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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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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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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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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
The T(EB4)Nta, T(IBj5)Nta, and T(B362i)NtA strains were constructed by introgressing the insertionaltranslocations EB4, IBj5, and B362i from Neurospora crassa into the related species N. tetrasperma. Theprogeny from crosses of T(IBj5)Nta and T(B362i)NtA with opposite mating-type derivatives of the standard N.tetrasperma strain 85 exhibited a unique and unprecedented transmission ratio distortion (TRD) that disfavoredhomokaryons produced following alternate segregation relative to those produced following adjacent-1 segregation. The TRD was not evident among the [mat A ? mat a] dikaryons produced following either segregation. Further, crosses of the T(IBj5)Nta and T(B362i)NtA strains with the Eight spore (E) mutant showed anunusual ascus phenotype called ‘max-4’. We propose that the TRD and the max-4 phenotype are manifestations of the same Bateson-Dobzhansky-Muller incompatibility (BDMI). Since the TRD selects against 2/3 ofthe homokaryotic progeny from each introgression cross, the BDMI would have enriched for the dikaryoticprogeny in the viable ascospores, and thus, paradoxically, facilitated the introgressions.
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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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Objective@#To investigate oxidative stress-mediated damage to the epididymal epithelial tight junction protein ZO-1 and its impact on epididymal function in varicocele rats.@*METHODS@#We randomly divided 45 male adolescent SD rats into three groups of equal number: sham operation (left renal vein exposed and isolated), experimental (left renal vein constricted and collaterals of the left spermatic vein fully ligated), and treatment (60-day intragastric administration of vitamin E at 150 mg/kg/d after modeling). At 60 days after modeling, we observed the histological changes in the left epididymis, detected the expressions of ZO-1 and other tight junction-related proteins by real-time quantitative PCR, immunohistochemistry, immunofluorescence staining and Western blotting, determined sperm motility, and measured the levels of superoxide dismutase (SOD), total antioxidant capacity (T-AOC), methylene dioxyamphetamine (MDA) and α-glucosidase (α-Glu) in the epididymal tissue of the rats.@*RESULTS@#Compared with the rats of the sham operation group, those of the experimental group showed disorganized epithelial structure and decreased number of epithelial cells in the left epididymis, with some epithelial cells desquamated into the lumen. The expression of ZO-1 was significantly lower in the experimental than in the sham operation group (P < 0.05) but markedly upregulated after VE treatment (P < 0.05). In comparison with the sham operation group, the animals in the experimental group exhibited remarkably increased content of MDA in the epididymal tissue (［0.41 ± 0.05］ vs ［1.21 ± 0.18］ nmol/mg prot, P < 0.05) but decreased levels of SOD (［814.65 ± 73.64］ vs ［298.62 ± 67.84］ U/mg prot, P < 0.05), T-AOC (［0.84 ± 0.07］ vs ［0.24 ± 0.04］ nmol/mg prot, P < 0.05) and α-Glu (［11.72 ± 2.72］ vs ［5.82 ± 1.24］ U/mg prot, P < 0.05). VE treatment, however, remarkably reduced the content of MDA (［0.69 ± 0.12］ nmol/mg prot) and elevated the levels of SOD (［497.73 ± 48.03］ U/mg prot), T-AOC (［0.42 ± 0.06］ nmol/mg prot) and α-Glu (［9.11 ± 1.91］ U/mg prot) as compared with those in the experimental group (all P < 0.05). The percentage of progressively motile sperm was significantly lower in the experimental than in the sham operation group (［31.33 ± 6.32］% vs ［71.21 ± 5.21］%, P < 0.05), but markedly increased after VE treatment (［60.68 ± 5.31］%, P < 0.05).@*CONCLUSIONS@#Varicocele reduces the expression of the EETJ protein ZO-1 and impairs epididymal function via oxidative stress, while vitamin E can effectively upregulate the ZO-1 expression and improve epididymal function by decreasing oxidative stress in the epididymis of varicocele rats.
