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The blood brain barrier (BBB) is a physical and metabolic barrier that maintains central nervous system homeostasis and protects brain tissues from potentially hazardous circulating substances. This article reviews the biological characteristics of caludin-5 during cerebral ischemia, its role in BBB integrity and permeability, as well as the research progress of related drug therapy based on calludin-5.
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Objective:To study the signaling pathway of the up-regulation of claudin-5 expression by Xuebijing injection.Methods:Animal and cell models of acute respiratory distress syndrome (ARDS) were induced by lipopolysaccharide (LPS). ① In vivo study, 20 male Sprague-Dawley (SD) rats were randomly divided into 4 groups: control group, LPS group (LPS injection 10 mg/kg for 12 hours), Xuebijing control group (Xuebijing injection 1 mg/kg, twice a day, for 3 days), and Xuebijing intervention group (LPS injection after pretreatment of Xuebijing injection), according to random number method with 5 rats in each group. The lung tissues were taken to detect lung dry/wet weight ratio (W/D) and the morphological changes in each group. Claudin-5, phosphorylated forkhead box transcription factor O1 (p-FOXO1), total FOXO1 (t-FOXO1), phosphorylated Akt (p-Akt) and total Akt (t-Akt) in lung tissues were detected by immunohistochemical staining (IHC) and Western blotting. ② In vitro study, human pulmonary microvascular endothelial cells (HPMECs) were divided into 6 groups (5 holes in each group): control group, Xubijing control group (incubated with 2 g/L Xubijing for 24 hours), phosphoinositide 3-kinases (PI3K) signaling pathway LY294002 control group (incubated with 10 μmol/L LY294002 for 1 hour), LPS group (incubated with 1 mg/L LPS for 12 hours), Xubijing intervention group (incubated with 2 g/L Xuebijing for 24 hours, then with 1 mg/L LPS for 12 hours) and LY294002 intervention group (incubated with 10 μmol/L LY294002 for 1 hour, then with 2 g/L and Xubijing for 24 hours, and then with 1 mg/L LPS for 12 hours). The expression levels of claudin-5, p-FOXO1, t-FOXO1, p-Akt and t-Akt of HPMECs in each group were assessed by Western blotting. Results:In vivo study: ① Compared with the control group, the lung W/D ratio increased significantly in LPS group (6.79±0.42 vs. 4.19±0.13), and decreased significantly after the intervention of Xuebijing (4.92±0.38 vs. 6.79±0.42, P < 0.01). ② Morphological changes of lung tissue: compared with the control group, the injury of lung tissue in LPS group was more serious, which was significantly improved after Xuebijing intervention. ③ Expression levels of claudin-5, p-Akt/t-Akt and p-FOXO1/t-FOXO1: the expression levels of claudin-5, p-Akt/t-Akt and p-FOXO1/t-FOXO1 in LPS group were significantly decreased as compared with the control group (claudin-5/GAPDH: 0.33±0.03 vs. 1.03±0.07, p-Akt/t-Akt: 0.18±0.02 vs. 1.01±0.13, p-FOXO1/t-FOXO1: 0.16±0.06 vs. 1.00±0.19, all P < 0.01). After the intervention of Xuebijing, the expression levels were significantly increased as compared with the LPS group (claudin-5/GAPDH: 0.53±0.05 vs. 0.33±0.03, p-Akt/t-Akt: 0.56±0.12 vs. 0.18±0.02, p-FOXO1/t-FOXO1: 0.68±0.10 vs. 0.16±0.06, all P < 0.01). In vitro study: compared with the control group, the expression level of claudin-5 in the LPS group was significantly decreased (claudin-5/β-actin: 0.45±0.03 vs. 1.01±0.15, P < 0.01), and the expression level of claudin-5 in Xuebijing intervention group was also significantly decreased (claudin-5/β-actin: 0.80±0.08 vs. 1.01±0.15, P < 0.01). After the intervention of LY294002, the expression of claudin-5 was significantly decreased as compared with the Xubijing intervention group (claudin-5/β-actin: 0.41±0.02 vs. 0.80±0.08, P < 0.01). Conclusion:Xuebijing injection improve pulmonary vascular barrier function in rats with ARDS by up-regulating claudin-5 expression through PI3K/Akt/FOXO1 signaling pathway.
