Objective: To investigate the association between adherens junction proteins E-cadherin and β-catenin and tight junction protein claudin-2 and clinical symptoms in patients with diarrhea predominant irritable bowel syndrome (IBS-D). Methods: Cecal biopsy tissues were collected from IBS-D patients (n=26) according to Rome Ⅲ criterion and healthy controls (n=26). The duration of symptoms, abdominal pain score and mean weekly bowel movements were recorded. Colorectal dilatation combined with restraint stress were applied to establish visceral hypersensitivity rat model. Abdominal contraction reflex (AWR) was applied to assess the visceral sensitivity in rats. The stool frequency within 1 hour was recorded after establishing the rat model. The expression of E-cadherin、β-catenin and claudin-2 were assessed by Western blot and immunofluorescence microscopy. Intercellular ultrastructure was observed by transmission electron microscopy. Results: Compared with the healthy controls, the protein expression of E-cadherin and β-catenin in cecal epithelium in IBS-D patients were significantly lower (P=0.015 and P=0.005, respectively), while claudin-2 was significantly higher (P=0.000). Reduced E-cadherin and β-catenin expression was associated high abdominal pain score (r=-0.463, P=0.017 and r=-0.407, P=0.039). The lower expression of β-catenin was associated with longer duration of symptoms (r=-0.458, P=0.019). The protein expression of E-cadherin and ß-catenin in the cecal epithelium of the visceral hypersensitivity rats were significantly lower (P=0.004 and P=0.003, respectively), while claudin-2 was significantly higher (P=0.008). Reduced E-cadherin and ß-catenin expression was associated high visceral sensitivity in IBS-D rats (r=-0.639, P=0.047 and r=-0.888, P=0.001). Conclusions: Intercellular ultrastructure alterations well as cecal β-catenin and E-cadherin protein expression decrease and are associated with high abdominal pain score in IBS-D patients and hypersensitivity rats. β-catenin is further associated with prolonged duration of symptoms in IBS-D patients. The expression of E-cadherin and β-catenin may play a vital role in visceral sensitivity and intestinal barrier dysfunction in IBS-D.

目的:探讨细胞角蛋白8(CK8)在促肾上腺皮质激素释放因子(CRF)诱导的肠上皮细胞间通透性改变中的作用及机制.方法:培养人结肠腺癌HT29细胞株建立肠上皮屏障模型,采用免疫荧光法检测HT29细胞表面CRF受体1(CRFR1)及CRFR2的表达情况,并将其分为对照组和CRF组,CRF组以100 nmol/L CRF处理细胞72 h.采用Transwell小室检测2组细胞FITC标记的dextran透过率,透射电镜观察2组细胞紧密连接结构变化,并用Western blot法检测2组细胞CK8及紧密连接相关蛋白(ZO-1和occludin)的表达,免疫荧光检测CK8表达微结构变化,ELISA检测加药5 min、10 min、30 min、1 h及2 h后蛋白激酶C(PKC)活性.采用sh-CK8慢病毒构建CK8低表达HT29细胞,检测给予CRF处理后相应蛋白表达量、FITC标记的dextran透过率及PKC活性的改变.结果:HT29细胞表面存在CRFR1及CRFR2受体,而CRF处理后,FITC标记的dextran透过率高于对照组(P<0.05),透射电镜观察可见对照组细胞紧密连接通道关闭,CRF处理后紧密连接开放.同时,CRF可引起HT29细胞CK8的荧光强度增高,呈颗粒样浓聚,其蛋白表达明显增加(P<0.05),而occludin和ZO-1表达下调(P<0.05).此外,CRF处理1 h时,PKC的活性下降(P<0.05).sh-CK8慢病毒转染HT29细胞后成功建立低表达CK8细胞株,与阴性对照组相比,CRF刺激后肠上皮细胞通透性并未明显降低,occludin蛋白表达仍下调(P<0.05),而ZO-1则无明显改变.同时,与阴性对照组相比,CK8低表达后,CRF刺激并未引起PKC活性的下降.结论:CK8可能通过抑制PKC活性参与CRF诱导的肠上皮通透性的增加,同时可能存在其它信号通路共同参与.

