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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 breakdown of blood-retinal outer barrier in the ischemia-reperfusion injury mice following acute intraocular hypertension.Methods:Fifty-seven SPF male C57BL/6J mice were selected and divided into the control group and high-intraocular pressure (IOP) group by using the random number table method.There were 25 mice in the control group and 32 mice in the high-IOP group.After the failure and poor modeling excluded, 20 mice were included in each group, and the left eyes were selected as the experimental eyes.The ischemia-reperfusion injury model of the high-IOP group was established following acute intraocular hypertension by anterior chamber perfusion of 0.9% sodium chloride solution, and the control group only received anterior chamber puncture.Optical coherence tomography was used to detect retinal thickness.Immunofluorescence staining was utilized to identify zonula occludens-1 (ZO-1) protein distribution in retina.Retinal capillary degeneration was identified by trypsin digestion.Inflammatory cell infiltration in retinal sections was observed by hematoxylin and eosin staining.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology, and the study protocol was approved by an Ethics Committee of Changsha Aier Eye Hospital (No.2018-KYPJ005).Results:Compared with the control group, the structure of the retinal pigment epithelium (RPE) layer was irregular with obvious exudation and local neuroepithelial detachment and elevation.The thickness of the full retinal layer of mice in the high-IOP group was (235.8±5.3)μm, which was significantly thicker than (213.3±3.9)μm in the control group ( t=3.427, P=0.009). ZO-1 staining results showed that ZO-1 was mainly located in cell membrane and a small part in cytoplasm in the RPE layer of mice.Two days after modeling, ZO-1 in the high-IOP group was significantly internalized with decrease in cell membrane and increase in cytoplasm, and its distribution was irregular.Seven days after modeling, retinal capillary degeneration was observed in the high-IOP group, and the number of degenerated retinal capillaries was 201.0±13.2, which was significantly larger than 11.2±1.7 in the control group ( t=14.280, P<0.01). Hematoxylin-eosin staining results showed that inflammatory cell infiltration, mainly neutrophils, could be observed in the mice retina with high IOP, and the infiltrated inflammatory cells were mainly located under the internal limiting membrane. Conclusions:Acute intraocular hypertension induced retinal ischemia-reperfusion injury in mice destroys the integrity of the outer retinal barriers, and causes granulocyte infiltration in the peripheral circulation, retinal edema as well as retinal capillary degeneration.
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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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Objective:To explore the protective effect of Gegen Qinliantang on the intestinal mucosal epithelial barrier function of ulcerative colitis （UC） mice， and to explore its mechanism of action in the treatment of ulcerative colitis via matrix metallopeptidase-9 （MMP-9）/p38 mitogen-activated protein kinase （p38 MAPK） signaling pathway. Method:The 48 female C57BL/6 mice were randomly divided into normal group， model group， sulfasalazine group （0.3 g·kg<sup>-1</sup>） and Gegen Qinliantang high， medium and low dose groups （2.84，1.42，0.71 g·kg<sup>-1</sup>）. The UC murine model was established by 3% dextran sulfate sodium （DSS）. Gegen Qinliantang and sulfasalazine were intragastrically administered on the 8<sup>th</sup> day after the model was established for 7 days， and the normal group was treated with the same amount of normal saline. Colon tissues were collected after the last administration， and the pathological changes of colon tissues were detected by hematoxylin-eosin （HE） staining. The expression of tight junction （TJ） proteins such as Occludin and zonula occludens-1（ZO-1） in colon tissues was detected by immunohistochemistry （IHC）， and the expression levels of tumor necrosis factor-alpha （TNF-<italic>α</italic>）， interleukin-1<italic>β</italic> （IL-1<italic>β</italic>）， and MMP-9 mRNA in colon tissues were detected by Real-time polymerase chain reaction （Real-time PCR）. The expression of phosphorylated p38 MAPK （p-p38 MAPK）， p38 MAPK and MMP-9 protein in colon tissues was detected by Western blot. Result:Compared with normal group， the body weight of mice decreased （<italic>P</italic><0.01） and disease activity index （DAI） score increased significantly （<italic>P</italic><0.01） in model group， the colon tissues of the model group were damaged more obviously， the expression of occludin and ZO-1 proteins in model group was significantly reduced （<italic>P</italic><0.01）， and the relative expression levels of TNF-<italic>α</italic>， IL-1<italic>β</italic>， and MMP-9 mRNA in model group were significantly increased （<italic>P</italic><0.01）， the expression of p-p38 MAPK and MMP-9 in model group was significantly increased （<italic>P</italic><0.01）. Compared with model group， the body mass and DAI score of the sulfasalazine group and Gegen Qinliantang group were significantly improved （<italic>P</italic><0.05，<italic>P</italic><0.01）， the colonic tissues damage were significantly improved， and the expression of Occludin and ZO-1 protein was significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）， the relative expression levels of TNF-<italic>α</italic>， IL-1<italic>β</italic>， and MMP-9 mRNA were significantly decreased （<italic>P</italic><0.01）， and the expression of p-p38 MAPK and MMP-9 was significantly decreased （<italic>P</italic><0.01）. The changes in the middle dose group were the most obvious among the various dose groups of Gegen Qinliantang. Conclusion:Gegen Qinliantang repairs the intestinal mucosal barrier function by inhibiting the expressions of MMP-9 and inflammatory cytokines such as TNF-<italic>α</italic> and IL-1<italic>β</italic>， blocking the activation of the p38 MAPK signaling pathway， and increasing the expressions of tight junction protein.
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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@#Previous studies have found abnormal proliferation and transdifferentiation of alveolar epithelial cells(AECs)in hyperoxic lung injury of neonatal rats.The purpose of this study was to clarify the expression of zonula occludens 1(ZO-1) and ZO-1 related nucleic acid binding protein(ZONAB)in AECs in hyperoxic lung injury model, in order to investigate its effect on the proliferation and transdifferentiation of AECs in the injured lung tissue.@*Methods@#Full-term neonatal Wistar rats were randomly divided into two groups within 12 h after birth, model group(inhaled oxygen concentration 85%)and control group(inhaled air). Lung specimens were collected at 7, 14 and 21 days after exposure.The expression of ZONAB in typeⅡalveolar epithelial cells(AECⅡ)was observed by double immunofluorescence staining.At the same time, AEC Ⅱ was isolated from lung tissues of animal models at these time points, and the expression levels of ZO-1, ZONAB protein and mRNA in lung tissues and AECⅡof the two groups were detected by Western blot and Real-Time PCR.In addition, AEC Ⅱ was isolated from lung tissue of normal newborn rats and then divided into model group(85% oxygen concentration)and control group(21% oxygen concentration). After 48 hours of culture in vitro, the expression levels of ZO-1, ZONAB protein and mRNA were detected, and the expression level and location of ZONAB were observed by immunofluorescence staining.@*Results@#Double immunofluorescence staining showed that the expression of ZONAB in AECⅡin model group was significantly lower than that in control group.The protein and mRNA expression levels of ZO-1 and ZONAB in AECⅡisolated from lung tissue of model group were both significantly lower than those from control group, starting from 7 d after hyperoxia exposure.AECⅡisolated from lung tissue of normal newborn rats, were then incubated for 48 hours under hyperoxia or normoxia in vitro, the protein and mRNA expression levels of ZO-1 and ZONAB significantly decreased in model group compared with those in control group.The results of immunofluorescence staining showed that the expression of ZONAB was higher in AECⅡof the control group, and ZONAB was mostly located in the junction and nucleus of cells, while the expression of ZONAB in the model group significantly decreased than that in the control group, and the expression sites were clustered in the cytoplasm, with little expression in the junction and nucleus.@*Conclusion@#ZO-1, as a tight junction-related protein, is down-regulated in hyperoxic lung injury model.In addition to destroying pulmonary epithelial barrier to mediate pulmonary edema, it also participated in the regulation of proliferation and differentiation of AECs by regulating transcription factor ZONAB, suggesting that this may be another pathway leading to hyperoxic lung injury.