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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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OBJECTIVE@#To investigate the protective effects and underlying mechanisms of Xuebijing Injection (XBJ) on the lung endothelial barrier in hydrogen sulfide (H@*METHODS@#Sprague-Dawley rats were exposed to H@*RESULTS@#The morphological investigation showed that XBJ attenuated H@*CONCLUSIONS@#XBJ ameliorated H
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Animals , Rats , Claudin-5 , Drugs, Chinese Herbal , Endothelial Cells , Hydrogen Sulfide , Phosphatidylinositol 3-Kinases , Rats, Sprague-Dawley , Respiratory Distress Syndrome, Newborn/drug therapyABSTRACT
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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Background@#The increased permeability of the blood-brain barrier (BBB) induced by ischemia/hypoxia is generally correlated with alteration of tight junctions (TJs). DL-3-n-butylphthalide (NBP) has been shown to exert neuroprotective effects after ischemic injury. However, few studies have assessed the correlation between NBP and TJs. This study aimed to investigate the potential effect of NBP on the TJ proteins claudin-5, zonula occludens-1 (ZO-1), and occludin during brain ischemia.@*Methods@#A chronic cerebral hypoperfusion (CCH) Sprague-Dawley rat model was established, and NBP (20, 40, or 80 mg/kg, gavage, once a day) treatment was performed for 14 days. NBP (0.1 or 1.0 μmol/L) pre-treatment was applied to an in vitro hypoxia microvascular endothelial cell model (1% O2, 24 h). BBB permeability was assessed by performing the Evans blue assay. The expressions and localization of claudin-5, ZO-1, occludin, phosphorylated/total protein kinase B (p-Akt/Akt), phosphorylated/total glycogen synthase kinase 3β (GSK-3β)/GSK-3β, and β-catenin/β-actin were evaluated by Western blotting or immunofluorescence. Reactive oxygen species (ROS) generation was measured by flow cytometry analysis. TJ ultrastructure was observed by transmission electron microscopy.@*Results@#In CCH rats, treatment with 40 and 80 mg/kg NBP decreased the Evans blue content in brain tissue (9.0 ± 0.9 μg/g vs. 12.3 ± 1.9 μg/g, P = 0.005; 6.7 ± 0.6 μg/g vs. 12.3 ± 1.9 μg/g, P < 0.01), increased the expression of claudin-5 (0.79 ± 0.08 vs. 0.41 ± 0.06, P < 0.01; 0.97 ± 0.07 vs. 0.41 ± 0.06, P < 0.01), and elevated the ZO-1 protein level (P < 0.05) in brain microvascular segments in a dose-dependent manner in comparison with the corresponding values in the model group. There was no significant difference in occludin expression (P > 0.05). In the hypoxia cell model, NBP pre-treatment improved TJ ultrastructure, decreased intracellular ROS level, and increased the expression of claudin-5 (P < 0.01) and ZO-1 (P < 0.01) in comparison with the corresponding values in the hypoxia group. NBP treatment also elevated the relative expression levels of p-Akt/Akt, p-GSK-3β/GSK-3β, and β-catenin/β-actin in comparison with the corresponding values in the hypoxia group (all P < 0.05).@*Conclusion@#NBP improves the barrier function of BBB against ischemic injury by upregulating the expression of TJ proteins, possibly by reducing oxidative stress and activating the Akt/GSK-3β/β-catenin signaling pathway.
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Objective To investigate the effects of single-hole and double-hole drilling and closed drainage on the expression of Hypoxia inducible factor-1α (HIF-1α),Claudin-5,transforming growth factor-β (TGF-β) and Smad2/3 in the epidermis of patients with chronic subdural hematoma.Methods 100 patients with chronic subdural hematoma were randomly divided into single hole and double hole drainage group according to random number table.Immunohistochemical streptavidin-peroxidase (SP) was used to detect the expression of TGF-β protein,and Western blot was used to detect the expression of HIF-1α,Claudin-5 and Smad2/3 in the epihematoma of the two groups before and after operation.Results Compared with pre-operation,the expression of HIF-1 α,TGF-β and Smad2/3 protein in adventitia of hematoma in both groups decreased after operation,especially in the double-hole group (P ＜ 0.05).The expression of Claudin-5 protein in adventitia of hematoma was significantly increased in both groups,especially in the double-hole group,with statistically significant difference (P ＜ 0.05).Conclusions Single-hole and double-hole drilling and sealing drainage is an effective method to treat chronic subdural hematoma.It can reduce the expression of HIF-1α,TGF-β and Smad2/3 protein in the epidermis of hematoma,and significantly increase the expression of Claudin-5,and the effect of double-hole drilling and sealing drainage is more significant.
