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Aim Excessive cerebral inflammation caused by chronic alcohol intake is an important risk factor for central nervous system injury. The purpose of this study was to explore the protective effect of konjac mannan oligosaccharide (KMOS) on central nervous system inflammation in alcohol-fed mice and its mechanism. Methods The chronic alcohol fed model of C57BL/6J mice was established using Gao-binge method. And the different doses of KMOS were gavaged every day for 6 weeks. The neuronal damage and microglia activation were evaluated in cerebral cortex and hippocampus. The damage of colon tissue was assessed and serum LPS concentrations were measured. In vitro, Caco-2 cells were stimulated with LPS to establish intestinal mucosal injury model. Results Chronic alcohol intake can cause brain neuron damage in mice, and different doses of KMOS effectively reduced the activation state of microglia, decreased the expression of inflammatory factors caused by the activation of the NLRP3 inflammasome and alleviated neuronal damage in the brain tissue of alcohol-fed mice. The results of colon tissue analysis showed that the use of KMOS effectively reduced the concentration of endotoxin LPS in serum of alcohol-fed mice, alleviated the pathological injury and inflammatory response of colon tissue, and enhanced the expression of Occludin in intestinal tissue. In vitro experiments also showed that KMOS significantly inhibited the inflammatory reaction of Caco-2 cells exposed to alcohol and increased the expression of Occludin protein. Conclusions KMOS treatment effectively inhibited intestinal inflammation caused by alcohol intake, repaired intestinal barrier to prevent the entry of intestinal LPS into brain tissue, decreased the activation of microglia, and then improved brain neuron damage. KMOS had the potential to prevent alcoholic nerve injury.
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Objective @#To ob serve the expression and trends of tight j unction proteins Occludin and zonula occluden-1 (ZO-1) in blood brain barrier (BBB) of rats with traumatic brain injury (TBI) , and to explore the intervention effect of cannabidiol (CBD) on the BBB . @*Methods @#The TBI model of rat was prepared by modified “Feeney free fall method ”and randomly divided into three groups : the sham operated group ( Sham group) , the model group (TBI + vehicle group) and the CBD intervention group ( TBI + CBD group) , with 24 rats in each group . Each group was subdivided into six time points : 8 h , 1 , 2 , 3 , 5 and 7 d after injury. The expression of Occludin and ZO-1 , which are closely related to the permeability of BBB , was detected by immunohistochemistry , immuno fluorescence staining and Western blot at different points . The permeability of BBB was detected by sodium fluores cein assay.@*Results @#The results of immunohistochemistry showed that compared with Sham group , the positive ex pression of Occludin and ZO-1 decreased with time after brain trauma ( P < 0.05) , and both reached the lowest level at 2 d . The expression levels of Occludin and ZO-1 were up regulated after 1 d of CBD intervention ( P < 0.05) . Immunofluorescence staining showed a similar trend to Western blot results , with Occludin and ZO-1 fluo rescence expression intensity and protein expression reduced after TBI compared with Sham group ( P < 0.05 ) . And the expression levels of Occludin and ZO-1 were up regulated after 2 d of CBD intervention (P < 0.05) . The results of fluorescein sodium experiment showed that the BBB integrity of brain tissue was destroyed after TBI , and the permeability increased after TBI (P < 0.01) . The permeability of BBB decreased after CBD intervention (P < 0.05) . @*Conclusion @#The expression of tight junction proteins O ccludin and ZO-1 decreases after TBI , and the permeability of BBB is disrupted , and CBD intervention reverses the disruption of the BBB by TBI .