Irritable bowel syndrome(IBS)is a common functional intestinal disease,and its etiology and pathogenesis are not completely clear. The pathogenesis of IBS involves disturbed gastrointestinal motility,gut hypersensitivity,intestinal inflammation,immune dysfunction and brain-gut axis abnormality. Cathepsin S(CTSS)is a proteolytic enzyme widely distributed in various cell lysosomes,and participates in a variety of pathophysiological processes. Recent studies have shown that CTSS may be involved in the pathogenesis of IBS. This article reviewed the advances in study on role of CTSS in IBS.

[目的]采用荆花胃康胶丸联合铋剂四联治疗幽门螺杆菌(Helicobacter pylor,Hp)阳性慢性胃炎初治患者,观察Hp根除率、临床症状改善情况以及用药安全性,探讨中西药联合对Hp感染的根除率以及Hp阳性慢性胃炎的疗效.[方法]采用前瞻性随机对照多中心临床研究,对象为4个中心经胃镜检查确诊为慢性胃炎并伴有Hp感染的初治患者,采用分层区组随机化分为4个治疗组:A组,艾司奥美拉唑20 mg+胶体果胶铋200 mg+阿莫西林1 000 mg+呋喃唑酮100 mg,疗程10 d;B组,荆花胃康胶丸240 mg+艾司奥美拉唑20 mg+胶体果胶铋200 mg+阿莫西林1 000 mg+呋喃唑酮100mg,疗程10 d;C1组:荆花胃康胶丸240 mg,前14 d;艾司奥美拉唑20 mg+胶体果胶铋200 mg+阿莫西林1 000 mg+呋喃唑酮100 mg,后10d,总疗程24 d;C2组:艾司奥美拉唑20 mg+胶体果胶铋200 mg+阿莫西林1 000 mg+呋喃唑酮100mg,前10 d;荆花胃康胶丸240 mg,后14 d,总疗程24 d;各组服药均为2次/d.治疗结束后评估4组患者Hp根除率、临床症状改善情况和药物不良反应发生情况.治疗结束停药至少28 d后以13.14 C-尿素呼气试验结果判断Hp是否根除.[结果]共纳入471例患者,A、B、C1、C2组分别入组1 24、11 4、114、119例.4组Hp根除率ITT分析结果分别为82.3％(102/124)、86.0％(98/114)、78.9％(90/114)、79.8％(95/119),组间比较差异无统计学意义(P=0.518);PP分析结果分别为87.9％(102/116)、90.7％(98/108)、86.5％(90/104)、85.6％(95/111),B组根除率高于其他治疗组,但组间比较差异无统计学意义(P =0.677).各组根除Hp治疗后症状均获明显改善(P＜0.01),其中B组症状改善明显优于A组(P=0.007).共有32例患者出现不良反应,其中A组10例(8.1％),B组7例(6.1％),C1组6例(5.3％),C2组9例(7.6％),组间差异无统计学意义(P =0.819).[结论]荆花胃康胶丸联合铋剂四联同时服用Hp根除率较高,且症状改善明显优于铋剂四联.中西药联合治疗Hp阳性慢性胃炎具有一定优势,值得进一步研究.

Background:Myosin light chain ( MLC ) is the initiating factor that regulating tight junction and intestinal permeability. Phosphorylated MLC( pMLC ) can cause redistribution of tight junction related protein,and break the structure and integrity of tight junction,thus induces the dysfunction of intestinal mucosal barrier. Aims:To explore the role of MLC phosphorylation in intestinal mucosal barrier dysfunction in diarrhea-predominant IBS ( IBS-D ) rats. Methods:Eight Spague-Dawley pregnant rats were randomly divided into model group and control group,IBS-D rat model was established by maternal separation. The character and particles of feces were record 1 hour after establishing the model. Abdominal withdrawal reflex( AWR)was used to evaluate visceral sensitivity. The distributions of fibrous actin( F-actin ), tight junction associated protein ZO-1 and claudin-1 in colon were assessed by immunofluorescence. The expressions of MLC,pMLC,F-actin,ZO-1 and claudin-1 were measured by Western blotting. Tight junction was observed by transmission electron microscope( TEM). Results:Compared with control group,visceral sensitivity was significantly increased. Immunofluorescence showed the decrease in fluorescence intensity,structural distortion and redistribution of claudin-1 and the fussy structure of F-actin and ZO-1 in model group. Western blotting showed that pMLC/MLC ratio and expression of pMLC were significantly increased and expression of claudin-1 was significantly decreased in model group,but no significant differences in expressions of MLC,F-actin and ZO-1 were found between the two groups. TEM showed that tight junction was broken and cell space was enlarged. Conclusions:The increased MLC phosphorylation may induce the redistribution of cell cytoskeletal protein F-actin,cause the change of structure and function of intercellular tight junction&nbsp;proteins and enlargement of intercellular space,thus may induce the dysfunction of intestinal mucosal barrier,and play an important role in the pathogenesis of IBS-D.