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Objective@#To investigate the regulation of hypoxia-inducible factor-1α (HIF-1α) on permeability of rat vascular endothelial cells and the mechanism.@*Methods@#Twelve male Sprague-Dawley rats aged 35 to 38 days were collected and vascular endothelial cells were separated and cultured. The morphology of cells was observed after 4 days of culture, and the following experiments were performed on the 2nd or 3rd passage of cells. (1) Rat vascular endothelial cells were collected and divided into blank control group, negative control group, HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group according to the random number table (the same grouping method below), with 3 wells in each group. Cells in negative control group, HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group were transfected with GV248 empty plasmid, recombinant plasmid respectively containing HIF-1α interference sequence 1, interference sequence 2, and interference sequence 3 with liposome 2000. Cells in blank control group were only transfected with liposome 2000. After transfection of 24 h, expression levels of HIF-1α mRNA and protein of cells in each group were respectively detected by reverse transcription real-time fluorescent quantitative polymerase chain reaction and Western blotting (the same detecting methods below) . The sequence with the highest interference efficiency was selected. (2) Another batch of rat vascular endothelial cells were collected and divided into blank control group, negative control group, and HIF-1α low expression group, with 3 wells in each group. Cells in blank control group were only transfected with liposome 2000, and cells in negative control group and HIF-1α low expression group were respectively transfected with GV248 empty plasmid and low expression HIF-1α recombinant plasmid selected in experiment (1) with liposome 2000. After 14 days of culture, the mRNA and protein expressions of HIF-1α in each group were detected. (3) Another batch of rat vascular endothelial cells were collected and divided into blank control group, negative control group, and HIF-1α high expression group, with 3 wells in each group. Cells in blank control group were transfected with liposome 2000, and cells in negative control group and HIF-1α high expression group were respectively transfected with GV230 empty plasmid and HIF-1α high expression recombinant plasmid with liposome 2000. After 14 days of culture, the mRNA and protein expressions of HIF-1α of cells in each group were detected. (4) After transfection of 24 h, cells of three groups in experiment (1) and three groups in experiment (2) were collected, and mRNA and protein expressions of myosin light chain kinase (MLCK), phosphorylated myosin light chain (p-MLC), and zonula occludens 1 (ZO-1) of cells were detected. Data were processed with one-way analysis of variance and t test.@*Results@#After 4 days of culture, the cells were spindle-shaped, and rat vascular endothelial cells were successfully cultured. (1) The interference efficiencies of HIF-1α of cells in HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group were 47.66%, 45.79%, and 62.62%, respectively, and the interference sequence 3 group had the highest interference efficiency. After transfection of 24 h, the mRNA and protein expression levels of HIF-1α of cells in interference sequence 3 group were significantly lower than those in blank control group (t=18.404, 9.140, P<0.01) and negative control group (t=15.099, 7.096, P<0.01). (2) After cultured for 14 days, the mRNA and protein expression levels of HIF-1α of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=21.140, 5.440, P<0.01) and negative control group (t= 14.310, 5.210, P<0.01). (3) After cultured for 14 days, the mRNA and protein expression levels of HIF-1α of cells in HIF-1α high expression group were significantly higher than those in blank control group (t=19.160, 7.710, P<0.01) and negative control group (t= 19.890, 7.500, P<0.01). (4) After transfection of 24 h, the mRNA expression levels of MLCK and p-MLC of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=2.709, 4.011, P<0.05 or P<0.01) and negative control group (t=2.373, 3.744, P<0.05 or P<0.01). The mRNA expression level of ZO-1 of cells in HIF-1α low expression group was significantly higher than that in blank control group and negative control group (t=4.285, 5.050, P<0.01). The mRNA expression levels of MLCK and p-MLC of cells in HIF-1α high expression group were significantly higher than those in blank control group (t=9.118, 11.313, P<0.01) and negative control group (t=9.073, 11.280, P<0.01). The mRNA expression level of ZO-1 of cells in HIF-1α high expression group was significantly lower than that in blank control group and negative control group (t=2.889, 2.640, P<0.05). (5) After transfection of 24 h, the protein expression levels of MLCK and p-MLC of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=2.652, 3.983, P<0.05 or P<0.01) and negative control group (t=2.792, 4.065, P<0.05 or P<0.01). The protein expression of ZO-1 of cells in HIF-1α low expression group was significantly higher than that in blank control group and negative control group (t=3.881, 3.570, P<0.01). The protein expression levels of MLCK and p-MLC of cells in HIF-1α high expression group were 1.18±0.24 and 0.68±0.22, which were significantly higher than 0.41±0.21 and 0.35±0.14 in blank control group (t=5.011, 3.982, P<0.05 or P<0.01) and 0.43±0.20 and 0.36±0.12 in negative control group (t= 4.880, 3.862, P<0.05 or P<0.01). The protein expression level of ZO-1 of cells in HIF-1α high expression group was 0.08±0.06, which was significantly lower than 0.20±0.09 in blank control group and 0.19±0.09 in negative control group (t=4.178, 3.830, P<0.05 or P<0.01).@*Conclusions@#HIF-1α up-regulates expressions of MLCK and p-MLC and down-regulates expression of ZO-1, thereby increasing the permeability of rat vascular endothelial cells.
