dawley (SD) rats were randomly divided into control group, ALI group and colquhounia root tablet pretreatment group, with 8 rats in each group. The model of ALI was reproduced by intravenous injection of oleic acid 0.04 mL/kg, and the rats in cont rol group were given the same amount of normal saline (NS) instead. The rats in colquhounia root tablet pretreatment group were intragastric administrated with colquhounia root tablet of 600 mg·kg-1·d-1 (2 mL) for 10 days before model reproduction, and the rats in control group and ALI group were given the same amount of NS. At 4 hours after model reproduction, the blood was drawn from abdominal aorta, and bronchoalveolar lavage fluid (BALF) was collected for determination of protein content in plasma and BALF, and the lung permeability index (LPI) was calculated. The rats were sacrificed to collect lung tissues for determination of lung wet/dry weight ratio (W/D), the changes in pathology of lung tissue were observed after hematoxylin and eosin (HE) staining with light microscope, and lung injury score (LIS) was evaluated. The immunohistochemic al staining was used to detect the expression and localization of claudin-2 and ZO-1 in bronchial epithelium tissues. The protein expressions of claudin-2 and ZO-1 in bronchial epithelium tissues were determined by Western Blot. Results Compared with control group, the lung injury in ALI group was more obvious including cellular edema and structural disorder of intercellular connection by optical microscope, and LIS, W/D ratio, and LPI were significantly increased (LIS: 3.81±0.42 vs. 0.40±0.08, W/D: 7.68±0.64 vs. 4.44±0.39, LPI: 0.89±0.15 vs. 0.38±0.05, all P < 0.01). Claudin-2 and ZO-1 were mainly expressed in the bronchial epithelium cell, and the expression degrees were significantly weakened in ALI group as compared with control group. It was shown by Western Blot results that compared with control group, the protein expressions of claudin-2 and ZO-1 were significantly down-regulated in ALI group [claudin-2 protein (gray value): 0.43±0.31 vs. 2.16±1.33, ZO-1 protein (gray value): 1.25±0.41 vs. 2.82±0.76, both P < 0.01]. Compared with ALI group, colquhounia root pretreatment could effectively diminish the degree of ALI (LIS: 1.22±0.39 vs. 3.81±0.42, W/D: 4.62±0.84 vs. 7.68±0.64, LPI: 0.46±0.07 vs. 0.89±0.15, all P < 0.01), and the protein expressions of claudin-2 and ZO-1 were significantly up-regulated [claudin-2 protein (gray value): 2.98±0.91 vs. 0.43±0.31, ZO-1 protein (gray value): 2.35±0.51 vs. 1.25±0.41, both P < 0.01]. Conclusion Administration of colquhounia root table could attenuate lung injury induced by oleic acid with improving epithelial barrier function via up-regulate the expression claudin-2 and ZO-1, which play a protective effect on the lung of rats with ALI.

The protective effect of Clostridium butyricum on intestinal injure in newborn rats with necrotizing enterocolitis / 临床儿科杂志

Objective To explore the influence of Clostridium butyricum on the expression of vascular endothelial cell growth factor (VEGF) and its receptor 2 (VEGFR-2), proliferating cell nuclear antigen (PCNA), and tight junction protein claudin-2 in intestinal tissue in newborn rat with necrotizing enterocolitis (NEC). Methods Forty-eight-hour-old Sprague-Dewley (SD) rats were randomly divided into model group, control group, low-dose group, mid-dose group, and high-dose group, 12 rats each. Rats in each group were fed with milk substitute. The NEC model were created by hypoxia and cold stimulation for 3 consecutive days in model group, low-dose group, mid-dose group, and high-dose group. Meanwhile, low-dose group, mid-dose group, and high-dose group were intervened by being fed with Clostridium butyricum 0.2, 0.4, and 0.8 g/(kg·d), respectively. All rats in each group were sacriifced on day 4 and the intestines tissue was obtained. The pathological changes had been observed. The expression of VEGF, PCNA, and claudin-2 were detected by immunohistochemistry. The expression of VEGFR-2 was detected by RT-PCR. Results The intestines pathological scores was signiifcantly different among ifve groups (P?0 . 05 ). Conclusion The expression of VEGF, VEGF-2 , and claudin-2 were higher in rats with NEC, while the expression of PCNA was lower. Supplementation of Clostridium butyricum may protect newborn rats by its act on these factors.