Objective To investigate whether there are changes in adherens junctions(AJ), including E-cadherin (E-cad) and β-catenin (β-cat), in the colonic mucosa of irritable bowel syndrome (IBS).Methods Colorectal dilatation combined with restraint stress was used to establish IBS SD rat model. There were 8 rats in the model group and 8 rats in normal control group. From each rat, the colon tissue 5 centimeters away from ileocecum (I) and the junction of the rectum and sigmoid colon (R) were taken, in which E-cad and β-cat mRNA,protein level and structural changes were detected using real-time quantitative-PCR, Western blot, and immunofluorescence. According to the Rome Ⅲ criteria, IBS with diarrhea (IBS-D) patients receiving colonoscopy (n=17) and healthy volunteers (n=17) were enrolled in Center of Endoscopy of the First Affiliated Hospital of Zhejiang Chinese Medicine University. The colon tissues of I and R locations were collected from them. Colonic epithelial cell AJ protein expression and AJ structural changes in the tissues were detected using Western blot, immunofluorescence and transmission electron microscope (TEM).Results (1)Both in I and R sites, the mRNA and protein expression of E-cad in IBS rats were both significantly lower than in normal SD rats (all P<0.05). The mRNA expression of β-cat showed no significant change in IBS SD rats compared with the normal SD rats;the protein expression of β-cat was decreased in I(P<0.05), no significantly different in R.(2)In I and R of IBS-D patients, the expression of E-cad protein were significantly decreased(I: 0.85±0.30 vs 1.24±0.34, P=0.00;R: 0.86±0.17 vs 1.14±0.48, P=0.05);the protein expression of β-cat also showed significant reduction both in I(0.85±0.39 vs 1.22±0.51, P=0.04)and R (0.92±0.22 vs 1.16±0.31, P=0.02) of IBS-D patients, compared with the healthy controls. Immunofluorescence results showed that E-cad and β-cat in colonic epithelial cells of I and R in the healthy controls were distributed along the cell membrane, demonstrating honeycomb linear fluorescence;however, in IBS-D patients, the structure of E-cad and β-cat were fuzzy, with disrupted distribution in the cytoplasm. Under TEM, the gaps of AJ in I and R of IBS-D patients appeared wider than in the healthy volunteers.Conclusions In IBS-D patients, there are wider gaps of AJ, and decreased expression with structural disorder of E-cad and β-cat in the colon. The change of AJ plays an important role in the damage of colonic mucosal barrier and the increase of intestinal epithelial permeability in IBS-D patients, but the mechanism is yet to be further explored.

Background/Aims: Intestinal permeability and stress have been implicated in the pathophysiology of irritable bowel syndrome (IBS). Cytokeratin 8 (CK8), for the first time, has been shown to mediate corticotropin-releasing factor (CRF)-induced changes in intestinal permeability in animal models of IBS. In this study, we investigated the regulatory effects of CRF on the permeability of human intestinal epithelial cells through the CK8-mediated tight junction. Methods: The expression levels of corticotropin-releasing factor receptor 1 (CRFR1) and corticotropin-releasing factor receptor 2 (CRFR2) on the HT29 cell surface were determined by immunofluorescence, RT-PCR, and Western blotting. After treatment with 100 nM CRF for 72 h, the translocation of FITC-labelled dextran was measured in a transwell chamber; the structural changes of tight junctions were observed under transmission electron microscopy; the expression levels of CK8, F-actin and tight junction proteins ZO-1, claudin-1, and occludin were detected by immunoblotting and immunofluorescence. The activity of RhoA was detected by immunoprecipitation. Furthermore, the effects of CRF on intestinal epithelial permeability were examined in CK8-silenced HT29 cells, which were constructed by shRNA interference. Results: CRF treatment increased FITC-labelled dextran permeability, caused the opening of tight junctions, induced increased fluorescence intensity of CK8 and decreased the intensities of ZO-1, claudin-1, and occludin, together with structural disruption. The expression levels of F-actin, occludin, claudin-1, and ZO-1 were downregulated. RhoA activity peaked at 30 min after CRF treatment. CRF-induced increased permeability, and downregulation of claudin-1 and occludin were not blocked by CK8 silencing. Nevertheless, CK8 silencing blocked the effects of CRF regarding the decrease in the expression levels of F-action and ZO-1 and increase in RhoA activity. Conclusion: CRF may increase intestinal epithelial permeability by upregulating CK8 expression, activating the RhoA signalling pathway, promoting intestinal epithelial actin remodelling, and decreasing the expression of the tight junction protein ZO-1. Other CK8-independent pathways may be involved in the downregulation of claudin-1 and occludin, which might also contribute to increased intestinal epithelial permeability.