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BACKGROUND:Diabetic nephropathy is an important cause of end-stage renal disease,and intestinal barrier damage plays an important role in the occurrence and development of diabetic nephropathy. OBJECTIVE:To observe the protective effect of human umbilical cord mesenchymal stem cells on the intestinal barrier in rats with diabetic nephropathy. METHODS:Thirty 8-week-old male SD rats were randomly assigned to healthy control group,model group and human umbilical cord mesenchymal stem cell group,with 10 rats in each group.Rats in the human umbilical cord mesenchymal stem cell group were injected with 1×106 human umbilical cord mesenchymal stem cells through the tail vein once a week for 4 weeks after the model establishment of diabetic nephropathy.Rats in the healthy control group and the model group were injected with an equal volume of PBS at the same time.1 week after the last injection,the histomorphological changes in the kidney and colon were observed under a light microscope.The expressions of ZO-1 and Occludin in the colon tissue of rats were detected by immunohistochemistry.Serum D-lactic acid and lipopolysaccharide levels were detected by ELISA.In addition,the distribution of human umbilical cord mesenchymal stem cells labeled with DiR dye in rats was observed by in vivo imaging system.The expression of human mesenchymal stem cell surface marker antigens CD44 and CD90 in colon tissue was detected by immunohistochemistry. RESULTS AND CONCLUSION:(1)Compared with the model group,human umbilical cord mesenchymal stem cell transplantation significantly inhibited the increase of urea nitrogen,serum creatinine,24-hour urine protein level and urinary albumin/creatinine ratio in diabetic nephropathy rats(all P<0.05).(2)The expression of human mesenchymal stem cell surface markers CD44 and CD90 was found in the colon of diabetic nephropathy rats.(3)Compared with the healthy control group,the expression levels of tight junction proteins Occludin and ZO-1 in the colon tissue of the model group were significantly reduced,while the expressions of Occludin and ZO-1 were significantly increased after treatment with human umbilical cord mesenchymal stem cells.(4)Compared with the model group,human umbilical cord mesenchymal stem cell transplantation significantly reduced serum D-lactic acid and lipopolysaccharide levels in diabetic nephropathy rats.(5)The results suggest that human umbilical cord mesenchymal stem cells may protect the intestinal barrier function by enhancing the expression of intestinal tight junction proteins in diabetic nephropathy rats.
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Aim To investigate the effects of cimifugin on mouse atopic dermatitis (AD) induced by fluorescein isothiocyanate (FITC) and further explore the mechanism of its action. Methods ICR mice were randomly divided into blank group, model group, positive group (dexamethasone),low dose group,high dose group and administration group of cimifugin. FITC solution was applied to the shaved abdomen of mice in the sensitization stage, and 0.6 % FITC solution was applied to attack the ears of mice in the stimulation stage. The administration groups were given medicine for seven consecutive days. The effects of cimifugin on body weight, thymus index and spleen index of mice were detected. Ear inflammatory cell infiltration was observed by HE staining. The ear swelling of mice was measured, and Th2 cytokines IL-5,IL-13 and the key promoter of allergy IL-33 were detected by ELISA. The epithelial barrier structural proteins, filaggrin, claudinl,occludin and E-cadherin,were detected by immunohistochemistry and Western blot. Results Compared with the blank group, the model group showed significant AD symptoms. Compared with the model group, cimifugin transdermal administration group significantly reduced ear inflammatory cell infiltration,ear swelling, IL-5,IL-13 and IL-33, and significantly increased the expression of filaggrin and occludin. Conclusions Transdermal administration of cimifugin could significantly inhibit AD in mice, and its mechanism involves repairing epithelial barrier function, restoring filaggrin and occludin, inhibiting allergy promoting factor IL-33, and finally inhibiting AD inflammation.
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Objective @#To study the effect of EFHD2 protein deletion in Sertoli cells on Occludin,a component of tight junction protein and the localization and expression of EF-hand domain family member D2 (EFHD2) in mouse testis.@*Methods @#Total RNA and protein were extracted from adult mice's heart ,liver ,spleen ,lung ,kidney, brain and testis tissues.The mRNA and protein levels of EFHD2 in each organ tissue were detected by qRT-PCR and Western blot.Immunofluorescence and immunohistochemistry detected the localization and expression of EF- HD2 in testicular tissues.SiRNA interference was used to reduce EFHD2 in Sertoli cells to detect Occludin protein expression. @*Results @#qRT-PCR showed that the expression of EFHD2 was the highest in the testis.Western blot results showed that the expression level increased in testis tissue.Indirect immunofluorescence and immunohisto- chemistry results showed that the protein was mainly distributed in Sertoli cells and co-localized with cytoskeletal Vimentin,indicating that the protein was expressed in Sertoli cells.After the decrease of EFHD2 protein expres- sion,Occludin protein expression also decreased.@*Conclusion @#The expression of EFHD2 protein in the testis is relatively high,mainly distributed in Sertoli cells of the testis,co-localized with Vimentin,and can affect the nor- mal expression of tight junction protein Occludin.It is suggested that EFHD2 can promote and maintain the junction structure of Sertoli cells and provide a stable microenvironment for spermatogenesis.
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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.
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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.
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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.
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Animals , Rats , Sucralfate/therapeutic use , Colitis/prevention & control , Colitis/drug therapy , Rats, Wistar , EnemaABSTRACT
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.
