Development of a CLDN18.2-targeting immuno-PET probe for non-invasive imaging in gastrointestinal tumors / 药物分析学报

Claudin18.2(CLDN18.2)is a tight junction protein that is overexpressed in a variety of solid tumors such as gastrointestinal cancer and oesophageal cancer.It has been identified as a promising target and a potential biomarker to diagnose tumor,evaluate efficacy,and determine patient prognosis.TST001 is a recombinant humanized CLDN18.2 antibody that selectively binds to the extracellular loop of human Claudin18.2.In this study,we constructed a solid target radionuclide zirconium-89(89Zr)labled-TST001 to detect the expression of in the human stomach cancer BGC823CLDN18.2 cell lines.The[89Zr]Zr-des-ferrioxamine(DFO)-TST001 showed high radiochemical purity(RCP,＞99％)and specific activity(24.15±1.34 GBq/μmol),and was stable in 5％human serum albumin,and phosphate buffer saline(＞85％RCP at 96 h).The EC50 values of TST001 and DFO-TST001 were as high as 0.413±0.055 and 0.361±0.058 nM(P＞0.05),respectively.The radiotracer had a significantly higher average standard uptake values in CLDN18.2-positive tumors than in CLDN18.2-negative tumors(1.11±0.02 vs.0.49±0.03,P=0.0016)2 days post injection(p.i.).BGC823CLDN18.2 mice models showed high tumor/muscle ratios 96 h p.i.with[89Zr]Zr-DFO-TST001 was much higher than those of the other imaging groups.Immunohistochemistry results showed that BGC823CLDN18.2 tumors were highly positive(+++)for CLDN18.2,while those in the BGC823 group did not express CLDN18.2(-).The results of ex vivo biodistribution studies showed that there was a higher distribution in the BGC823CLDN18.2 tumor bearing mice(2.05±0.16％ID/g)than BGC823 mice(0.69±0.02％ID/g)and blocking group(0.72±0.02％ID/g).A dosimetry estimation study showed that the effective dose of[89Zr]Zr-DFO-TST001 was 0.0705 mSv/MBq,which is within the range of acceptable doses for nuclear medicine research.Taken together,these re-sults suggest that Good Manufacturing Practices produced by this immuno-positron emission tomog-raphy probe can detect CLDN18.2-overexpressing tumors.

Advances in research and development of molecular targeted drugs for advanced gastric cancer / 中国肿瘤生物治疗杂志

@#[摘 要] 近年来，化疗基础上加用抗表皮生长因子受体2药物、抗血管生成药物及免疫检查点抑制剂，不仅可提高晚期胃癌（GC）患者治疗的有效率，而且可明显改善患者预后。然而，目前晚期GC患者治疗后的生存获益远落后于肺癌、结直肠癌及乳腺癌等实体肿瘤，靶向治疗仍需进一步探索。随着分子生物学技术的发展，新的治疗靶点如Claudin 18.2、基质金属蛋白酶（MMP）、Dickkopf相关蛋白1（DKK-1）、RAD3相关蛋白激酶（ATR）被发现在GC细胞中表达，针对这些靶点的药物逐渐在临床治疗中崭露头角，并显示出较好的临床应用前景。阐述晚期GC治疗中靶点的作用机制及靶向药物的研究进展，对提高GC的临床治疗疗效具有重要意义。

Research progress of tight junction protein Claudin of salivary glands / 口腔医学

@#Tight junction(TJ) is complex dynamic system involved in protein interactions in the paracellular secretory pathway, with both barrier and fence functions. Claudin family, the main section of tight junction strands, will be abnormal in expression pattern in the circumstances of radiation injuries, inflammation, Sj?gren's syndrome, diabetes and other pathological conditions in salivary glands. This change leads to abnormal structure and function of tight junctions, indirectly manifested as salivary gland dysfunction. In addition, the difference of Claudin expression in salivary gland tumors can also be used as an indicator of tumor type and prognosis. This review focuses on the progress of research on common Claudin in salivary glands, including the structure, function, expression patterns of related diseases and their applications. It is believed that the review may provide new ideas for clinical and basic research on Claudin protein-related diseases.

Correlation of REG1A, Claudin 7 and Ki67 expressions with tumor recurrence and prognostic factors in superficial urothelial urinary bladder carcinomas