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Objective@#To investigate the effects of single-hole and double-hole drilling and closed drainage on the expression of Hypoxia inducible factor-1α (HIF-1α), Claudin-5, transforming growth factor-β (TGF-β) and Smad2/3 in the epidermis of patients with chronic subdural hematoma.@*Methods@#100 patients with chronic subdural hematoma were randomly divided into single hole and double hole drainage group according to random number table. Immunohistochemical streptavidin-peroxidase (SP) was used to detect the expression of TGF- β protein, and Western blot was used to detect the expression of HIF-1α, Claudin-5 and Smad2/3 in the epihematoma of the two groups before and after operation.@*Results@#Compared with pre-operation, the expression of HIF-1α, TGF-β and Smad2/3 protein in adventitia of hematoma in both groups decreased after operation, especially in the double-hole group (P<0.05). The expression of Claudin-5 protein in adventitia of hematoma was significantly increased in both groups, especially in the double-hole group, with statistically significant difference (P<0.05).@*Conclusions@#Single-hole and double-hole drilling and sealing drainage is an effective method to treat chronic subdural hematoma. It can reduce the expression of HIF-1α, TGF-β and Smad2/3 protein in the epidermis of hematoma, and significantly increase the expression of Claudin-5, and the effect of double-hole drilling and sealing drainage is more significant.
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Objective To explore the effect of Dexmedetomidine (Dex) on acute brain edema in mice in condition with targeted temperature management (TTM) following traumatic brain injury (TBI).Methods A total of 180 male C57BL/6J mice were divided into control group,sham operation group,TBI group,TBI + Dex group,TBI + TTM group,and TBI + Dex + TTM group according to the random number table (n =30 per group).The sham operation group only opened the bone window but did not hit it,and the control group did not open the bone window.The TBI + Dex,TBI + TIM,and TBI + Dex + TTM groups were intraperitoneally injected with Dex (60 μg/kg once every 2 h for 3 times) and/or hypothermia after TBI.The brain tissue injury volume,EB extravasation and brain water content of each group were determined by toluidine blue,Evans blue staining and dry-wet weight method at 24 hours after injury.Real-time quantitative PCR and Western blot were used to detect the expression of Claudin-5 in the injured brain tissue.At 24,48,and 72 hours after injury,the neurological deficiency degree was assessed using the modified neurological severity scores (mNSS).Results Compared with the sham operation group,TBI mice showed significant increase in brain tissue injury volume [(0.49 ± 0.04)mm3 vs.(1 1.57 ± 1.01)mm3],blood-brain barrier permeability [(16.4 ± 0.8) μg/g vs.(54.3 ± 1.7) μg/g],brain tissue water content [(76.7 ± 0.9) ％ vs.(83.1 ± 0.8) ％],and mNSS score [(1.6 ± 0.7) points vs.(13.4 ± 0.7) points] at 24 hour after TBI (all P ＜ 0.01).However,Dex or TTM treatment reduced brain tissue injury volume [(7.20±0.18)mm3 and (5.94 ±0.18)mm3],blood-brain barrier permeability [(32.7 ± 1.2) μg/g and (27.6 ± 1.0) μg,/g],brain tissue water content [(78.5 ± 0.4) ％ and (78.2 ± 0.6) ％],and neurological function [mNSS:(7.3 ± 1.1) points and (5.8 ± 1.3) points] (all P＜0.01).Moreover,Dex + TTM group showed better neuroprotection [reduced brain tissue injury volume:(3.92 ± 0.05) mm3,reduced BBB permeability:(21.6 ± 0.7) μg/g,reduced brain water content:(77.7 ±0.3)％,and reduced mNSS:(4.3 ± 1.2) points] compared with Dex or TTM alone (all P ＜ 0.01).Additionally,the mRNA expression of Claudin-5 (0.23 ± 0.01) decreased significantly at 24 hours after TBI compared with sham group (0.93 ± 0.04,P ＜ 0.01),but Dex or TTM could increase the expression of Claudin-5 (0.47 ± 0.01,and 0.54 ± 0.09) compared with TBI group (P ＜0.01),especially that of TBI + Dex + TTM group (0.64 ± 0.02,P ＜ 0.01).Furthermore,the protein expression of Claudin-5 was in accordance with the result of its mRNA expression.Conclusion Dex in condition with targeted temperature management can up-regulate Claudin-5 expression in early TBI,protect the integrity of blood-brain barrier,attenuate acute brain edema and neurological damage,and improve neurological function recovery.