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Objective:To explore the protective effect of Gegen Qinliantang on the intestinal mucosal epithelial barrier function of ulcerative colitis (UC) mice, and to explore its mechanism of action in the treatment of ulcerative colitis via matrix metallopeptidase-9 (MMP-9)/p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. Method:The 48 female C57BL/6 mice were randomly divided into normal group, model group, sulfasalazine group (0.3 g·kg<sup>-1</sup>) and Gegen Qinliantang high, medium and low dose groups (2.84,1.42,0.71 g·kg<sup>-1</sup>). The UC murine model was established by 3% dextran sulfate sodium (DSS). Gegen Qinliantang and sulfasalazine were intragastrically administered on the 8<sup>th</sup> day after the model was established for 7 days, and the normal group was treated with the same amount of normal saline. Colon tissues were collected after the last administration, and the pathological changes of colon tissues were detected by hematoxylin-eosin (HE) staining. The expression of tight junction (TJ) proteins such as Occludin and zonula occludens-1(ZO-1) in colon tissues was detected by immunohistochemistry (IHC), and the expression levels of tumor necrosis factor-alpha (TNF-<italic>α</italic>), interleukin-1<italic>β</italic> (IL-1<italic>β</italic>), and MMP-9 mRNA in colon tissues were detected by Real-time polymerase chain reaction (Real-time PCR). The expression of phosphorylated p38 MAPK (p-p38 MAPK), p38 MAPK and MMP-9 protein in colon tissues was detected by Western blot. Result:Compared with normal group, the body weight of mice decreased (<italic>P</italic><0.01) and disease activity index (DAI) score increased significantly (<italic>P</italic><0.01) in model group, the colon tissues of the model group were damaged more obviously, the expression of occludin and ZO-1 proteins in model group was significantly reduced (<italic>P</italic><0.01), and the relative expression levels of TNF-<italic>α</italic>, IL-1<italic>β</italic>, and MMP-9 mRNA in model group were significantly increased (<italic>P</italic><0.01), the expression of p-p38 MAPK and MMP-9 in model group was significantly increased (<italic>P</italic><0.01). Compared with model group, the body mass and DAI score of the sulfasalazine group and Gegen Qinliantang group were significantly improved (<italic>P</italic><0.05,<italic>P</italic><0.01), the colonic tissues damage were significantly improved, and the expression of Occludin and ZO-1 protein was significantly increased (<italic>P</italic><0.05,<italic>P</italic><0.01), the relative expression levels of TNF-<italic>α</italic>, IL-1<italic>β</italic>, and MMP-9 mRNA were significantly decreased (<italic>P</italic><0.01), and the expression of p-p38 MAPK and MMP-9 was significantly decreased (<italic>P</italic><0.01). The changes in the middle dose group were the most obvious among the various dose groups of Gegen Qinliantang. Conclusion:Gegen Qinliantang repairs the intestinal mucosal barrier function by inhibiting the expressions of MMP-9 and inflammatory cytokines such as TNF-<italic>α</italic> and IL-1<italic>β</italic>, blocking the activation of the p38 MAPK signaling pathway, and increasing the expressions of tight junction protein.
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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.
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Animals , Male , Rats , Colitis, Ulcerative/genetics , Disease Models, Animal , Intestinal Mucosa , Rats, Sprague-Dawley , Signal Transduction , Tight Junction Proteins/genetics , p38 Mitogen-Activated Protein Kinases/geneticsABSTRACT
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.
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OBJECTIVE: To observe the effect of electroacupuncture (EA) on intestinal epithelial mucosal barrier function in diarrhea-predominant irritable bowel syndrome(IBS-D) rats, so as to explore its mechanisms underlying improvement of IBS-D. METHODS: Forty SD rats (half male and half female) were randomly divided into control, model, EA and medication (Pinaverium Bromide, PB) groups, with 10 rats in each group. The IBS-D model was established by chronic unpredictable mild stress combined with gavage of Senna-leaf solution. EA (2 Hz/15 Hz,0.1-1 mA) was applied to unilateral "Zusanli"(ST36),"Tianshu" (ST25), "Sanyinjiao"(SP6) and "Taichong"(LR3) alternatively for 15 min, once daily for 14 days. Rats of the medication group was treated by gavage of PB (10 mL·kg-1·d-1) for 14 days. The visceral sensitivity (pain) was assessed by using the pressure threshold which the inserted rectal balloon catheter air-inflation (connected to a blood pressure gauge) induced stronger abdominal muscular contraction to force the rat's abdomen to lift the experimental stand surface. The diarrhea index was used to evaluate loose stool grade. The expression of Claudin-1 and Occludin (intestinal epithelial tight junction proteins) of colon tissue was detected by immunohistochemistry. The activity of plasma diamine oxidase (DAO) was assayed by using spectrophotometry. RESULTS: Compared with the control group, the diarrhea index and plasma DAO activity in the model group were significantly increased (P0.05). CONCLUSION: Electroacupuncture can significantly improve abdominal pain and diarrhea in IBS-D model rats, which may be closely associated with its effects in up-regulating the expression of intestinal epithelial tight junction proteins Claudin-1 and Occludin to restore the function of intestinal epithelial mucosal barrier.
