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.

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.

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.

Gegen Qinliantang Regulates MMP-9/p38 MARK Pathway to Repair Intestinal Mucosal Barrier Function in Mice with Ulcerative Colitis / 中国实验方剂学杂志

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^-1） and Gegen Qinliantang high， medium and low dose groups （2.84，1.42，0.71 g·kg^-1）. The UC murine model was established by 3% dextran sulfate sodium （DSS）. Gegen Qinliantang and sulfasalazine were intragastrically administered on the 8^th 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.

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.

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.

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

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

Effect of electroacupuncture on intestinal epithelial mucosal barrier function in rats with diarrhea-predominant irritable bowel syndrome / 针刺研究

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.

Modified Da-chai-hu Decoction regulates the expression of occludin and NF-κB to alleviate organ injury in severe acute pancreatitis rats / 中国天然药物

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.

Effect of Modified Sijunzi Tang on Protein and mRNA Expressions of Occludin, ZO-1 and Claudin-1 in Cerebral Ischemia Rats / 中国实验方剂学杂志

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.

Study on Improvement Effects of Fuling Gancao Decoction on Functional Dyspepsia Model Rats / 中国药房

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.

Effect of scalp acupuncture stimulation on expression of pentraxin 3 in striatum in acute ische-mic cerebrovascular disease rats / 针刺研究

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.

MicroRNA-21 up-regulates expression of occludin by targeting ROCK1 in NCM460 cells / 中国病理生理杂志

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.

Expressions and Significance of Occludin and Zonula Occluden-1 in Chronic Gastritis / 胃肠病学

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.

Dynamic Changes of IL-17, occludin, and ZO-1 in Mice with Post-infectious Irritable Bowel Syndrome / 中山大学学报(医学科学版)

[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.

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.

Role of TGF-β1 in Sertoli cells and tight junction / 中华男科学杂志

Objective@#To explore the role of TGF-β1 in the proliferation and apoptosis of Sertoli cells and its effect on the expressions of tight junction-related proteins and genes in rats.@*METHODS@#Rat Sertoli cells were isolated in vitro, primarily cultured, and divided into groups A (blank control), B (TGF-β1 receptor blocker), C (TGF-β1), and D (TGF-β1 + receptor blocker). The proliferation and apoptosis of the cells were detected by CCK-8 and flow cytometry, respectively. After establishment of the dual-chamber model for the primary culture of Sertoli cells, the trans-epithelia electrical resistance (TER) value was measured and the relative expressions of Occludin, ZO-1 and Claudin Ⅱ determined by RT-PCR and Western blot.@*RESULTS@#The OD value of the proliferation of the Sertoli cells was markedly higher in group C than in groups A and D (0.79 ± 0.04 vs 0.66 ± 0.05 and 0.68 ± 0.02, P0.05). The TER value was dramatically decreased in group C as compared with groups A and D (［176.37 ± 16.61］ vs ［281.42 ± 9.83］ and ［254.37 ± 13.55］ /cm2, P0.05) or their protein expressions (F = 0.28 and 1.31, P>0.05). Both the mRNA and protein expressions of Occludin were markedly lower in group C than in A and D (P<0.01 and P<0.05), with statistically significant differences among the four groups (F = 6.86 and 6.87, P<0.01).@*CONCLUSIONS@#TGF-β1 can promote the proliferation of Sertoli cells in rats and act on the tight junction of the cells by regulating the expression of Occludin.

Protective effect of Zhenrenyangzang decoction on intestinal mucosal bar-rier function in ulcerative colitis rats / 中国病理生理杂志