A?m: Superficial urothelial urinary bladder tumors are neoplasms that frequently recur and have a potential for invasion and metastasis. REG gene family is composed of various acute phase reactants, lectins, antiapoptotic factors, and growth factors that are effective on pancreatic island cells, neural cells, and epithelial cells. REG1A and REG1B are two forms of the human REG1 gene. It is reported that they are expressed in several cancers and are correlated with the prognosis of the patient. Claudins are integral transmembrane proteins that interconnect cells. However, their role in human tumorigenesis is extremely controversial. The aim of this study is to evaluate the relationship of REG1A, claudin 7 protein expressions, and Ki67 proliferation index in superficial urothelial urinary bladder tumors with well-known parameters of prognosis and tumor recurrence, and also to clarify whether these parameters are independent prognostic factors or not. Materials and Methods: A hundred and eleven patients diagnosed with superficial urothelial carcinoma between 2011 and 2016 years in our hospital and followed up in our urology clinic were included in this study. The slides prepared from paraffin blocks were immunohistochemically stained with REG1A, claudin 7, and Ki67 antibodies. Results: REG1A showed positive staining in 37 (33%) and negative staining in 74 (67%) of urothelial tumors. Claudin 7 was positive in 24 (22%) and negative in 87 (78%) cases. REG1A expression showed a positive correlation with tumor stage and tumor recurrence; a high Ki67 proliferation index was positively correlated with tumor stage and grade. The loss of claudin 7 expression was related to tumor recurrence. Besides, tumors with REG1A expression and claudin 7 loss had a shorter survival independent of recurrence. Conclus?on: In urothelial tumors, REG1A expression and loss of claudin 7 might be significant markers of prognosis that predict tumor recurrence.

The protective role of raltegravir in experimental acute lung injury in vitro and in vivo

Disruption of pulmonary endothelial permeability and associated barrier integrity increase the severity of acute respiratory distress syndrome (ARDS). This study investigated the potential ability of the human immunodeficiency virus-1 (HIV-1) integrase inhibitor raltegravir to protect against acute lung injury (ALI) and the underlying mechanisms. Accordingly, the impact of raltegravir treatment on an in vitro lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cell (HPMEC) model of ALI and an in vivo LPS-induced two-hit ALI rat model was examined. In the rat model system, raltegravir treatment alleviated ALI-associated histopathological changes, reduced microvascular permeability, decreased Evans blue dye extravasation, suppressed the expression of inflammatory proteins including HMGB1, TLR4, p-NF-κB, NLRP3, and MPO, and promoted the upregulation of protective proteins including claudin 18.1, VE-cadherin, and aquaporin 5 as measured via western blotting. Immunohistochemical staining further confirmed the ability of raltegravir treatment to reverse LPS-induced pulmonary changes in NLRP3, claudin 18.1, and aquaporin 5 expression. Furthermore, in vitro analyses of HPMECs reaffirmed the ability of raltegravir to attenuate LPS-induced declines in VE-cadherin and claudin 18.1 expression while simultaneously inhibiting NLRP3 activation and reducing the expression of HMGB1, TLR4, and NF-kB, thus decreasing overall vascular permeability. Overall, our findings suggested that raltegravir may represent a viable approach to treating experimental ALI that functions by maintaining pulmonary microvascular integrity.

Protective Effect and Mechanism of Sanhuatang and Its Modifications on Cerebral Ischemia-reperfusion Injury in Rats / 中国实验方剂学杂志

ObjectiveTo observe the protective effect of Sanhuatang and its modifications on the brain tissue of rats exposed to cerebral ischemia-reperfusion injury （CIRI） and explore its action mechanism and compatibility characteristics. MethodOne hundred and forty SD male rats of clean grade were randomly divided into the control group， sham-operation group， and operation group. The Longa suture method was employed to establish the CIRI model. The successfully modeled CIRI rats were further divided into five groups， namely the model group， nimodipine group， Sanhuatang without Notopterygii Rhizoma et Radix group， Notopterygii Rhizoma et Radix group， and Sanhuatang group， and treated with the corresponding medicines by gavage for five days. The cerebral infarct size in each group was examined by 2，3，5-triphenyltetrazolium chloride （TTC） staining， and the pathological changes in the brain tissue were observed by hematoxylin-eosin （HE） staining and electron microscopy. The mRNA and protein expression levels of Claudin-5， Occludin， and zonula occludens-1 （ZO-1） in brain tissues were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. ResultCompared with the control group， the model group exhibited markedly increased infarct size， obvious changes in brain morphology and ultrastructure， and down-regulated mRNA and protein expression of Claudin-5， Occludin， and ZO-1 （P<0.01）. Compared with the model group， both nimodipine and Sanhuatang significantly decreased the infarct size （P<0.01） and relived the pathological changes. The infarct sizes in the Sanhuatang without Notopterygii Rhizoma et Radix group and Notopterygii Rhizoma et Radix group were reduced without exhibiting a statistically significant difference. The mRNA and protein expression levels of Claudin-5， Occludin， and ZO-1 in the nimodipine group， Sanhuatang group， and Notopterygii Rhizoma et Radix group were up-regulated significantly in comparison with those in the model group （P<0.01， P<0.01）. The mRNA and protein expression levels of Claudin-5 and ZO-1 were higher in the Notopterygii Rhizoma et Radix group than in the Sanhuatang without Notopterygii Rhizoma et Radix group （P<0.01， P<0.01）. ConclusionSanhuatang exerts the protective effect against CIRI in rats possibly by regulating the expression of Claudin-5， Occludin， and ZO-1 and improving the blood-brain barrier function. Notopterygii Rhizoma et Radix in Sanhuatang may play an important role in the protection of rats from CIRI.