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PURPOSE: The tight junction protein claudin-5 (CLDN5) is critical to the control of endothelial cellular polarity and pericellular permeability. The role of CLDN5 in chronic obstructive pulmonary disease (COPD) remains unclear. The aim of this study was to investigate the association between CLDN5 levels and clinical variables in patients with COPD. METHODS: In total, 30 patients with COPD and 30 healthy controls were enrolled in the study. The plasma CLDN5 level was checked in patients with stable or exacerbated COPD and in healthy controls. RESULTS: The mean plasma CLDN5 level of patients with COPD was 0.63 ± 0.05 ng/mL and that of healthy controls was 6.9 ± 0.78 ng/mL (P = 0.001). The mean plasma CLDN5 level was 0.71 ± 0.05 ng/mL in exacerbated COPD patients and 0.63 ± 0.04 ng/mL in patients with stable COPD (P < 0.05). The plasma CLDN5 level among COPD subjects was correlated with the smoking amount (r = −0.530, P = 0.001). The plasma CLDN5 level in stable COPD patients was correlated with forced expiratory volume in one second (FEV1, %pred.) (r = −0.481, P = 0.037). CONCLUSIONS: The plasma CLDN5 level was not correlated with age. CLDN5 may be involved in the pathogenesis of COPD. Further studies having a larger sample size will be needed to clarify CLDN5 in COPD.
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Humans , Claudin-5 , Forced Expiratory Volume , Permeability , Plasma , Pulmonary Disease, Chronic Obstructive , Sample Size , Smoke , Smoking , Tight JunctionsABSTRACT
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 effect of high mobility group box-1 protein (HMGB1) on the permeability of human umbilical vein endothelial cells, and the protective effect of unfractionated heparin on HMGB1-mediated, endothelial cell tightly junction-related protein Claudin-5. Methods The human umbilical vein endothelial cells after trypsin digestion were subcultured in culture flasks and divided into 4 groups: blank control group (addition of PBS equivalent), rhHMGB1 treatment group (100 ng/mL), unfractionated heparin control group (UFH, 10 U/mL) and rhHMGB1+ unfractionated heparin-treated group (100 ng/mL rhHMGB1 + UFH 10 U/mL). After human umbilical vein endothelial cells were cultured: the viability of endothelial cells was determined by MTT assay; transwell method was used to measure the permeability of endothelial cells; the expression and distribution of Claudin-5 were determined by immunofluorescence; and Claudin-5 protein expression was detected by Western blotting. Results After treated with rhHMGB1 (100 ng/mL), the viability of endothelial cells was notsignificantly different from that of the blank control group (P> 0.05). After treated with rhHMGB1 (100 ng/mL) for 3 and 6 h respectively, the permeability of endothelial cells was not significantly different from that of the blank control group (P> 0.05). After 12 h and 24 h treatment of rhHMGB1, the permeability of endothelial cells was significantly increased compared with the blank control group (P< 0.05). However, after endothelial cells were incubated with unfractionated heparin and rhHMGB1 for 12 and 24 h, the permeability of endothelial cell was lower than that of rhHMGB1 treatment group (P< 0.05). Immunofluorescence and Western-blot showed that after treatment of rhHMGB1 for 24 h, the distribution and expression of claudin-5, a tightly junction-associated protein, was decreased. After incubation with unfractionated heparin and rhHMGB1, the expression of tightly junction-associated protein Claudin-5 was increased, as well as the fluorescence intensity of Claudin-5. Conclusions HMGB1 can increase the permeability of endothelial cells by mediating the abnormal distribution and decreased expression of Claudin-5. Unfractionated heparin can improve the expression and distribution of Claudin-5, improving the permeability of endothelial cells.