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Purpose To clarify the effect of adenosine on brain metastasis of lung cancer and the possible mechanism of adenosine promoting brain metastasis of lung cancer. Methods Western blot was used to dynamically detect the expression level of hypoxia inducible factor-1 (HIF-1) in lung cancer cells and tight junction protein ZO-1 in brain microvascular endothelial cells on blood-brain barrier. The content of adenosine in lung cancer cell culture was determined by ELISA. Fluorescence analysis was used to detect the changes of permeability of the blood-brain barrier model in vitro. Hemocytometer counts the number of A549 lung cancer cells in Transwell's lower chamber. Results The expression level of HIF-1 in lung cancer cells and the content of adenosine in lung cancer cell culture reached the highest level when lung cancer cells were deprived of oxygen for 12 hours. At the same time, the expression level of ZO-1 protein in the blood-brain barrier was the lowest, the blood-brain barrier permeability was the highest (7.11), and the number of lung cancer cells passing through the blood-brain barrier model was the highest (84.6). The permeability of the blood-brain barrier model increased after the action of adenosine, and its change trend was consistent with the effect of hypoxic lung cancer cell culture solution. Conclusion Hypoxia can induce the lung cancer cell to release adenosine, the increased adenosine can reduce the expression of tight junction protein ZO-1 in blood brain barrier, which leads to the increase of permeability of blood-brain barrier and eventually lead to brain metastasis of lung cancer.
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Objective To explore the effect of astragaloside IV on the damaged blood-brain barrier (BBB) in vitro model induced by amyloid-beta protein (Aβ1-42) and the underlying mechanism. Methods Firstly, the non-contact co-culture blood-brain barrier (BBB) model was established by Aβ1-42-induced mouse brain microvascular endothelial cell (bEnd.3) and primary rat astrocyte (As). Then the mice were divided into four groups: control, model (Aβ1-42 30 μmol/L), astragaloside IV low and high dose groups (Aβ1-42 30 μmol/L with Astragaloside IV 50 and 200 μmol/L). The effect of astragaloside IV on the vitality of bEnd.3 induced by Aβ1-42 was detected by methyl thiazolyl tetrazolium (MTT) assay. The permeability of BBB in vitro was determined by detecting the quantity of fluorescein sodium through BBB in various groups. In order to explore the mechanism of its protection, the apoptosis related proteins Caspase-3, cleaved Caspase-3 and tight junction proteins ZO-1, Claudin-5 and Occludin were detected by Western blotting. Results Compared with model group, the astragaloside IV groups improved the activity of bEnd.3 cells significantly (P < 0.001). The protective effect was positively correlated with the concentration of astragaloside IV. Astragaloside IV with low and high dose decreased the permeability of BBB model in vitro (P < 0.001). According to the results of Western blotting, the ratio of cleaved Caspase-3/Caspase-3 was significantly declined, and the expression levels of ZO-1, Claudin-5 and Occludin were significantly increased in astragaloside IV groups (P < 0.05). Conclusion Astragaloside IV may play a BBB protective role by inhibiting the apoptosis of bEnd.3 cells induced by fibrous Aβ1-42 and increasing the expression of tight junction proteins.
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Objective To explore the effects of N-butylphthalide on the expressions of ZO-1 and claudin-5 in blood-brain barrier (BBB) in rats with acute carbon monoxide (CO) poisoning. Methods A total of 144 adult healthy male Sprague-Dawley (SD) rats were randomly divided into normal control group, CO poisoning group, and NBP treatment group, with 48 rats in each group. The acute CO poisoning model was reproduced in hyperbaric oxygen chamber, and all model rats were given hyperbaric oxygen therapy once daily. The rats in the normal control group were free to breathe fresh air. The rats in NBP treatment group were administered orally NBP 60 mg/kg twice a day at 2 hours after poisoning until death. The rats in normal control group and CO poisoning group were treated with equal amount of pure olive oil. Four rats were sacrificed from each group at 1, 3, 7, 14 days after model reproducing, respectively. The changes in ultrastructure of BBB were observed under transmission electron microscope. The expressions of ZO-1 and claudin-5 proteins were determined by immunofluorescence staining and Western Blot. The localization of the two target proteins was observed by immunofluorescence double staining. The correlation between the two proteins was analyzed by linear regression. Results The ultrastructure of BBB was normal in normal