Xuebijing injection improve pulmonary vascular barrier function in ARDS by up-regulating claudin-5 expression through PI3K/Akt/FOXO1 signaling pathway / 中华危重病急救医学

Objective:To study the signaling pathway of the up-regulation of claudin-5 expression by Xuebijing injection.Methods:Animal and cell models of acute respiratory distress syndrome (ARDS) were induced by lipopolysaccharide (LPS). ① In vivo study, 20 male Sprague-Dawley (SD) rats were randomly divided into 4 groups: control group, LPS group (LPS injection 10 mg/kg for 12 hours), Xuebijing control group (Xuebijing injection 1 mg/kg, twice a day, for 3 days), and Xuebijing intervention group (LPS injection after pretreatment of Xuebijing injection), according to random number method with 5 rats in each group. The lung tissues were taken to detect lung dry/wet weight ratio (W/D) and the morphological changes in each group. Claudin-5, phosphorylated forkhead box transcription factor O1 (p-FOXO1), total FOXO1 (t-FOXO1), phosphorylated Akt (p-Akt) and total Akt (t-Akt) in lung tissues were detected by immunohistochemical staining (IHC) and Western blotting. ② In vitro study, human pulmonary microvascular endothelial cells (HPMECs) were divided into 6 groups (5 holes in each group): control group, Xubijing control group (incubated with 2 g/L Xubijing for 24 hours), phosphoinositide 3-kinases (PI3K) signaling pathway LY294002 control group (incubated with 10 μmol/L LY294002 for 1 hour), LPS group (incubated with 1 mg/L LPS for 12 hours), Xubijing intervention group (incubated with 2 g/L Xuebijing for 24 hours, then with 1 mg/L LPS for 12 hours) and LY294002 intervention group (incubated with 10 μmol/L LY294002 for 1 hour, then with 2 g/L and Xubijing for 24 hours, and then with 1 mg/L LPS for 12 hours). The expression levels of claudin-5, p-FOXO1, t-FOXO1, p-Akt and t-Akt of HPMECs in each group were assessed by Western blotting. Results:In vivo study: ① Compared with the control group, the lung W/D ratio increased significantly in LPS group (6.79±0.42 vs. 4.19±0.13), and decreased significantly after the intervention of Xuebijing (4.92±0.38 vs. 6.79±0.42, P < 0.01). ② Morphological changes of lung tissue: compared with the control group, the injury of lung tissue in LPS group was more serious, which was significantly improved after Xuebijing intervention. ③ Expression levels of claudin-5, p-Akt/t-Akt and p-FOXO1/t-FOXO1: the expression levels of claudin-5, p-Akt/t-Akt and p-FOXO1/t-FOXO1 in LPS group were significantly decreased as compared with the control group (claudin-5/GAPDH: 0.33±0.03 vs. 1.03±0.07, p-Akt/t-Akt: 0.18±0.02 vs. 1.01±0.13, p-FOXO1/t-FOXO1: 0.16±0.06 vs. 1.00±0.19, all P < 0.01). After the intervention of Xuebijing, the expression levels were significantly increased as compared with the LPS group (claudin-5/GAPDH: 0.53±0.05 vs. 0.33±0.03, p-Akt/t-Akt: 0.56±0.12 vs. 0.18±0.02, p-FOXO1/t-FOXO1: 0.68±0.10 vs. 0.16±0.06, all P < 0.01). In vitro study: compared with the control group, the expression level of claudin-5 in the LPS group was significantly decreased (claudin-5/β-actin: 0.45±0.03 vs. 1.01±0.15, P < 0.01), and the expression level of claudin-5 in Xuebijing intervention group was also significantly decreased (claudin-5/β-actin: 0.80±0.08 vs. 1.01±0.15, P < 0.01). After the intervention of LY294002, the expression of claudin-5 was significantly decreased as compared with the Xubijing intervention group (claudin-5/β-actin: 0.41±0.02 vs. 0.80±0.08, P < 0.01). Conclusion:Xuebijing injection improve pulmonary vascular barrier function in rats with ARDS by up-regulating claudin-5 expression through PI3K/Akt/FOXO1 signaling pathway.

Caludin-5: target of regulating blood brain barrier in the treatment of ischemic stroke / 国际脑血管病杂志

The blood brain barrier (BBB) is a physical and metabolic barrier that maintains central nervous system homeostasis and protects brain tissues from potentially hazardous circulating substances. This article reviews the biological characteristics of caludin-5 during cerebral ischemia, its role in BBB integrity and permeability, as well as the research progress of related drug therapy based on calludin-5.