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Objective To study the expression difference and the meaning of interleukin (IL)-1β and Claudin-5 in the kidney and kidney's membrane between lupus mice and the control mice. Methods Gene expression difference of IL-1βand claudin-5 between lupus mice and control mice in their kidney and kidney's membrane was detected with quantitative polymerase chain reaction (Q-PCR). The location of the expression was identified by immunohistochemistry. One-way analysis of variance (ANOVA) was used to compare the means of each group, pair-wise comparison was used to compare the difference between multiple sample means. LSD method was used when the variance was equal, and Tamhane's T2 method was used when the variance was different. Results Q-PCR test results showed that IL-1β expression in lupus mice's kidney membrane (0.0095±0.0052) was statistically higher than lupus mice's kidney parenchyma (0.0057±0.0013) (t=2.137, P=0.0458) and control mice's kidney membrane (0.0045±0.0033) (t=2.709, P=0.0131), however, there's no statistical significant difference between control mice's kidney membrane and parenchyma (0.0065± 0.0011) (P>0.05), and there's no statistical difference between control and lupus mice's kidney parenchyma (P>0.05). Claudin-5 expression was statistically higher in control mice kidney membrane (0.0192 ±0.0048) than its kidney parenchyma (0.01156 ±0.002190) (t=4.009, P=0.0015) but statistically lower in lupus mice kidney membrane (0.0069±0.0004) than its kidney parenchyma (0.0098±0.0027) (t=2.727, P=0.0173);there's no statistical significant difference between control mice's kidney parenchyma and lupus mice's kidney parenchyma (P>0.05), and lupus mice's kidney membrane expression was statistically lower than control mice's kidney membrane (t=6.018, P=0.0001). Immun-ohistochemistry showed that IL-1β expression was mainly around glomerulus and membrane, but not renal tu-bule. Claudin-5 expression was mainly around glomerulus and membrane. Conclusion Immune inflammation induced by IL-1β has mainly shown in blood vessels, while claudin-5 has protective effect on lupus immune inflammation.
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Objective To observe the effects of keratinocyte growth factor-2 (KGF-2) on the expressions of chemokine FKN and tight junction protein claudin-5 in lung tissue of rats with acute lung injury (ALI). Methods Healthy male Sprague-Dawley (SD) rats were randomly divided into normal saline (NS) control group, ALI model group and KGF-2 pretreatment group, with 10 rats in each group. The rat ALI model was reproduced by injection of 0.01 mL/kg oleic acid into the tail vein, and the rats in NS control group were injected with the same amount of NS. The rats in KGF-2 pretreatment group were instilled with 5 mg/kg KGF-2 in the airway at 72 hours before the model reproduction, and the rats in the NS control group and the ALI model group were instilled with the same amount of NS. The abdominal aortic blood of rats was collected at 8 hours after model reproduction, and then the rats were sacrificed, bronchoalveolar in left lung was lavage, and the bronchoalveolar lavage fluid (BALF) was collected for determination of protein levels in plasma and BALF, and the lung permeability index (LPI) was calculated. The lung tissues were harvested, after hematoxylin-eosin (HE) staining, the histopathological changes were observed under light microscope, and the ALI pathology score (LIS) was calculated. The lung wet/dry weight (W/D) ratio was determined. Immunohistochemistry and Western Blot were used to qualitatively and quantitatively analyze the expressions of FKN and claudin-5 in the lung tissue. The correlation between two variables was analyzed by linear or curve fitting correlation analysis. Results In the ALI model group, the lung tissue was severely damaged, and obvious pathological changes were observed, including thickened alveolar space and inflammatory cell infiltration, and LIS score, lung W/D ratio and LPI were significantly higher than those of the NS control group (LIS: 3.56±0.28 vs. 0.62±0.36, lung W/D ratio: 6.37±0.29 vs. 4.39±0.33, LPI: 3.46±0.69 vs. 0.98±0.17, all P < 0.01). Compared with the NS control group, the positive expression of FKN in the lung tissue of the ALI model group was significantly increased, and the expression level was significantly increased (FKN/GAPDH: 0.97±0.18 vs. 0.62±0.04, P < 0.01); the positive expression of claudin-5 was significantly decreased, and the expression level was significantly decreased (claudin-5/GAPDH: 0.56±0.11 vs. 1.06±0.13, P < 0.01). There was a significant negative correlation between FKN and claudin-5 protein expression (r = -0.817, P = 0.025). After pretreatment with KGF-2, the degree of lung tissue damage was significantly reduced, and the pathological changes were significantly improved, and the LIS score, lung W/D ratio and LPI were significantly lower than those of the ALI model group (LIS: 2.41±0.17 vs. 3.56±0.28, lung W/D ratio: 5.45±0.55 vs. 6.37±0.29, LPI: 2.42±0.19 vs. 3.46±0.69, all P < 0.01). Compared with the ALI model group, the positive expression of FKN in the lung tissue of KGF-2 pretreatment group was significantly decreased, and the expression level was significantly decreased (FKN/GAPDH: 0.79±0.03 vs. 0.97±0.18, P < 0.01); the positive expression of claudin-5 was significantly increased, and the expression level was significantly increased (claudin-5/GAPDH: 0.80±0.05 vs. 0.56±0.11, P < 0.01). There was still a significant negative correlation between FKN and claudin-5 protein expression (r = -0.847, P = 0.012). Conclusion KGF-2 may restore the expression of tight junction protein claudin-5 by down-regulating the expression of chemokine FKN, thereby reducing the damage of blood barrier in ALI.