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OBJECTIVE: To observe the influence of scalp-acupuncture on the expression of pentraxin 3 (PTX3), Interleukin (IL)-1β, zonula occludens-1(ZO-1) mRNA and Occludin mRNA in striatum in acute ischemic cerebrovascular disease (AICD) rats, so as to investigate its mechanisms underlying improvement of AICD. METHODS: Forty-eight male SD rats were randomly allocated to control, model, IL-1Ra and IL-1Ra+scalp-acupuncture groups (n=12 rats in each group). The AICD model was established by occlusion of the middle cerebral artery (MCAO). Rats of the IL-1Ra group and IL-1Ra+scalp-acupuncture group received intraperitoneal injection of IL-1Ra (0.05 mg·kg-1·d-1), once daily for 6 days. Scalp acupuncture stimulation was applied to bilateral "Dingnieqianxiexian" (MS6) once daily for 6 days for rats in IL-1Ra+scalp-acupuncture group. Before and after intervention, the neurologic deficit score (NDS) was evalua-ted according to Longa's method. The expression of striatum PTX3 and IL-1β was detected by immunohistochemistry, and ZO-1 mRNA and Occludin mRNA in the striatum tissue were detected by fluorescence quantitative real-time PCR. The Evans Blue (EB) tracer method was used to monitor the degree of blood-brain barrier (BBB) damage. RESULTS: Following modeling, the NDS, EB content and the expression of PTX3 and IL-1β in the striatum tissue were significantly increased, and the ZO-1 mRNA and Occludin mRNA expression was considerably decreased in the model group compared with the control group (P0.05). The effects of scalp acupuncture combined with IL-1Ra were obviously superior to that of IL-1Ra in down-regulating NDS, EB content and IL-1β expression level, and in up-regulating PTX3, ZO-1 mRNA and Occludin mRNA expression levels (P<0.05). CONCLUSION: Scalp acupuncture can improve neurological function and reduce the degree of BBB injury in AICD rats, which may be associated with its function in up-regulating the expression of PTX3 and in promoting the expression of ZO-1 mRNA and Occludin mRNA.
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In the central nervous system, gap junctions exist between neurons and glial cells. Among them, connexin 43 (Cx43) is one of the most abundant connexin proteins in the central nervous system,involved in the metabolic coupling of intercellular substance exchange and electrical coupling of electrical signaling. It plays an important role in regulating cell metabolism, homeostasis, and cell differentiation. After cerebral ischemia, the uncoupling of gap junctions and abnormal hemichannel activity cause a steady-state imbalance of the internal and external environment of the cells, eventually leading to brain tissue damage.Therefore, maintaining the normal function of Cx43 is essential for protecting brain tissue from neuronal damage induced by cerebral ischemia-reperfusion.