Effect and Mechanism of "Liver-Soothing and Spleen-Invigorating" Therapies Against Liver Injury in Rats： An Exploration Based on TGR5 and Intestinal Mucosal Barrier Function / 中国实验方剂学杂志

ObjectiveTo explore the effects of different treatment methods of "soothing liver， invigorating spleen， soothing liver and invigorating spleen， soothing liver first and then soothing liver and invigorating spleen， as well as invigorating spleen first and then soothing liver and invigorating spleen" on liver depression combined with liver injury in rats and their action mechanisms. MethodA six-week rat model of liver depression combined with liver injury was established by restraint stress and subcutaneous injection of carbon tetrachloride （CCl4， 5.89 g·kg-1， once every three days）. At the same time， the drugs were given by gavage. Forty-eight male SD rats of clean grade were randomly divided into eight groups， namely the normal group， model group， bicyclol （0.2 g·kg-1） group， Sinisan （4.32 g·kg-1） group， Liu Junzitang （9.26 g·kg-1） group， Chaishao Liu Junzitang A （Chai A， soothing liver and invigorating spleen，13.57 g·kg-1） group， Chaishao Liu Junzitang B （Chai B， soothing liver first and then soothing liver and invigorating spleen， 13.57 g·kg-1） group， and Chaishao Liu Junzitang C （Chai C， invigorating spleen first and then soothing liver and invigorating spleen， 13.57 g·kg-1） group， with six rats in each group. The pathological changes in liver and colon tissues of each group were observed under light microscope and electron microscope. The serum biochemical indexes of the liver were detected using an automatic biochemical analyzer. The relative mRNA expression levels of Takeda G protein-coupled receptor 5 （TGR5） and intestinal mucosal zona occluden-1 （ZO-1）， Occludin， and Claudin-1 in the liver and colon were detected by reverse-transcription polymerase chain reaction （RT-PCR）. The positive expression rate of proliferating cell nuclear antigen （PCNA） in the colon was detected by immunohistochemistry. ResultCompared with normal group， the model group exhibited significantly elevated serum alkaline phosphatase （ALP）， alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， total bilirubin （TBIL）， and direct bilirubin （DBIL） （P<0.01）， lowered TGR5 mRNA expression in liver tissue， up-regulated TGR5 mRNA expression in the colon tissue （P<0.05，P<0.01）， and down-regulated ZO-1， Occludin， and tight junction protein-1 (Claudin-1) mRNA expression and PCNA in the colon tissue （P<0.01）. Compared with the model group， bicyclol and Chai C remarkably decreased the levels of serum ALP， ALT， AST， TBIL， and DBIL （P<0.05，P<0.01）， while Liu Junzitang， Chai A， Chai B， and Chai C significantly up-regulated the TGR5 mRNA expression in the liver and down-regulated its expression in the colon （P<0.01）. Bicyclol， Chai A， Chai B， and Chai C enhanced the ZO-1 and Claudin-1 mRNA expression in the colon （P<0.05，P<0.01）. Bicyclol， Sinisan， and Chai C increased PCNA expression （P<0.01）. The comparison with the Chai C group showed that the TGR5 mRNA expression in the liver and ZO-1 mRNA expression in the colon of the bicyclol and Sinisan groups were lower， whereas the TGR5 mRNA expression in the colon was higher （P<0.01）. However， the PCNA expression in the colon of the Liu Junzitang and Chai B groups declined significantly （P<0.05）. ConclusionIn the presence of liver injury， invigorating spleen first helps to relieve the liver injury， and the efficacy of "spleen-invigorating" therapy in increasing the intestinal mucosal tight junction proteins and improving the gastrointestinal function is related to its activation of TGR5 to improve the intestinal mucosal barrier function， promote the renewal of intestinal stem cells， and drive the regeneration after injury.

Expression and clinical significance of CLDN-7 in pancreatic cancer / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the expression of tight junction protein claudin-7 (CLDN-7) in pancreatic cancer and its correlation with the clinicopathological features and prognosis of pancreatic cancer patients. Methods: Oncomine, GEPIA and GEO databases were used to comprehensively analyze the mRNA expression level of CLDN-7 in pancreatic cancer, and Kaplan-Meier Plotter database was used to analyze the relationship between the expression of CLDN-7 and the survival prognosis of pancreatic cancer patients. Immunohistochemical staining was used to detect the protein level of CLDN-7 in 44 cases of pancreatic cancer tissues and 31 cases of para-cancerous tissues resected in the Department of General Surgery of the Second Hospital of Lanzhou University from 2015 to 2018, and the relationship between CLDN-7 expression and clinicopathological characteristics and prognosis of patients was also analyzed. GO (Gene Ontology) analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis were conducted to analyze the possible signaling pathways that CLDN-7 may involve in and their main functions, which were further verified in TCGA and GEPIA databases. Results: Analysis of both the databases and the clinical samples showed that CLDN-7 was significantly over-expressed in pancreatic cancer tissues, and its high expression was correlated with clinical prognosis of pancreatic cancer patients; moreover, CLDN-7 expression was an independent factor affecting the overall survival time of pancreatic cancer patients (all P<0.05). GO analysis and KEGG pathway enrichment analysis confirmed that CLDN-7 was involved in DNA damage repair and glucose metabolism in pancreatic cancer patients. TCGA and GEPIA database validation showed that CLDN-7 expression in pancreatic cancer was significantly and positively correlated with the expression of DNA damage repair related genes (POLD4, SMUG1, NTHL1) and glucose metabolism related genes (ALDOA, TALDO1, PGLS) (all P<0.01). Conclusion: CLDN-7 is highly expressed in pancreatic cancer and indicates a worse clinical prognosis; moreover, CLDN-7 is associated with DNA damage repair and intratumoral glucose metabolism in pancreatic cancer.