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Objective:To investigate the effect of alteplase on the expressions of Claudin-1 and Claudin-5 proteins in the vascular endothelial cells after thrombolysis in the rats with acute cerebral infarction and its mechanism,and to provide experimental evidence for its clinical application. Methods: The models of acute thrombosis of middle cerebral artery of the rats were established.Fifty-four SD rats were randomly divided into sham operation group,model group and alteplase group (n= 18).The ultrastructure of brain endothelial cells of the rats was observed under transmission electron microscope.The expression levels of Claudin-1 and Claudin-5 proteins in the vascular endothelial cells of the rats were detected by immunoflurorescence and Western blotting methods.Results:The transmission electron microscope results showed that the brain volume in the ischemic area of the rats in model group was significantly increased and the endothelial cells were swollen,some of the cortical and surrounding brain tissues had obvious boundaries,and the thickness of the basement membrane was uniform and the tightly connected structure was very loose and disappeared;the swelling condition of the capillary endothelial cells in infarcted area of the rats in alteplase group was significantly reduced and the thickness of the basement membrane was improved,and most of the tightly connected structures between the brain capillary endothelial cells and the endothelial cells were loose and the fracture was lost and the structure disappeared.The immunofluorescence results showed that the expressions of Claudin-1 and Claudin-5 proteins in the vascular endothelial cells of the rats in alteplase group were significantly improved compared with model group. The Western blotting results showed the expression levels of Claudin-1 and Claudin-5 proteins in the vascular endothelial cells of the rats in model group were significantly decreased compared with sham operation group (P < 0.01 ); compared with model group, the expression levels of Claudin-1 and Claudin-5 proteins in the vascular endothelial cells of the rats in alteplase group at different time points were increased (P < 0.01 ). Conclusion: Alteplase can improve the structure of brain endothelial cells in the rats with acute cerebral infarction,and the mechanism may be related to decreasing the expressions of Claudin-1 and Claudin-5 proteins in the vascular endothelial cells of the rats induced by alteplase.