The effect of corticotropin-releasing factor (CRF) on gastrointestinal tract inflammation via modulation of the immune system cells such as dendritic cells (DCs) is not known. We investigated the expression of CRF and its receptors CRFR1 and CRFR2 in a colonic DC model (JAWSII cells) in a pro-inflammatory and anti-inflammatory milieu. The mRNA expression of CRF and protein expression of CRFR1 and CRFR2 was assessed, and lipopolysaccharide (LPS) was used to induce the maturation of JAWSII cells. JAWSII cells were divided into four groups: control, CRF, LPS, and LPS + CRF. The levels of secreted cytokines IL-6, IL-4, TNF- α , and MIP-1 α were determined, both in JAWSII cells and in the culture supernatant, by qRT-PCR and ELISA. The expression of CRFR1 and CRFR2 in JAWSII cells was accompanied by a low CRF expression. Compared with control group, CRF group did not affect the expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and MIP-1 α , but LPS treatment significantly increased the expression of these cytokines, indicating maturation of JAWSII cells. CRF further augmented the production of IL-6, TNF-α, and MIP-1 α in mature JAWSII cells, with no increase in TNF- α mRNA expression. However, the expression of anti-inflammatory factor IL-4 did not change after LPS treatment. CRF treatment decreased the expression of IL-4 in both mature and immature JAWSII cells. JAWSII cells produce low level of CRF and express CRFR1 and CRFR2 surface receptors. CRF promotes immune/inflammatory responses in mature JAWSII cells when induced by LPS treatment.

Intestinal barrier is formed by intestinal mucous layer,epithelial cells,cellular tight junction,enterocyte membrane,submucosal lamina propria and immunologic factors,and plays a pivotal role in maintaining gastrointestinal function. Different types of stress can induce intestinal barrier dysfunction and increased intestinal permeability,leading to a series of gastrointestinal diseases. This article reviewed the progress of research on pathological changes and mechanism of stress-related intestinal barrier dysfunction.

Objective To investigate the expression of corticotropin-releasing factor (CRF) and its receptors including CRFR1 and CRFR2 on mouse mesenteric lymph nodes dendritic cells (MLNDC), and to analyze their effects on the biological phenotypes of intestinal dendritic cells .Methods The MLNDCs were isolated from C57BL/6 mice by using magnetic bead sorting .The purity of CD11c+DCs was identified by flow cytometry .The double-labeling immunofluorescence and the reverse transcriptase-polymerase chain reaction (RT-PCR) were performed to detect the expression of CRF , CRFR1 and CRFR2 on MLNDCs.The MLNDCs were exposed to CRF with or without the interference of CRFR 1 and CRFR2 antagonists .Flow cy-tometry was used to measure the changes of surface molecules ( MHCⅠ and MHCⅡ) and co-stimulatory molecules (CD80 and CD86).Results The CD11c+DCs accounted for (80.12±6.34)% of the isolate cells with a high cell viability of more than 90%.The expression of CRF , CRFR1 and CRFR2 at mRNA lev-el were detected in MLNDCs by RT-PCR.Results of the immunofluorescent staining assay indicated that both CRFR1 and CRFR2 were expressed on the surface of MLNDCs .The expression of CD86 on MLNDCs was inhibited by the treatment of MLNDCs with CRFR 1 antagonist , but enhanced by the treatment with CRFR2 antagonist .Conclusion Both CRF and CRFRs were detected in the MLNDCs isolated from the C57BL/6 mice.The CRF could alter the biological phenotypes of MLNDCs through binding to different CRFRs (CRFR1 and CRFR2), which affected the phenotypes of MLNDCs in opposite ways .