Xuebijing Injection Ameliorates H / 中国结合医学杂志

OBJECTIVE@#To investigate the protective effects and underlying mechanisms of Xuebijing Injection (XBJ) on the lung endothelial barrier in hydrogen sulfide (H@*METHODS@#Sprague-Dawley rats were exposed to H@*RESULTS@#The morphological investigation showed that XBJ attenuated H@*CONCLUSIONS@#XBJ ameliorated H

Claudin-1 and Claudin-3 as Molecular Regulators of Myelination in Leukoaraiosis Patients

OBJECTIVES: Leukoaraiosis is described as white matter lesions that are associated with cognitive dysfunction, neurodegenerative disorders, etc. Myelin depletion is a salient pathological feature of, and the loss of oligodendrocytes is one of the most robust alterations evident in, white matter degeneration. Recent studies have revealed that claudin proteins are aberrantly expressed in leukoaraiosis and regulate oligodendrocyte activity. However, the roles of claudin-1 and claudin-3 in oligodendrocytes and leukoaraiosis are still not well-defined. METHODS: Quantitative polymerase chain reaction was used to measure the expression of claudin-1 (CLDN1), claudin-3 (CLDN3), and myelinogenesis-related genes such as myelin basic protein (MBP), proteolipid protein (PLP), oligodendrocyte transcription factor 2 (OLIG2), and SRY-box transcription factor 10 (SOX10) in leukoaraiosis patients (n=122) and healthy controls (n=122). The expression of claudin-1 and claudin-3 was either ectopically silenced or augmented in Oli-neu oligodendrocytes, and colony formation, apoptosis, and migration assays were performed. Finally, the expression of myelin proteins was evaluated by western blotting. RESULTS: Our results revealed that in addition to SOX10, the expression levels of claudin-1, claudin-3, and myelinogenesis-related proteins were prominently downregulated in leukoaraiosis patients, compared to those in healthy controls. Furthermore, the growth and migration of Oli-neu cells were downregulated upon silencing claudin-1 or claudin-3. However, the overexpression of claudin-1 or claudin-3 resulted in the reduction of the degree of apoptosis in Oli-neu cells. In addition, claudin-1 and claudin-3 promoted the expression of MBP, OLIG2, PLP, and SOX10 at the translational level. CONCLUSION: Our data has demonstrated that the abnormal expression of claudin-1 and claudin-3 regulates the pathological progression of leukoaraiosis by governing the viability and myelination of oligodendrocytes. These findings provide novel insights into the regulatory mechanisms underlying the roles of claudin-1 and claudin-3 in leukoaraiosis.

Effect of ionizing radiation on the secretion of the paracellular pathway in rat submandibular glands / 华西口腔医学杂志

OBJECTIVES@#This study aims to investigate the effects of ionizing radiation on the secretion of the paracellular pathway in rat submandibular glands (SMGs) and reveal the changes in the tight junction (TJ) protein claudin-4.@*METHODS@#A total of 24 Wistar rats were randomly divided into control and irradiation groups. The irradiation groups were further divided into 1, 4, and 12 weeks groups after irradiation. One-time 20 Gy irradiation was given to the SMG area on the experimental side of the irradiation group. At 1, 4, and 12 weeks after irradiation, the secretion of SMGs was measured using the Schirmer's test. The pathological changes in the gland tissues were observed under light microscopy after hematoxylin⁃eosin (HE) staining. The changes in the TJ ultrastructure were observed under transmission electron microscopy. The immunofluorescence staining and Western blot were used to detect the expression levels of muscarinic acetylcholine M3 receptor, aquaporin 5 (AQP5), and claudin-4 protein.@*RESULTS@#At 1, 4, and 12 weeks after irradiation, the secretion of SMGs in the irradiation group was significantly decreased and lower than that in the control group (@*CONCLUSIONS@#The changes in the TJ structure, the upregulation of the claudin-4 expression, and the damage in the paracellular pathway were involved in the hyposecretion of SMGs after irradiation.