control group, some ZO-1 and a large number of claudin-5 positive cells were observed. The ultrastructure of BBB was seriously injured, ZO-1 and claudin-5 positive cells in brain tissue were significantly decreased, and the expressions of ZO-1 and claudin-5 proteins in brain tissue at 1 day after poisoning in CO poisoning group were significantly lower than those of normal control group (ZO-1 protein:3.38±0.30 vs. 24.50±5.62, claudin-5 protein: 11.38±0.93 vs. 46.35±6.88, both P < 0.05), and although gradually restored, they were maintained at relatively lower levels until 14 days as compared with those in normal control group (ZO-1 protein: 10.35±0.80 vs. 24.63±3.57, claudin-5 protein: 32.35±3.11 vs. 46.43±7.20, both P < 0.05). NBP treatment could significantly alleviate the ultrastructure injury of BBB induced by acute CO poisoning, the amount of ZO-1 and claudin-5 positive cells in brain tissue were significantly increased, as well as the expressions of ZO-1 and claudin-5 proteins were significantly increased, which were significantly higher than those of CO poisoning group from 1 day and 3 days on, respectively (1-day ZO-1 protein: 7.57±0.69 vs. 3.38±0.30, 3-day claudin-5 protein:20.46±1.42 vs. 11.43±0.86, both P < 0.05), and which showed an increase tendency with time prolongation. The results of immunofluorescence double staining showed that ZO-1 and claudin-5 proteins could not only coexist in the same cell, but also could be expressed separately in different cells. Linear regression analysis showed the positive correlation between the expressions of ZO-1 and claudin-5 proteins in brain tissue of rats with acute CO poisoning (R2= 0.917, P = 0.022). Conclusion NBP could markedly improve the ultrastructure and functional integrity of BBB through up-regulating the expressions of ZO-1 and claudin-5 proteins, and then reduce brain damage caused by CO poisoning.
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Objective To investigate the effects of bradykinin (BK) on the proliferation of rabbit corneal endothehal ceils (RCECs) and the expression of tight junction-related proteins zonula occludens-1 (ZO-1) and zonula occludens-1-associated nucleic-acid-binding protein (ZONAB),and to explore the underlying mechanisms of BK on cell proliferation in corneal endothelium.Methods RCECs at logarithmic growth phase were treated with different concentrations of BK (0.01,0.1,1,10 μmol · L-1) BK group,with the controls left untreated.Morphological changes of cells in each group were examined under phase-contrast microscope,and MTT assays were used to detect cell proliferation at 24 h,48 h,72 h,96 h after BK treatment.And,at 72 h,the expression levels of ZO-1 and ZONAB protein were determined by Western blot.Results After 72 h of treatment,the cells in each group were fused into pieces and closely linked into a monolayer;but after 96 h,the growth of the cells was restricted,with the intercellular space become larger and the cells exfoliated.Compared with the control group,BK induced a significant increase of absorbance value and cell viability,and the differences were statistically significant (all P ＜ 0.001),and the promoting effects showed a concentration-dependent manner,and 1 μmol · L-1 BK demonstrated the strongest regulative effect (P ＜ 0.001).Western blot results showed that BK upregulated the expression of ZO-1 and ZONAB protein in a concentration-dependent manner.Conclusion BK can stimulate the proliferation of RCECs in a time-and concentration-dependent manner,and the mechanisms are probably associated with ZO-1/ZONAB-mediated signaling pathway.
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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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OBJECTIVE To investigate the expression of zonula occluden-1(ZO-1) in hypopharyngeal squamous cell carcinoma(HSCC) and its clinical significance.METHODS Immunohistochemistry with tissue microarrays was used to detect the expression of the ZO-1 protein in 52 cases of HSCC.The correlation of prognosis and clinicopathological features was analyzed.RESULTS The expression of ZO-1 protein in HSCC was lower than that in adjacent tissues,its expression was closely related with tumor differentiation,tumor stage,tumor location and lymphatic metastasis.The Log-rank univariate analysis showed that the prognosis of HSCC was related to tumor differentiation,tumor stage,lymphatic metastasis and tumor location.The patients with lower expression of ZO-1 showed the worse prognosis than patients with higher expression of ZO-1.As shown in the Cox regression analyses,tumor differentiation,lymphatic metastasis,the ZO-1 expression and tumor stage were all independent prognostic factors.CONCLUSION The ZO-1 can serve as an independent prognostic factor of HSCC,and the low expression of ZO-1 is closely related to poor prognosis.