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Background:In the pathogenesis of chronic gastritis,the changes of biological factors can affect the physiological barrier of gastric mucosa,increase the risk of injury of intercellular junctions,leading to disruption of mucosal barrier. Aims:To investigate the expressions of occludin and zonula occluden-1 (ZO-1)in chronic gastritis and their significance. Methods:A total of 82 patients with chronic gastritis from October 2014 to October 2017 at the First People's Hospital of Xiaochang were enrolled and divided into chronic non-atrophic gastritis (CNAG)group and chronic atrophic gastritis (CAG)group according to the endoscopic biopsy pathology. Forty healthy subjects were served as normal controls. Expressions of occludin and ZO-1 were examined by immunohistochemistry. Patients positive for Helicobacter pylori (Hp) infection received eradication therapy,and expressions of occludin and ZO-1 were reexamined after Hp eradication. Results:The positivity rates of occludin and ZO-1 proteins in CNAG and CAG groups were significantly lower than those in normal control group (P＜0.05). The positivity rates of occludin and ZO-1 proteins in CNAG group were significantly higher than those in CAG group (P＜0.05). The positivity rates of occludin and ZO-1 proteins in Hp-positive CNAG patients were significantly lower than those in Hp-negative CNAG patients (P＜0.05). The positivity rate of ZO-1 protein in Hp-positive CAG patients was significantly lower than that in Hp-negative CAG patients (P＜0.05). Expressions of occludin and ZO-1 proteins showed a linear trend correlation with activity of gastric inflammation (P ＜0.05);their expressions decreased significantly with the increase in activity of inflammation. The positivity rates of occludin and ZO-1 proteins in CNAG patients increased significantly after Hp eradication (P＜0.05). Conclusions:Expressions of occludin and ZO-1 are decreased in chronic gastritis,especially in CAG,and are correlated with Hp infection and activity of gastric inflammation.
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AIM:To investigate the 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.
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Objective To investigate the effects of exogenous hydrogen sulfide on brain edema and injury and their mechanisms. Methods Sixty male SD rats were randomly divided into a sham operation group, an ischemia-reperfusion group, a 30 ppm hydrogen sulfide group, and a 60 ppm hydrogen sulfide group (n =15 in each group; 1 ppm =1 mg/L). A model of focal cerebral ischemia for 2 h and reperfusion for 24 h was induced by middle cerebral artery occlusion. The neurological scores were observed after 24 h cerebral ischemia-reperfusion. The cerebral infarction volume, the degree of brain edema, and the changes of blood-brain barrier permeability were measured. Western blotting was used to detect the expressions of occludin and zonula occludens-1 protein (ZO-1) in ischemic penumbra. Results Compared with the ischemia-reperfusion group, the neurological function scores in the 30 ppm and 60 ppm hydrogen sulfide group significantly increased in a dose-dependent manner (al P brain edema aleviated (al P < 0.05). The content of Evans blue in the ischemic brain tissue in the ischemia-reperfusion group increased significantly compare with the sham operation group (0.74 ±0.14 μg/100 mg vs. 0.19 ±0.06 μg/100 mg; P <0.05). The content of Evans blue in the brain tissue in the 30 ppm hydrogen sulfide group (0.55 ±0.10 μg/100 mg ) and the 60 ppm hydrogen sulfide group (0.35 ±0.08 μg/100 mg ) decreased significantly compared with the ischemia-reperfusion group (al P < 0.05), among them the 60 ppm hydrogen sulfide group was significantly lower than the 30 ppm hydrogen sulfide group (P <0.05). Western blot analysis showed that expression levels of occludin in penumbra (0.621% ±0.101% vs.0.787% ±0.087% vs.0.453% ± 0.127%; P <0.05) and ZO-1 (0.602% ±0.118% vs.0.778% ±0.805% vs.0.426% ±0.146; P <0.05) in the 30 ppm and 60 ppm hydrogen sulfide groups increased significantly compared with the ischemia-reperfusion group, among them, the expression levels of occludin and ZO-1 in the 60 ppm hydrogen sulfide group were significantly higher than those in the 30 ppm hydrogen sulfide group (al P < 0.05). Conclusions Inhalation of exogenous hydrogen sulfide can significantly attenuate brain edema after ischemia-reperfusion in a dose dependent manner, reduce infarct volume, and improve neurological function.Their mechanisms may be associated with inhibiting the downregulated expressions of occludin and ZO-1 and maintaining the integrity of the blood-brain barrier.