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。

Research of the change of blood-brain barrier permeability in septic rats / 中华急诊医学杂志

Objective:To explore the change of blood-brain barrier (BBB) permeability in septic rats.Methods:A rat model of sepsis was established by cecal ligation and puncture. Rats were randomly (random number) grouped according to the intervention time: sham-operated group, sepsis 1-day group, sepsis 4-day group, and sepsis 7-day group. Fluorescein sodium was used to test the permeability of the BBB. Western blot and immunofluorescence methods were applied to detect the expression of tight junction proteins including Claudin-5, Occludin and ZO-1.Results:Compared with the sham-operated group, rats in the sepsis group presented quick breath, slow response, decreased intake of food and water, obvious abdominal distension and loose stools. After abdominal anatomy of sepsis rats, we found mesenteric adhesions, dilatation of proximal intestinal, black cecum ligation site with purulent exudate, enlarged liver and diffused bloody exudate. Compared with the sham-operated group, body weight of sepsis rats was reduced remarkably ( P < 0.05). The body weight of rats of sepsis 7-day group was the lowest, which was significantly lower than that of rats of sepsis 4-day group ( P< 0.05) and 1-day group ( P< 0.05). Compared with the sham-operated group, the content of fluorescein sodium in sepsis 1-day rats was increased remarkably ( P< 0.05). The content of fluorescein sodium in rats of sepsis 7-day group was the highest, which was significantly higher than that in rats of sepsis 4-day group ( P< 0.05) and 1-day group ( P< 0.05). Compared with the sham-operated rats, the expression of Claudin-5, Occludin and ZO-1 in sepsis rats were decreased remarkably (all P < 0.05). The expression of Claudin-5, Occludin and ZO-1 were the lowest in rats of the sepsis 7-day group, which were significantly decreased than those of rats in the sepsis 4-day group (all P< 0.05) and rats in sepsis 1-day group (all P < 0.05). Conclusions:Sepsis rats showed increased permeability of the BBB, and the permeability of BBB increased continuously along with the duration of sepsis.

Relationship between serum claudin-1 level and papillary thyroid carcinoma / 中华内分泌代谢杂志

Objective:To analyze the correlation between serum claudin-1(Cla-1) level and the risk of papillary thyroid carcinoma(PTC) in patients with thyroid nodules.Methods:The clinical data of 345 patients with thyroid nodules were retrospectively analyzed. According to the pathological results, they were divided into PTC group and benign thyroid nodule(BTN) group. The difference of serum Cla-1 level between 2 groups and its correlation with the risk of PTC were analyzed.Results:In groups of PTC( n=225) and BTN( n=120), the median value of serum Cla-1 level was 14.03(10.30, 20.40) ng/mL. The differences in the median value[17.90(14.00, 22.93)ng/mL vs 9.40(8.15, 11.20) ng/mL] of serum Cla-1 level in the PTC and BTN were statistically significant by Wilcoxon signed-rank test. The prevalence of PTC in thyroid nodules increased gradually with increasing of serum Cla-1 level. Receiver operated characteristic curve analysis showed that the best diagnostic cut-off value of the PTC was 13.02 ng/ml of which the sensitivity was 81.8%, the specificity was 89.2%, and the area under curve(AUC) was the largest(AUC=0.944, P<0.01, 95% CI 0.922-0.965). Logistic regression analysis showed that elevated serum Cla-1 level increased the risk of PTC, and it was statistically significant( OR=4.334, 95% CI 1.662-11.303, P=0.003). There was a significant correlation among the serum Cla-1 level and gender, age, location of involvement, number and diameter of cancer nodules, extracapsular invasion of thyroid, lymph node metastasis, tumor stage and combined with Hashimoto′s thyroiditis( P<0.01). Conclusion:The serum level of Cla-1 may be one of risk factors to predict PTC, and it is related to the total amount of PTC tumor cells in vivo, but it was not related to the aggressive behavior of tumor.

Jiawei Baitouweng Decoction affects intestinal mucosal tight junction proteins in rats with ulcerative colitis through p38 MAPK-MLCK signaling pathway / 中国中药杂志

The aim of this paper was to explore the effect and mechanism of Jiawei Baitouweng Decoction(JWBTW) against ulcerative colitis(UC) from the perspective of intestinal mucosal tight junction proteins. From 60 SPF-grade male SD rats, 10 were randomly selected as the blank control, and the remaining 50 were treated with 3% dextran sodium sulfate(DSS) solution to induce UC and then randomized into the model group, mesalazine group, and low-, medium-, and high-dose JWBTW( L-JWBTW, M-JWBTW and H-JWBTW) groups, with 10 rats in each group. After successive medication for 14 days, the rat general conditions like body weight and stool were observed and the disease activity index(DAI) was calculated. The pathological changes in colon tissue was observed under a microscope for injury severity scoring and histopathological scoring. The serum endotoxin content was determined by limulus assay, followed by the measurement of protein expression levels of ZO-1, occludin, claudin-1, p38 MAPK, MLCK, MLC2 and p-MLC in colon tissue by Western blot. The results showed that compared with the blank group, the model group exhibited significantly reduced body weight, elevated DAI, injury severity and histopathological scores and serum endotoxin content, up-regulated protein expression levels of p38 MAPK, MLCK, MLC2 and p-MLC, and down-regulated ZO-1, occludin and claudin-1. Compared with the model group,mesalazine and JWBTW at each dose obviously increased the body weight, lowered the DAI, injury severity and histopathological scores and serum endotoxin content, down-regulated the protein expression levels of p38 MAPK, MLCK, MLC2 and p-MLC, and up-regulated the ZO-1, occludin and claudin-1, with the most obvious changes noticed in the H-JWBTW group. All these have indicated that JWBTW exerts the therapeutic effect against UC by inhibiting the activation of p38 MAPK/MLCK pathway, reversing the protein expression levels of occludin, claudin-1 and ZO-1, decreasing the serum endotoxin content, promoting the repair of intestinal mucosal mechanical barrier, maintaining the integrity of tight junctions, and reducing the permeability of intestinal mucosa.