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Objective To investigate the alterations of tight junction proteins in duodenal mucosa from 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease(PD) model. Methods Rats were subjected with 6-OHDA in bilateral substantia nigra (SN) as PD model. The location and quantitative detecting techniques inclu-ding immunohisto- chemistry and western blotting were used to determine the expressions and alterations of tight junction proteins (claudin-1, occludin, ZO-1). Results Claudin-1, occludin and ZO-1 were highly expressed in the duodenal mucosa of PD models, where the expressions of occluding (P<0.001) and ZO-1 (P<0.001) were much lower, while the expression of claudin-1 had no alteration. Conclusions The down-regulated expres-sion of occludin and ZO-1 was detected in duodenal mucosa of PD models, which might be related to duodenal ulcer in PD.
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Objective To investigate the alterations of tight junction proteins in duodenal mucosa from 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease(PD) model. Methods Rats were subjected with 6-OHDA in bilateral substantia nigra (SN) as PD model. The location and quantitative detecting techniques inclu-ding immunohisto- chemistry and western blotting were used to determine the expressions and alterations of tight junction proteins (claudin-1, occludin, ZO-1). Results Claudin-1, occludin and ZO-1 were highly expressed in the duodenal mucosa of PD models, where the expressions of occluding (P<0.001) and ZO-1 (P<0.001) were much lower, while the expression of claudin-1 had no alteration. Conclusions The down-regulated expres-sion of occludin and ZO-1 was detected in duodenal mucosa of PD models, which might be related to duodenal ulcer in PD.
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OBJECTIVE To establish an in vitro blood-cerebrospinal fluid barrier (BCB) model to investigate the underlying mechanism of lead-induced BCB injuries.METHODS The in vitro BCB model was established by Z310 cells.Different concentrations of Pb(AC)2 (2.5,5.0 and 10.0 mmol·L-1) were used for 24,48 and 72 h.Transendothelial electrical resistance (TEER) and flux of FITC-dextran were performed to determine the permeability of the in vitro BCB model.Western blotting and immunofluorescence methods were used to observe the expression of tight junction protein ZO-1 and occludin.RESULTS Compared with control group,Pb(AC)2 2.5,5.0 and 10.0 mmol· L-1 exposure for 48 h to Z310 cells had no significant effect on survival rate and density.TEER in different groups was gradually increasing.At the 12th day after Pb(AC)2 exposure,the values of TEER and flux of FITC-dextran in Pb(AC)2 5 and 10 mmol· L-1 groups were significantly decreased (P＜0.05).Western blotting and immunofluorescence images showed that the expression of ZO-1 and occludin were significantly decreased (P＜0.05) after Pb(AC)2 exposure for 48 h.CONCLUSION Lead exposure can cause the breakdown of BCB barriers,and this effect may be mediated by reducing the expression of ZO-1 and occludin proteins.
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Objective To study the impact of tumor necrosis factor-α (TNF-α) and its antagonist on the expressions of intestinal mucosa claudin-1,Zonula Occludens-1 (ZO-1) and myosin light chain kinase (MLCK) in rat models of acute liver failure.Methods Fifty four healthy male SpragueDawley (SD) rats were randomly divided into normal control group,model group and intervention group according to a random number table.Rats in normal control (n=6) group were intraperitoneally injected with 0.9％ saline (12 mL/kg).Rats in model group (n=24) and intervention group (n=24) were intraperitoneally injected with a full dose of D-galactosamine (D-GalN) at a dose of 1 200 mg/kg to establish model of acute liver failure,while rats in intervention group were intraperitoneally injected with TNF-α antagonists (rhTNFR∶Fc) at a dose of 12.5 mg/kg before 24 hours given D-GalN.At each time point of hour 8,24,48 and 72,six rats in both model group and the intervention group were sacrificed,respectively,while the normal control group were all anesthetized and sacrificed at 72 h.Models were repeated five times.Serum liver function was detected by biochemical method,and serum TNF-α level was detected by enzyme-linked immunosorbent assay (ELISA).Hematoxylin-eosin (HE) stained sections of liver and terminal ileum were examined under an optical microscope for pathological changes;and protein expression