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Objective:To investigate the effect of modified Sijunzi Tang on protein and mRNA expressions of Occludin, Claudin-1 and zonula occludens-1(ZO-1) in cerebral ischemia rats. Method:Totally 60 male Sprague-Dawley rats were randomly divided into sham operation group, model group, edaravone group, small-dose modified Sijunzi Tang group, middle-dose modified Sijunzi Tang group and high-dose modified Sijunzi Tang group. The middle cerebral artery occlusion (MCAO) model was prepared by suture method. After 7 days of treatment, the modeling group was put to death. Western blot was used to detect the contents of Occludin, Claudin-1 and ZO-1 in the ischemic cerebral cortex of rats. Detection of Occludin, ZO-1, Claudin-1 mRNA expression in the ischemic cortex of rats by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR). Result:Compared with the sham operation group, protein expressions of Occludin, Claudin-1, and ZO-1 in the model group were significantly down-regulated (PPPPConclusion:Modified Sijunzi Tang may protect the blood-brain barrier and reduce brain edema in ischemic stroke rats by increasing the expression of tight junction proteins Occludin, ZO-1 and Claudin-1.
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Modified Da-chai-hu Decoction (MDD), a traditional Chinese medicinal formulation, which was empirically generated from Da-chai-hu decoction, has been utilized to treat severe acute pancreatitis (SAP) for decades. The aim of the present study was to explore its potential organprotective mechanism in SAP. In the present study, rat SAP model was induced by retrograde injection of 3.5% sodium taurocholate into the biliopancreatic duct, MDD (23.35 g/kg body weight, twelve times the clinical dose) were orally given at 2 h before and 10 h after injection. At 12 h after model induction, blood was taken from vena cava for analysis of amylase, diamine oxidase (DAO), pulmonary surfactant protein-A (SP-A), and C-reactive protein (CRP). Histopathological change of pancreas, ileum and lung was assayed by H&E staining, myeloperoxidase (MPO) activity were determinated using colorimetric assay, and the expressions of occludin and nuclear factor-κB (NF-κB) were detected by real-time RT-PCR and western blot, respectively. In addition, the tissue concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1) were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that in SAP rats, MDD significantly alleviated histopathological damage, depressed the MPO activity and the concentrations of TNF-α, IL-1β, and MCP-1 of pancreas, ileum and lung, and reduced the serum levels of amylase [(3283.4 ± 585.5) U·Lvs (5626.4 ± 795.1)U·L], DAO [(1100.1 ± 334.3) U·Lvs (1666.4 ± 525.3) U·L] and CRP [(7.6 ± 1.2) μg·mLvs (17.8 ± 3.8) μg·mL]. However, the serum SP-A concentration [(106.1 ± 16.6) pg·mLvs (90.1 ± 14.9) pg·mL] was elevated when treated SAP rats with MDD. Furthermore, MDD increased the occludin expression and reduced the NF-κB expression in pancreas, ileum and lung of SAP rats. Our findings suggested that MDD administration was an effective therapeutic approach for SAP treatment. It could up-regulate occludin expression to protect intercellular tight junction and down-regulate NF-κB expression to inhibit inflammatory reaction of pancreas, ileum and lung.