AIM:To investigate the protective effect of Zhenrenyangzang decoction (ZRYZ) on the intestinal mucosal barrier function and the expression of tight junction proteins,zonula occludens-1 (ZO-1) and occludin,in the co-lon of rats with trinitrobenzenesulfonic acid (TNBS)-induced ulcerative colitis in order to explore the possible mechanism involved. METHODS:Male Wistar rats were randomly divided into normal group, model group, salazosulfapyridine (SASP) positive control group,high-dose ZRYZ(ZRYZ-H) group and low-dose ZRYZ(ZRYZ-L) group. Except the nor-mal group,the rats in other groups were given the TNBS/50% ethanol mixed solution by enema to make the ulcerative coli-tis model. The rats in positive control group were given SASP suspension liquid. The rats in ZRYZ-H group and ZRYZ-L group were orally administered with ZRYZ at doses of 30.4 g/kg and 15.2 g/kg, respectively, while the rats in normal group and model group were orally administered with saline. All the drugs were administered to the rats for consecutive 21 d. Disease activity index (DAI) was investigated during the drug treatment, and colon samples were collected after drug administration for evaluating morphological damage score. Intestinal mucosal permeability was measured by detection of lac-tulose/mannitol (L/M) in urine. Colonic myeloperoxidase(MPO) activity and goblet cell number,serum diamine oxidase (DAO) and D-lactic acid (D-LA) levels, and the colonic expression of ZO-1 and occludin were also measured. RE-SULTS:Compared with normal group,DAI,morphological damage score,L/M value,colonic MPO activity as well as se-rum D-LA and DAO levels in model group significantly increased(P<0.05),while goblet cell number as well as the ex-pression levels of ZO-1 and occludin in model group were significantly reduced(P<0.05). Compared with model group, DAI,morphological damage score,L/M value,colonic MPO activity as well as serum DAO and D-LA levels in ZRYZ-H group and ZRYZ-L group were reduced significantly(P<0.05),while goblet cell number as well as the expression of ZO-1 and occludin in ZRYZ-H group and ZRYZ-L group increased significantly(P<0.05). CONCLUSION:ZRYZ protects the intestinal mucosal barrier function in ulcerative colitis rats by reducing intestinal mucosal permeability,and its mecha-nisms may be related to increasing the expression of ZO-1 and occludin.

Effects of miR-122a on blood-spinal cordbarrier after spinal cord ischemia-reperfusion injury in rats / 中国药理学通报

Aim To investigate the effects of miR-122a on blood-spinal cord barrier after spinal cord ischemia-reperfusion injury in rats.Methods Thirty-six SD rats were randomly divided into three groups:group of sham(S group),group of control(C group)and group of miR-122a antagomir(M group).Rats in S group were subjected to exposure of aorta arch but without occlusion.Spinal ischemia-reperfusion injury was induced by clamping the aorta arch for 14 min in C group and M group.Rats in M group and C group were intrathecally injected with miR-122a antagomir or antagomir control daily for three times after injury.The miR-122a expression in injured spinal cord tissue was detected by real-time PCR.The occludin expression in injured spinal cord tissue was detected by Western blot.The permeability of blood-spinal cord barrier was examined using evans blue as a vascular tracer.The neurological motor function was evaluated by Basso Beattie Bresnahan score.Results Compared with S group,the expression of miR-122a was increased,the expression of occludin was decreased,the permeability of blood-spinal cord barrier was increased,and neurological motor function score was decreased significantly in C group(P<0.05).Compared with C group,the expression of miR-122a was decreased,the expression of occludin was increased,the permeability of blood-spinal cord barrier was decreased,and neurological motor function score was increased significantly in M group(P<0.05).Conclusion miR-122a can regulate the expression of occludin and change the permeability of blood-spinal cord barrier.

Inhibition Effect of Zoledronic Acid on Metastasis of Esophagus Squamous Cell Carcinoma / 医药导报

Objective To examine the inhibition effect of zoledronic acid (ZOL) on malignant metastasis of human esophagus squamous cell carcinoma (ESCC) cells and to analyze its molecular mechanisms.Methods EC9706 and EC109 cells were treated with ZOL,and then MTT assay,adhesion and invasion assay were performed to observe the inhibitory effect of As2O3 on proliferation and metastasis of esophagus carcinoma cells.The expression of metastasis-related proteins was detected by Western blotting.Results Exposure to ZOL significantly presented suppressive functions on growth and metastasis of both kinds of cancer cells,in a dose-dependent manner(P＜ 0.05).Additionally,the expression level of occludin was increased after ZOL treatment by suppressing transcriptional factor Slug.Transfection of Slug could reverse anti-metastasis of ZOL.Conclusion ZOL possesses a significant anti-metastasis function on ESCC cells,mainly through repressing Slug to restore occludin expression.