Claudin 14/15 play important roles in early wallerian degeneration after rat sciatic nerve injury / 中华创伤杂志(英文版)

PURPOSE@#Wallerian degeneration (WD) is an antegrade degenerative process distal to peripheral nerve injury. Numerous genes are differentially regulated in response to the process. However, the underlying mechanism is unclear, especially the early response. We aimed at investigating the effects of sciatic nerve injury on WD via CLDN 14/15 interactions in vivo and in vitro.@*METHODS@#Using the methods of molecular biology and bioinformatics analysis, we investigated the molecular mechanism by which claudin 14/15 participate in WD. Our previous study showed that claudins 14 and 15 trigger the early signal flow and pathway in damaged sciatic nerves. Here, we report the effects of the interaction between claudin 14 and claudin 15 on nerve degeneration and regeneration during early WD.@*RESULTS@#It was found that claudin 14/15 were upregulated in the sciatic nerve in WD. Claudin 14/15 promoted Schwann cell proliferation, migration and anti-apoptosis in vitro. PKCα, NT3, NF2, and bFGF were significantly upregulated in transfected Schwann cells. Moreover, the expression levels of the β-catenin, p-AKT/AKT, p-c-jun/c-jun, and p-ERK/ERK signaling pathways were also significantly altered.@*CONCLUSION@#Claudin 14/15 affect Schwann cell proliferation, migration, and anti-apoptosis via the β-catenin, p-AKT/AKT, p-c-jun/c-jun, and p-ERK/ERK pathways in vitro and in vivo. The results of this study may help elucidate the molecular mechanisms of the tight junction signaling pathway underlying peripheral nerve degeneration.

Enema de sulcralfato pode prevenir colite em segmentos colônicos sem trânsito fecal? / Can sucralfate enema prevent colitis in colonic segments without fecal transit?

RESUMO - RACIONAL: O estresse oxidativo é um dos principais mecanismos associados à ruptura dos mecanismos de defesa que formam a barreira epitelial cólica e reduz o conteúdo tecidual das proteínas claudina-3 e ocludina principais constituintes das junções de oclusão intercelulares. O sucralfato, possui atividade antioxidante e tem sido usado para tratar diferentes formas de colite. OBJETIVO: Mensurar o conteúdo tecidual de claudina-3 e ocludina da mucosa do cólon sem trânsito fecal, submetido à intervenção com sucralfato. MÉTODO: Trinta e seis ratos foram submetidos à colostomia do cólon esquerdo e fístula mucosa distal. Os animais foram divididos em dois grupos de acordo com a eutanásia ser realizada duas ou quatro semanas após a intervenção. Cada grupo foi dividido em três subgrupos de acordo com o tipo de intervenção realizada diariamente: solução salina isolada; sucralfato a 1 g/kg/dia ou sucralfato a 2g/kg/dia. A colite foi diagnosticada por análise histológica adotando escala de validação prévia. A expressão tecidual de ambas as proteínas foi identificada por imunoistoquímica. O conteúdo das proteínas foi quantificado por análise de imagem assistida por computador. RESULTADOS: O escore inflamatório foi maior nos segmentos cólicos sem trânsito fecal e os enemas com sucralfato reduziram o escore inflamatório nesses segmentos, principalmente nos animais submetidos à intervenção com sucralfato em maior concentração e por período mais longo de intervenção. Houve aumento no conteúdo tecidual das proteínas claudina-3 e ocludina, relacionado com a concentração de sucralfato. O conteúdo tecidual de ambas as proteínas não se modificou com a duração da intervenção. CONCLUSÃO: Enemas com sucralfato reduzem a inflamação e aumentam o conteúdo tecidual de claudina-3 e ocludina na mucosa cólica sem trânsito intestinal.

ABSTRACT - BACKGROUND: Oxidative stress is one of the main mechanisms associated with the rupture of the defense mechanisms of the colonic epithelial barrier; it reduces the tissue content of the claudin-3 and occludin proteins, which are the main constituents of intercellular tight junctions. Sucralfate (SCF) has antioxidant activity and has been used to treat different forms of colitis. AIM: This study aimed to measure the tissue claudin-3 and occludin content of the colon mucosa without fecal transit, subjected to intervention with SCF. METHODS: Thirty-six rats were subjected to left colon colostomy and distal mucous fistula. They were divided into two groups according to euthanasia that was performed 2 or 4 weeks after the intervention. Each group was divided into three subgroups according to the enema applied daily: saline alone, SCF at 1 g/kg/day, or SCF at 2 g/kg/day. Colitis was diagnosed by the histological analysis adopting the previous validate scale. The tissue expression of both proteins was identified by immunohistochemical technique. The content of proteins was quantified by computer-assisted image analysis. RESULTS: The inflammatory score was high in colonic segments without fecal transit, and enemas with SCF reduced the inflammatory score in these segments, mainly in those animals submitted to intervention with SCF in greater concentration and for a longer period of intervention. There was an increase in tissue content of claudin-3 and occludin, related to SCF concentration. The tissue content of both proteins was not related to the intervention time. CONCLUSION: Enemas with SCF reduced the inflammation and increased the tissue content of claudin-3 and occludin in colonic mucosa without fecal stream.