OBJECTIVE To study the phenotypic alteration of intestinal dendritic cells (DC) in a rat model of irritable bowel syndrome (IBS) and the change of mitogen-activated protein kinase (MAPK) signaling pathway, in order to explore the potential mechanism of ERK1/2 pathway mediation in abnormal DC immune response. METHODS IBS rat model was established by combining neonatal maternal separation and colorectal distension in 10 SD rats, and 10 healthy rats served as controls. Visceral sensitivity was evaluated with abdominal withdrawal reflex (AWR). Mesenteric lymph node DC (MLNDC) was isolated and purified by magnetic label-based technique after modeling. Expression of surface major histocompatibility complex (MHC)-Ⅱin control rats was determined by flow cytometric analyses. Western-blot was used to determine the expression of MHC-Ⅱ, p-p38, p38, phosphorylated extracellular regulated protein kinase (p-ERK1/2), ERK1/2, phosphorylated c-Jun N-terminal kinase (p-JNK), and JNK in MLNDC. RESULTS Visceral sensitivity was significantly higher in the IBS group than in the control group. The purity of the OX62 positivity MLNDC following magnetic sorting was about 85.57%±7.67%. MLNDC in the control group expressed high level of MHC-Ⅱ. The expression of MHC-Ⅱ and p-ERK1/2 in MLNDC in the IBS group were higher than those in the control group (1.05±0.13 vs 0.67±0.18, t=-2.973, P=0.041; 3.21±0.48 vs 2.34±0.85, t=-3.130, P=0.035); while there was no significant difference in the expressions of p-JNK and p-p38 compared with the control groups (0.95±0.17 vs 0.76±0.36, t=0.808, P=0.464; 1.07±1.13 vs 1.19±0.91, t=0.137, P=0.897). CONCLUSION The intestinal DC in IBS rats show a upregulated expression of MHC-Ⅱ, which may be related to the activation of intracellular ERK1/2 pathway.

Epithelial-mesenchymal transition( EMT ) and mesenchymal-epithelial transition are involved in the process of development of tissues and organs in embryonic stage. Currently studies are focused on the relationship between EMT and tumor invasion and metastasis. This article reviewed the role of EMT in the occurrence and development of gastric cancer by analyzing the relationship between EMT and gastritis,gastric mucosal barrier,initiation of gastric carcinogenesis, metastasis and invasion,drug resistance and immune escape which may provide insights to the pathologic mechanism and approaches to prevent and treat gastric cancer.

OBJECTIVE:To study the mechanism of reversal effect of Curcuma Wenyujin n-Butyl alcohol extract (CWNAE) on multiple drugs resistance (MDR) of SGC7901/VCR cells.METHODS:SGC7901/VCR cells were co-culured with different concentrations CWNAE (80, 40, and 20 μg/mL) and Verapamil (VP, 10 μg/mL) for 24 h, and then acted with Adriamycin (ADM) for 1, 2, and 4 h, respec- tively. SGC7901/VCR cells with no intervention were taken as the vehicle control group. SGC7901/VCR cells treated with ADM alone were taken as the control group. The effect of CWNAE on intracellular ADM concentration was detected by flow cytometry (FCM). Cells were treated as mentioned before without any intervention of ADM. SGC7901/VCR with no ADM intervention were taken as the control group. The effect of CWNAE on the expression of P-glycoprotein (P-gp), lung resistance protein (LRP), and glu- cosylceramide synthase (GCS) was studied by Western blot. The effect of CWNAE on the location and expression quantity of P-gp was further illustrated by immunohistochemistry (IHC).RESULTS:Compared with the ADM group, the expression ratio obviously increased in the W80, W40, W20, and VP10 groups with statistical difference (all P < 0.05). The comparative expression quantity of P-gp, GCS, and LRP in SGC7901/VCR cells was obviously higher than that of non-MDR with statistical difference (all P < 0.05). The expression quantity of P-gp and GCS could be obviously down-regulated by 80 and 40 μg/mL CWN- AE, and 10 μg/mL VP, with no effect on the expression of LRP. Results of IHC proved that P-gp was mainly expressed on the cytomembrane or in the plasma, and it was also expressed on the nuclear membrane. P-gp in different locations could all be down-regulated by CWNAE.CONCLUSIONS:CWNAE could reverse the MDR of SGC7901/VCR cell line probably by inhibiting the expression of P-gp and GCS. CWNAE had no effect on LRP that also highly expressed on SGC7901/VCR. So we supposed that CWNAE could become a potential drug to reverse MDR of highly expressed P-gp and GCS.