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OBJECTIVE: To study improvement effects of Fuling gancao decoction on functional dyspepsia (FD) model rats. METHODS: Sixty SD rats were randomly divided into normal group, model group, Fuling gancao decoction high-dose group (20 g/kg), Fuling gancao decoction low-dose group (10 g/kg), drug combination group (Fuling gancao decoction 10 g/kg+domperidone 0.004 g/kg), domperidone group (0.004 g/kg), with 10 rats in each group. Except for normal group, other groups were givenicy dilute hydrochloric acid intragastrically to establish FD model. After modeling, normal group and model group were given constant volume of distilled water at room temperature intragastrically, and other groups were given relevant drug solution (1 mL/100 g) intragastrically, once a day, for consecutive 7 d. The amount of gastric residue was measured to evaluate the gastric emptying function. Immunohistochemistry SP method was used to detect the protein expression of AQP3, Ghrelin and Substance P in gastric mucosa tissue. The protein expression of Claudin-1, Occludin and VIP were detected by Western blot assay. RESULTS: Compared with normal group, the amount of gastric residue was increased significantly in model group (P<0.01); positive expression of AQP3 and Ghrelin protein in gastric mucosa tissue was decreased significantly, while the positive expression of Substance P protein tended to increase; the protein expression levels of AQP3, Ghrelin, Claudin-1 and Occludin were decreased significantly, while those of Substance P and VIP were increased significantly (P<0.05 or P<0.01). Compared with model group, the amount of gastric residue was decreased significantly in administration groups (P<0.05 or P<0.01); the positive expression of AQP3 and Ghrelin protein tended to increase, while less positive expression of Substance P was found; the protein expression levels of AQP3 and Occludin in gastric mucosa tissue were increased significantly in administration groups, while those of VIP were decreased significantly (P<0.05 or P<0.01); the protein expression levels of Ghrelin and Claudin-1 in gastric mucosa tissue were increased significantly in Fuling gancao decoction high-dose group, Fuling gancao decoction low-dose group and drug combination group, while those of Substance P were decreased significantly (P<0.05). CONCLUSIONS: Fuling gancao decoction may improve the symptom of fluid retention in FD model rats by up-regulating the protein expression of AQP3, Ghrelin, Claudin-1 and Occludin and down-regulating the protein expression of Substance P and VIP in gastric mucosa tissue of rats.
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OBJECTIVE: To observe the influence of scalp-acupuncture on the expression of pentraxin 3 (PTX3), Interleukin (IL)-1β, zonula occludens-1(ZO-1) mRNA and Occludin mRNA in striatum in acute ischemic cerebrovascular disease (AICD) rats, so as to investigate its mechanisms underlying improvement of AICD. METHODS: Forty-eight male SD rats were randomly allocated to control, model, IL-1Ra and IL-1Ra+scalp-acupuncture groups (n=12 rats in each group). The AICD model was established by occlusion of the middle cerebral artery (MCAO). Rats of the IL-1Ra group and IL-1Ra+scalp-acupuncture group received intraperitoneal injection of IL-1Ra (0.05 mg·kg-1·d-1), once daily for 6 days. Scalp acupuncture stimulation was applied to bilateral "Dingnieqianxiexian" (MS6) once daily for 6 days for rats in IL-1Ra+scalp-acupuncture group. Before and after intervention, the neurologic deficit score (NDS) was evalua-ted according to Longa's method. The expression of striatum PTX3 and IL-1β was detected by immunohistochemistry, and ZO-1 mRNA and Occludin mRNA in the striatum tissue were detected by fluorescence quantitative real-time PCR. The Evans Blue (EB) tracer method was used to monitor the degree of blood-brain barrier (BBB) damage. RESULTS: Following modeling, the NDS, EB content and the expression of PTX3 and IL-1β in the striatum tissue were significantly increased, and the ZO-1 mRNA and Occludin mRNA expression was considerably decreased in the model group compared with the control group (P0.05). The effects of scalp acupuncture combined with IL-1Ra were obviously superior to that of IL-1Ra in down-regulating NDS, EB content and IL-1β expression level, and in up-regulating PTX3, ZO-1 mRNA and Occludin mRNA expression levels (P<0.05). CONCLUSION: Scalp acupuncture can improve neurological function and reduce the degree of BBB injury in AICD rats, which may be associated with its function in up-regulating the expression of PTX3 and in promoting the expression of ZO-1 mRNA and Occludin mRNA.