of the terminal ileum Claudin-1,ZO-1 protein and MLCK were determined by immunohistochemistry and Western blot.Means among groups were compared with t test.Results Acute liver failure was successfully induced in the D-GalN injected rats.In the model group,alanine aminotransferase (ALT) began to decline,total bilirubin continued to rise,and enzyme-jaundice separation developed at hour 72.But total bilirubin in intervention group at hour 72 was decreased.Light microscope showed that at hour 72,villus lodged at terminal ileum in the model group with part of villus tip failing off in the model group.Villus mucosa and submucosa interstitial were edema and infiltrated with numerous neutrophils.The terminal ileum kept integrate in the intervention group,and villus mucosa and submucosa were mild edema and only infiltrated with a small amount of neutrophil.Expressions of tumor necrosis necrosis factor (TNF)-α in rats of model group and intervention group were gradually increased and peaked at hour 24 ([239.83 ± 15.81] and [182.71± 17.08] ng/L,respectively),which were significantly higher than that of the control group ([24.19±3.57] ng/L,t=22.68and 15.73,respectively;both P＜0.01).Expression of serumTNF-α in the intervention group was significantly lower than that of model group (t=4.58,P＜0.01).Expressions of Claudin-1 and ZO-1 in model group decreased gradually at an early stage and reached the lowest level at hour 24 (0.355 ± 0.068 and 0.387 ± 0.091,respectively),which were both significantly lower than that of control group (1.640±0.188 and 1.015±0.150,respectively;t=12.87 and 7.14,respectively;both P＜0.01).In the intervention group,expressions of Claudin-1 and ZO-1 also decreased to the lowest level at hour 24 (1.051 ± 0.370 and 0.642 ± 0.082,respectivley),which were both significantly lower than that of control group (t =2.84 and 4.36,respectively;both P＜0.05),but significantly higher than model group with stastically difference (t =3.70 and 4.15,respectively;both P＜0.01).MLCK protein levels in the model and intervention group were gradually increased,which peaked at hour 24 (1.298±0.194 and 1.033 ± 0.073,respectively),significantly higher than the control group (0.460±0.069,t=8.16 and 11.44,both P＜0.01);and MLCK in the intervention group was lower than that in the model group with statistically difference (t=2.56,P＜0.05).Conclusions Expression of serum TNF-α in rat model of acute liver failure increases,which leads to decreased expression of Claudin-1 and ZO-1,and increased expression of MLCK,makes cell shrunk and cell gap increased.TNF-α antagonist could significantly reduce the inflammation and liver cell apoptosis,improve liver function by inhibiting MLCK expression and preventing decrease of Claudin-1 and ZO-1 proteins.
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Objective To establish and evaluate intestinal epithelial barrier model using Caco-2 cell so as to play a foundation for next study of barrier permeability.Methods Caco-2 cells were cultured in vitro then seeded into Transwell cell culture inserts.The permeability of the intestinal epithelial barrier was detected by transepithelial electrical resistance(TEER)and lucifer yellow flux,and verified by transmission electron micro-scope.Different concentrations of PAF(0,50,100,and 200 nmol /L)were exposed for 24 hours to Caco-2 mono-layer when cultured 21 days.The tight junction was observed under transmission electron microscope.Assess-ment of ZO-1 protein localization and expression were detected by immunofluorescence and Western blot analy-sis.Results Cultured Caco-2 cell confluencd as monolayer with time passed.From 5th day,TEER increased, then reached 600Ω?cm2 at 15th day and lasted to 21 st day,there was little flux of lucifer yellow,transmission e-lectron microscopy also found cells differentiated better,had well-arranged villi and polarity alined as monolayer, forming completed tight junction which was the marker of intestinal epithelial barrier model in vitro.TEER de-creased and lucifer yellow flux increased in cells exposed to PAF.The permeability reached the peak when ex-posed to 100 nmol /L PAF(P <0.01 ),tight junction disrupted,ZO-1 protein expression downregulated,abnor-mal localization and distribution was assessed by immunofluorescence staining.Conclusion Cultured Caco-2 cells for 2-3w can be used to study intestinal epithelial barrier as a model in vitro.PAF increased intestinal epi-thelial permeability,which would correlate to the decreased protein expression and abnormal distribution of ZO-1.