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Objective To investigate whether the inhibition of caveolin-1 (Cav-1) phosphorylation will regulate effectively nuclear factor-erythroid 2-related factor (Nrf2) signal pathway and downstream effector molecules and protest against ventilation induced lung injury (VILI) in an animal model in vivo. Methods Ninety male Sprague-Dawley (SD) rats were randomly divided into nine groups (each n = 10): sham group in which rats did not receive ventilation but received tracheotomy; lung protective ventilation (PV) for 1 hour or 2 hours group; mechanical ventilation (MV) at high volume tidal (VT, 40 mL/kg) for 1 hour or 2 hours group; protein tyrosine kinase inhibitor PP2 or rosiglitazone (Rsg) pretreatment + high VT ventilation for 1 hour or 2 hours groups. The two pretreatment groups were given intraperitoneal injection PP2 15 mg/kg or intragastric administration of Rsg 5 mg/kg 1 hour before ventilation respectively. The rats were sacrificed after model reproduction, and bronchoalveolar lavage fluid (BALF) was collected. Pulmonary vascular permeability was measured by Evans blue (EB). The levels of tumor necrosis factor-α (TNF-α), activator protein-1 (AP-1), nuclear factor-κB (NF-κB), and interleukin-8 (IL-8) in BALF were determined by enzyme linked immunosorbent assay (ELISA). Then the lung tissues were collected, the lung wet/dry ratio (W/D) was calculated, the changes in pathology was observed with light microscope, and myeloperoxidase (MPO) activity was determined by colorimetric analysis. Nrf2 mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR). The expressions of Cav-1 tyrosine residues 14 phosphorylation (pCav-1-Y14), Cav-1, peroxisome proliferators-activated receptor γ (PPARγ) and claudin-5 as well as Nrf2 in cytoplasm and nucleus were determined by Western Blot. The positive expressions of PPARγ and claudin-5 in lung tissues were assayed with immunohistochemistry staining. Results There were no obvious pathological changes in the lung tissue in sham group and PV groups, and there were no significant differences in all the parameters between the two groups either. However, the injury in lung tissue was severe in the high VT groups in which W/D ratio, EB contents, MPO activity, and TNF-α, AP-1, IL-8, NF-κB levels in BALF as well as the protein expressions of Cav-1 and pCav-1-Y14 were significantly higher than those of sham group and PV groups, and the protein expressions of PPARγ and claudin-5 were significant lower than those of sham group and PV groups with a dose-dependent manner; but Nrf2 expressions in cytoplasm and nucleus did not show a statistical increase. After pretreatment of PP2 or Rsg, W/D ratio, MPO activity, EB contents, TNF-α, AP-1, IL-8, and NF-κB in BALF were significantly decreased as compared with those of high VT group, and RT-PCR showed significant up-regulation of Nrf2 mRNA in lung tissues too. Moreover, there was a statistically significant increase in expressed Nrf2 proteins in nucleus in PP2 or Rsg groups as compared with those of high VT groups [Nrf2 in nucleus (gray value): 0.61±0.06, 0.56±0.06 vs. 0.31±0.02 at 1 hour, 0.38±0.06, 0.43±0.07 vs. 0.22±0.03 at 2 hours; all P < 0.05], but no significant difference was found in the expression of Nrf2 protein in the cytoplasm among all groups. The protein expressions of pCav-1-Y14 in PP2 pretreatment groups were significantly lower than those of high VT groups (gray value: 0.89±0.04 vs. 1.48±0.02 at 1 hour, 0.86±0.02 vs. 1.31±0.01 at 2 hours; both P < 0.05); but expressed PPARγ proteins and expressed claudin-5 proteins in PP2 or Rsg pretreatment groups were significantly higher than those of high VT groups [PPARγ (gray value): 0.34±0.07, 0.42±0.13 vs. 0.17±0.07 at 1 hour, 0.38±0.09, 0.33±0.07 vs. 0.16±0.03 at 2 hours; claudin-5 (gray value): 0.33±0.05, 0.38±0.07 vs. 0.14±0.03 at 1 hour; 0.30±0.06, 0.31±0.04 vs. 0.17±0.04 at 2 hours; all P < 0.05]. Conclusions The inhibition of Cav-1-Y14 phosphorylation can increase the expression of Nrf2 in the nucleus, then result in an increase in the protein expressions of PPARγ and claudin-5 of its effector molecules. This effect can reduce the inflammation and capillary permeability of lung tissue in the model of VILI.
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Objective To investigate the effects of PJ34,a poly ADP-ribose polymerase(PARP)inhibitor,on the expression of matrix metallopro-teinases-9( MMP-9 )and Claudin-5 in ischemic cortex of rats with focal cerebral ischemia-reperfusion(I/R)injury. Methods The focal cerebral ischemia-reperfusion model of middle cerebral artery occlusion(MCAO)was established by intraluminal suture . PJ34 was injected intraperitoneal-ly. Blood-brain barrier(BBB)permeability was quantitatively determined by Evans blue assay. Infarct volume changes were observed by 2,3,5-tri-phenyltetrazolium chloridedyeing(TTC)staining. The expression of the MMP-9 and Claudin-5 in rats of cerebral cortex were measured by immuno-histochemistry assay and western blot analysis . Results Compared with sham group,the expression of MMP-9,the contents of EB and infarct vol-ume increased progressively over time after I/R,and reached maximum levels at 48 h(P<0.05);The expression of Claudin-5,the contents of EB and infarct volume reduced significantly over time after I/R,and reached the minimum levels at 48 h in model group(P<0.05). Compared with model group,the expression of MMP-9,the contents of EB and infarct volume was reduced significantly and the expression of Claudin-5,the con-tents of EB and infarct volume was increased at the same time point in PJ34 group(P<0.05). Conclusion PJ34 maintained the blood-brain barri-er permeability by inhibiting the expression of MMP-9,and increasing the expression of Claudin-5,which had neuroprotection on cerebral ischemia-reperfusion injury.
