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.

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.

Molecular engineering of antibodies for site-specific conjugation to lipid polydopamine hybrid nanoparticles

Conjugation of antibodies to nanoparticles allows specific cancer targeting, but conventional conjugation methods generate heterogeneous conjugations that cannot guarantee the optimal orientation and functionality of the conjugated antibody. Here, a molecular engineering technique was used for site-specific conjugation of antibodies to nanoparticles. We designed an anti-claudin 3 (CLDN3) antibody containing a single cysteine residue, h4G3cys, then linked it to the maleimide group of lipid polydopamine hybrid nanoparticles (LPNs). Because of their negatively charged lipid coating, LPNs showed high colloidal stability and provided a functional surface for site-specific conjugation of h4G3cys. The activity of h4G3cys was tested by measuring the binding of h4G3cys-conjugated LPNs (C-LPNs) to CLDN3-positive tumor cells and assessing its subsequent photothermal effects. C-LPNsspecifically recognized CLDN3-overexpressing T47D breast cancer cells but not CLDN3-negative Hs578T breast cancer cells. High binding of C-LPNs to CLDN3-overexpressing T47D cells resulted in significantly higher temperature generation upon NIR irradiation and potent anticancer photothermal efficacy. Consistent with this, intravenous injection of C-LPNsin a T47D xenograft mouse model followed by NIR irradiation caused remarkable tumor ablation compared with other treatments through high temperature increases. Our results establish an accurate antibody-linking method and demonstrate the possibility of developing therapeutics using antibody-guided nanoparticles.

ZDHHC12-mediated claudin-3 -palmitoylation determines ovarian cancer progression

The membrane protein claudin-3 (CLDN3) is critical for the formation and maintenance of tight junction and its high expression has been implicated in dictating malignant progression in various cancers. However, the post-translational modification of CLDN3 and its biological function remains poorly understood. Here, we report that CLDN3 is positively correlated with ovarian cancer progression both and Of interest, CLDN3 undergoes -palmitoylation on three juxtamembrane cysteine residues, which contribute to the accurate plasma membrane localization and protein stability of CLDN3 Moreover, the deprivation of -palmitoylation in CLDN3 significantly abolishes its tumorigenic promotion effect in ovarian cancer cells. By utilizing the co-immunoprecipitation assay, we further identify ZDHHC12 as a CLDN3-targating palmitoyltransferase from 23 ZDHHC family proteins. Furthermore, the knockdown of ZDHHC12 also significantly inhibits CLDN3 accurate membrane localization, protein stability and ovarian cancer cells tumorigenesis Thus, our work reveals -palmitoylation as a novel regulatory mechanism that modulates CLDN3 function, which implies that targeting ZDHHC12-mediated CLDN3 -palmitoylation might be a potential strategy for ovarian cancer therapy.

Claudin-3 and occludin content in the glands of colonic mucosa devoid from fecal stream submitted to topical intervention with oil extract of Curcuma longa

Abstract Purpose: To evaluate the inflammatory intensity and measure the tissue content of the proteins claudin-3 and occludin in the colonic mucosa without fecal stream submit to intervention with curcumin. Methods: Thirty-six rats were submitted to a proximal colostomy and a distal mucous fistula and divided into two groups according to sacrifice to be performed two or four weeks. Each group was divided into three subgroups according daily application of enemas containing saline, curcumin at 50 mg/kg/day or 200 mg/kg/day. Colitis was diagnosed by histological analysis. Claudin-3 and occludin were determined by immunohistochemistry. The tissue content of claudin-3 and occludin were quantified by computer-assisted image analysis. Mann-Whitney, Student t and ANOVA tests were used to analyze the results establishing the level of significance of 5% for both (p<0.05). Results: Curcumin at both concentrations reduces the inflammation and preserves the tissue content of the proteins claudin-3 and occludin, which was related to the concentration used and to the time of the intervention. Conclusion: The application of enemas with curcumin reduces inflammation and preserves the tissue content of the proteins claudin-3 and occludin in the colonic mucosa devoid from the fecal stream.

Expression of claudin-3 and β-catenin in borderline ovarian tumors and its clinicopathological significance / 第二军医大学学报

Objective To investigate the expression of claudin-3 and β-catenin in borderline ovarian tumors (BOTs) and its clinicopathological significance. Methods We examined the expressions of claudin-3 and β-catenin and their relationship with the clinicopathological features in 17 normal ovarian tissues, 31 epithelial benign ovarian tumors, 39 epithelial malignant ovarian tumors and 69 BOTs by SP immuno histochemistry method. Results The positive expression rate of claudin-3 protein in BOTs (59. 4%, 41/69) was significantly lower than that in the malignant ovarian tumors (89. 7%, 35/39), and higher than that in normal ovaries (5.9%, 1/17) and benign ovarian tumors (22. 6%, 7/31)(P0. 05). The positive expression rate of β-catenin protein in BOTs (52.2%, 36/39) was significantly lower than that in the malignant ovarian tumors (84. 7%, 33/39), and higher than that in normal ovaries (11. 8%, 2/17) and benign ovarian tumors (29. 0%, 9/31; P0. 05). The expression of claudin-3 and β-catenin was correlated with the clinciopathological stage and intraperitoneal metastases (P0. 05). Expression of claudin-3 and β-catenin was positively correlated in BOTs (r = 0. 439, P = 0. 000). Conclusion Claudin-3 and β-catenin may play a synergistic role in the oncogenesis and progression of BOTs. Simutaneous detection of claudin-3 and β-catenin is valuable for diagnosis and treatment of BOTs.

Expression and significance of Claudin 3 in adenocarcinoma of esophagus and cardia adenocarcinoma / 第三军医大学学报

Objective To investigate the expression and significance of Claudin 3 in adenocarcinoma of esophagus and gastric cardia.Methods We subjected pathologically confirmed 16 cases of esophageal adenocarcinoma(EAC),20 cases of esophageal squamous cell carcinoma(ESC),and 40 cases of gastric cardiac adenocarcinoma(CAC,including 15 diffuse and 25 intestinal cases).Tissue samples were collected with aid of gastroscope.Fifteen samples of normal esophageal mucosa and 15 samples of normal cardiac mucosa served as control.RT-PCR and Western blotting were used respectively to detect the mRNA and protein expression of Claudin 3 in these tissue samples.Results The mRNA and protein expressions of Claudin 3 were increased in EAC and intestinal CA compared with those in normal tissues,ESC and diffuse CAC.There was no significant difference of the expression of Claudin 3 between normal group and ESC and diffuse CAC group.Conclusion Claudin 3 might be related to the development of EAC and intestinal CAC,and expected to be the tumor maker and therapeutic target of this kind of carcinoma.