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[Objective] To observe the dynamic changes of intestinal IL-17,occludin,and ZO-1 in mice with postinfectious irritable bowel syndrome (PI-IBS).[Methods] Forty C57BL/6 mice were randomly divided into 5 groups:control group and infection groups (2 weeks,4 weeks,6 weeks,and 8 weeks after trichinella infection).Infection groups were given by gavaging of 400~500 Trichinella spiralis in 0.2 mL of normal saline.The body weight of mice were recorded at week 2,4,6,and 8 after infection.The visceral sensitivity of mice was measured by the abdominal withdrawal reflex (AWR).The stool was collected continuously for 8 hours to calculate the percentage of fecal water content.Pathological changes of gut were observed by HE staining.The expressions of IL-17,occludin,and ZO-1 in ileocecus and colon were detected by immunohistochemistry and Western blotting.[Results] At week 2 after infection,the acute inflammation of the intestinal tract was observed and the body weight of mice were significantly decreased (P=0.000).Until week 8 after infection,the intestinal inflammation and body weight of mice recovered to normal.When the colorectal dilatation capacity was 0.35 and 0.5 mL,the AWR scores in the infection groups were significantly higher than those in the control group (P<0.01).The percentages of fecal water content in the infection groups were significantly higher than those in the control group (P<0.05).Compared with the control group,the expressions of IL-17 were significantly decreased in week 2 group (P<0.05) and increased in week 8 group (P<0.05).The expressions of occludin and ZO-1 in the infection groups were significantly lower than those in the control group (P<0.05).[Conclusion] The dynamic changes of IL-17 and the decrease of Tight junction proteins may be one of the mechanisms of visceral hypersensitivity and increased percentages of fecal water content.They may be involved in the development of PI-IBS.
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Background:In the pathogenesis of chronic gastritis,the changes of biological factors can affect the physiological barrier of gastric mucosa,increase the risk of injury of intercellular junctions,leading to disruption of mucosal barrier. Aims:To investigate the expressions of occludin and zonula occluden-1 (ZO-1)in chronic gastritis and their significance. Methods:A total of 82 patients with chronic gastritis from October 2014 to October 2017 at the First People's Hospital of Xiaochang were enrolled and divided into chronic non-atrophic gastritis (CNAG)group and chronic atrophic gastritis (CAG)group according to the endoscopic biopsy pathology. Forty healthy subjects were served as normal controls. Expressions of occludin and ZO-1 were examined by immunohistochemistry. Patients positive for Helicobacter pylori (Hp) infection received eradication therapy,and expressions of occludin and ZO-1 were reexamined after Hp eradication. Results:The positivity rates of occludin and ZO-1 proteins in CNAG and CAG groups were significantly lower than those in normal control group (P<0.05). The positivity rates of occludin and ZO-1 proteins in CNAG group were significantly higher than those in CAG group (P<0.05). The positivity rates of occludin and ZO-1 proteins in Hp-positive CNAG patients were significantly lower than those in Hp-negative CNAG patients (P<0.05). The positivity rate of ZO-1 protein in Hp-positive CAG patients was significantly lower than that in Hp-negative CAG patients (P<0.05). Expressions of occludin and ZO-1 proteins showed a linear trend correlation with activity of gastric inflammation (P <0.05);their expressions decreased significantly with the increase in activity of inflammation. The positivity rates of occludin and ZO-1 proteins in CNAG patients increased significantly after Hp eradication (P<0.05). Conclusions:Expressions of occludin and ZO-1 are decreased in chronic gastritis,especially in CAG,and are correlated with Hp infection and activity of gastric inflammation.
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AIM:To investigate the role of microRNA-21(miR-21)in regulation of tight junction(TJ)-asso-ciated protein occludin in human normal colon mucosal epithelial cell line NCM 460, and to analyze related target genes. METHODS:Using miR-21 over-expression lentivirus, the NCM460 cells with miR-21 overexpression were established. The expression level of miR-21 was detected by qPCR.The expression of occludin was determined by Western blot.The target genes of miR-21 were predict by bioinformatic method.According to the scores and the appropriate literature search, a target gene was selected for further study.The miR-21 mimic and inhibitor were transfected into NCM 460 cells,and the expression levels of miR-21 and ROCK1 were measured.Dual-luciferase reporter assay was also performed to verify ROCK1 as the target gene of miR-21.RESULTS:In miR-21-overexpressing NCM460 cells,the protein expression level of occlu-din was upregulated.Bioinformatics and literature analysis predicted that the target gene of miR-21 was ROCK1.The mR-NA and protein levels of ROCK1 were down-regulated in the NCM460 cells transfected with miR-21 mimic.Accordingly, the mRNA and protein levels of ROCK1 were up-regulated in the NCM460 cells transfected with miR-21 inhibitor.Lucifer-ase assay confirmed that miR-21-binding sequence was on the 3'UTR of ROCK1,indicating that ROCK1 is the target gene of miR-21.CONCLUSION: In NCM460 cells, miR-21 up-regulates the expression of occludin, which functions in the maintenance of intestinal epithelial mechanical barrier.ROCK1 is a target gene of miR-21,and is involved in the miR-21 regulation of occludin.