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Objective To investigate the protective effect of Limb ischemic postconditioning ( LIPostC) on blood brain barrier (BBB) and explore the influence on the the expression of tight junction protein claudin-5,occludin in cerebral ischemia-reperfusion injury of mice.Methods Ninety male CD1 mice were divided into three groups randomly:Sham-operated group(sham group), ischemia-reperfusion group(I/R group) and LIPostC group.Mouse middle cerebral artery cerebral ischemia-reperfusion model was established by modified-occlusion method. Neurological deficit scores, brain water content, infarct volume, BBB dysfunction were measured at 24 h after reperfusion.Western blot and RT-qPCR were used to analyze the expressions of claudin-5, occludin.Results Compared with I/R group, neurological deficits scores were significantly decreased, brain edema was relieved, infarct volume was reduced, and the expression of claudin-5, occludin up-regulated, the differences were statistically significant(all P<0.05).Conclusion LIPostC can protect the BBB of cerebral ischemia-reperfusion injury mice by increasing the expression of claudin-5,occludin, and that will provide a theoretical basis for its clinical application.
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Objective To study the effects of methylprednisolone on the permeability of blood-spinal cord barrier (BSCB) and claudin-5 expression after spinal cord injury in rats. Methods The rat model of spinal cord injury was estab?lished using modified Allen method. SD rats were randomly divided into sham-operated group, spinal cord injury group and methylprednisolone pretreatment group. The permeability of BSCB and expression of claudin–5 were assessed at 12 h, 1, 3, 5, and 7 d after the onset of spinal cord injury (five animals per each time point). RT-PCR and Western blot were used to detect the expression of claudin-5. Results The success rate of the model was 84.0%. EB content was sig?nificantly higher in spinal cord injury group than in sham-operated group at each time point (F value 27.732,P was lower in spinal cord injury group than in sham operated group at each time point (Fvalue 18.108,P<0.05). The pro?tein expression of Claudin-5 was higher in methylprednisolone pretreatment group than spinal cord injury group at each time point (F value 20.247,P<0.05). Conclusions Methylprednisolone improves permeability of BSCB after spinal cord injury probably through enhancing claudin-5 expression in rats.
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Objective To examine the expression of tight junction protein claudin-5 in blood-spinal cord barrier (BSCB) after spinal cord injury about rat. Methods One hundred-twenty adult male SD rats were randomly divided into blank group (60) and injured group (60). The animal model of spinal cord injury was established using modified Allen method. The expression of claudin-5 in BSCB was examined at 6 h, 12 h, 1 d, 3 d, 5 d and 7 d (five rats per time point). Western blot and RT_PCR were used to detect protein and mRNA expression levels of claudin-5, respectively. Results The success rate of spinal cord injury molding was 81.7%. In injured group, EB content increased gradually over time, reached the peak at the third day(0.9435 ± 0.0813)μg/g and then reduced gradually (P<0.05), EB content was signifi-cantly higher in injured group than in blank group. Claudin-5 mRNA expression in injured group reduced gradually over time and reached the lowest point at the third day(2.871 ± 0.527)and then increased gradually(P<0.05). Claudin-5 mRNA expression was significantly lower in injured group than in blank group(P<0.05). Claudin-5 protein expression in injured group reduced gradually over time, reached the lowest at the third day(0.072 ±0.008)and then increased gradually (P<0.05). Claudin- 5 protein expression was significantly lower in injured group than in blank group(P<0.05). Con-clusions The alteration of claudin-5 expression after SCI may lead to the permeability of BSCB, which may in turn con-tribute to the secondary spinal cord injury.