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Objective To investigate the effects of traditional Chinese medicine colquhounia root tablet on the expression of tight junction protein claudin-2 and ZO-1 in bronchial epithelium tissues of rats with acute lung injury (ALI), and to study the mechanism of protective effect of colquhounia root tablet on ALI. Methods Twenty-four healthy male Sprague-Dawley (SD) rats were randomly divided into control group, ALI group and colquhounia root tablet pretreatment group, with 8 rats in each group. The model of ALI was reproduced by intravenous injection of oleic acid 0.04 mL/kg, and the rats in cont rol group were given the same amount of normal saline (NS) instead. The rats in colquhounia root tablet pretreatment group were intragastric administrated with colquhounia root tablet of 600 mg·kg-1·d-1 (2 mL) for 10 days before model reproduction, and the rats in control group and ALI group were given the same amount of NS. At 4 hours after model reproduction, the blood was drawn from abdominal aorta, and bronchoalveolar lavage fluid (BALF) was collected for determination of protein content in plasma and BALF, and the lung permeability index (LPI) was calculated. The rats were sacrificed to collect lung tissues for determination of lung wet/dry weight ratio (W/D), the changes in pathology of lung tissue were observed after hematoxylin and eosin (HE) staining with light microscope, and lung injury score (LIS) was evaluated. The immunohistochemic al staining was used to detect the expression and localization of claudin-2 and ZO-1 in bronchial epithelium tissues. The protein expressions of claudin-2 and ZO-1 in bronchial epithelium tissues were determined by Western Blot. Results Compared with control group, the lung injury in ALI group was more obvious including cellular edema and structural disorder of intercellular connection by optical microscope, and LIS, W/D ratio, and LPI were significantly increased (LIS: 3.81±0.42 vs. 0.40±0.08, W/D: 7.68±0.64 vs. 4.44±0.39, LPI: 0.89±0.15 vs. 0.38±0.05, all P < 0.01). Claudin-2 and ZO-1 were mainly expressed in the bronchial epithelium cell, and the expression degrees were significantly weakened in ALI group as compared with control group. It was shown by Western Blot results that compared with control group, the protein expressions of claudin-2 and ZO-1 were significantly down-regulated in ALI group [claudin-2 protein (gray value): 0.43±0.31 vs. 2.16±1.33, ZO-1 protein (gray value): 1.25±0.41 vs. 2.82±0.76, both P < 0.01]. Compared with ALI group, colquhounia root pretreatment could effectively diminish the degree of ALI (LIS: 1.22±0.39 vs. 3.81±0.42, W/D: 4.62±0.84 vs. 7.68±0.64, LPI: 0.46±0.07 vs. 0.89±0.15, all P < 0.01), and the protein expressions of claudin-2 and ZO-1 were significantly up-regulated [claudin-2 protein (gray value): 2.98±0.91 vs. 0.43±0.31, ZO-1 protein (gray value): 2.35±0.51 vs. 1.25±0.41, both P < 0.01]. Conclusion Administration of colquhounia root table could attenuate lung injury induced by oleic acid with improving epithelial barrier function via up-regulate the expression claudin-2 and ZO-1, which play a protective effect on the lung of rats with ALI.
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Purpose Investigating the significance of ZO-1 different domains in the invasion and metastasis of gastric carcinoma (GC).Methods A tissue microarray that simulates the invasion and metastasis process of GC was created,and immunohistochemistry was performed to detect the expression of ZO-1 (α-pan),ZO-1 (α +) and ZO-1 (ZU5).Results The GC cell exhibited aberrant expression of ZO-1 (α-pan),ZO-1 (α +) and ZO-1 (ZU5) from membrane translocated to cytoplasm or no expression.The aberrant degree was increased with the invasion,however,was decreased in metastatic lymph node.The aberrant expression was associated with histological types.Conclusion The aberrant expression of ZO-1 (α-pan),ZO-1 (α +) and ZO-1 (ZU5),from membrane translocated to cytoplasm or no expression suggest that domains of PDZ3,GUK,SH3,ZU5 and alpha motif in ZO-1 might be involved in the invasion and metastasis of GC and maintaining of GC phenotype.The aberrant expression of these domains may be the one mechanism of ZO-1 involved in EMT or MET.