Expressão de e-caderina e claudina-3 no epitélio cólico após terapia com infliximabe: modelo experimental de colite de exclusão / Expression of e-cadherin and claudin-3 in the colonic epithelium after the infliximab therapy: experimental model of disuse colitis

RESUMO - RACIONAL: A etiopatogenia da colite por desuso (DC) ainda não foi totalmente elucidada. As principais teorias consideram que a doença pode estar relacionada ao aumento de bactérias anaeróbias, falta de suprimento de ácidos graxos de cadeia curta (AGCC) e distúrbios imunológicos que se desenvolvem em segmentos colorretais desprovidos de trânsito fecal. OBJETIVO: Verificar se a aplicação de infliximabe modifica o conteúdo tecidual das proteínas E-caderina e claudina-3 no epitélio cólico de ratos sem trânsito intestinal. MÉTODOS: Vinte dois ratos foram submetidos a derivação do trânsito intestinal pelo procedimento de Hartmann. Eles permaneceram com o ostoma por 12 semanas para permitir o desenvolvimento da colite de exclusão. Em seguida, foram divididos em três grupos experimentais: seis animais receberam 2,0 ml de solução salina/semana, oito infliximabe na dose de 5 mg/Kg/semana e, os demais, infliximabe na dose de 10 mg/Kg/semana por 5 semanas consecutivas. Em seguida, os animais foram eutanasiados e os segmentos cólicos com e sem trânsito intestinal foram removidos. A colite por desuso foi diagnosticada pelas alterações histológicas definidas por uma escala previamente validada. Expressão tecidual de E-caderina e claudina-3 foi avaliada por imuno-histoquímica, e o conteúdo tecidual de ambas as proteínas foi quantificado por análise de imagem assistida por computador. RESULTADOS: Segmentos cólicos exclusos de trânsito fecal apresentaram maior grau de inflamação do que os expostos ao trânsito fecal. Inflamação foi menor nos animais tratados com infliximabe, independente da dose utilizada. Níveis de E-caderina e claudina-3 estavam reduzidos no cólon excluso. O tratamento com infliximabe aumentou os níveis das proteínas em segmentos do cólon sem trânsito intestinal, principalmente nos animais que receberam a dose de 10mg/kg/semana. CONCLUSÃO: Infliximabe reduz inflamação nos segmentos do cólon excluso e aumenta o conteúdo tecidual de E-caderina e claudina-3, especialmente na concentração de 10mg/kg/semana.

ABSTRACT - BACKGROUND: The etiopathogenesis of disuse colitis (DC) has not yet been fully elucidated. The main theories consider that the disease may be related to an increase in anaerobic bacteria, the lack of short-chain fatty acid (SCFA) supply, and immunological disorders that develop in the colorectal segments devoid of fecal transit. AIM: The aim of this study was to verify whether the application of infliximab modifies the tissue content of E-cadherin and claudin-3 proteins in colonic epithelium of rats devoid of intestinal transit. METHODS: A total of 22 rats underwent intestinal transit bypass using Hartmann's procedure. They remained with the shunt for 12 weeks to allow the development of DC. Later, they were divided into three experimental groups: six animals received 2.0 mL saline solution/week, eight received infliximab at a dose of 5 mg/kg/week, and eight received infliximab at a dose of 10 mg/kg/week for 5 consecutive weeks. At the end of this period, the animals were euthanized, and the colonic segments with and without intestinal transit were removed. DC was diagnosed based on the histological changes defined by a previously validated scale. The tissue expression of E-cadherin and claudin-3 was assessed by immunohistochemistry, and the tissue content of both proteins was quantified by computer-aided image analysis. RESULTS: The colonic segments excluded from fecal transit showed a higher degree of inflammation than those exposed to fecal transit. The degree of inflammation was lower in animals treated with infliximab, regardless of the dose used. The levels of E-cadherin and claudin-3 were reduced in the excluded colon. Treating animals with infliximab increased the levels of both proteins in the colonic segments without intestinal transit, especially in animals receiving a dose of 10 mg/kg/week. CONCLUSION: Infliximab therapy reduces inflammation in the colonic segments excluded from intestinal transit and increases the tissue content of E-cadherin and claudin-3 proteins, especially when used at a concentration of 10 mg/kg/week.