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Objective Disorder of intestinal barrier function is associated with the non-alcohol fatty liver disease ( NAFLD) . The present paper aimed to explore the intestinal barrier function in the rat model of NAFLD . Methods Sixteen SD rats were ran-domly divided into an NAFLD and a normal control group of equal number .The NAFLD models were constructed by high-fat feeding . HE staining was used for pathologic examination of the liver , the levels of TNF-α, IL-1, and endotoxin (ET ) were measured by ELISA and the limulus reagent method , and the expressions of intestinal ZO-1 and Occluding were determined by real time PCR . Resu lts Compared with the normal controls , the NAFLD rats showed typical hepatic lipid deposition , with significantly increased levels of serum TNF-αand IL-1 and plasma ET and decreased expressions of intestinal ZO-1 and Occluding (P<0.05). Conclusion Intestinal barrier function is decreased in NAFLD rats .
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Objective To observe the time-course expression of zonula occludens-1 (ZO-1) in cerebral cortex after traumatic brain injury (TBI). Methods The TBI model of mouse was established. The mice were divided in 1 h, 3 h, 6 h, 12 h, 24 h, 3 d, 7 d after TBI, shamand control groups. The permeability of the blood brain barrier was evaluated by measuring the extravasation of Evans blue (EB) dye. The expression of Z O-1 in cerebral cortex in the injured area was detected by western blotting and im-munohistochemistry. Results The extravasation of EBdye of injured cortex gradually increased from 1 h, peaked at 1-3 d and approximately decreased to normal at 7 d after TBI. western blotting revealed that the expression of Z O-1 gradually decreased after 1 h, was at the lowest at 1-3 d, and then significantly increased after 7 d but was still lower than that of normal and shamgroups. The result of immunohisto-chemistry showed that Z O-1 had strong expression in vessel of normal cortex, gradually decreased after TBI, and almost disappeared at 3 d after TBI and gradually recovered to normal level later. Conclusion The expression of Z O-1 in the injured cortex after TBI initially decreases and then increases. The nega-tive correlation between Z O-1 expression and EBextravasation after TBI could be used as a newindi-cator for wound age estimation.
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AIM:To investigate the effect of Xingnaojing (XNJ) injection on the permeability of blood-brain barrier ( BBB) and zonula occludens-1 ( ZO-1) protein expression after global ischemia-reperfusion in rats.METHODS:Improved Pulsinelli four-vessel occlusion method was adopted to establish the global ischemia-reperfusion model in the rats. Male Wistar rats were randomly divided into sham group, model group, solvent group and XNJ group.The observations were conducted at the time points of 24 h, 48 h and 72 h after ischemia reperfusion.The water content of the brain tissues was determined by dry-wet weight method, while the Evans blue ( EB) content of brain tissue was detected by spectropho-tometry.The protein levels of ZO-1 in the cerebral cortex were analyzed by Western blot.RESULTS:The water contents in the brain tissues in model group, solvent group and XNJ group were significantly higher than those in sham group ( P<0.05) 24 h after ischemia reperfusion.However, the brain water contents in model group and solvent group were signifi-cantly higher than those in XNJ group and sham group (P<0.05) 48 h and 72 h after ischemia reperfusion.The EB con-tents in the brain tissues in model group, solvent group and XNJ group were entirely higher than that in sham group 24 h af-ter ischemia reperfusion (P<0.05).The EB contents in sham group and XNJ group were significantly lower than those in model group and solvent group 48 h and 72 h after ischemia reperfusion (P<0.05).The protein expression of ZO-1 in the rat cerebral cortex in model group, solvent group and XNJ group was significantly lower than that in sham group 24 h after ischemia-reperfusion (P<0.05).Similarly, 48 h and 72 h after ischemia reperfusion, ZO-1 protein level in the cortex in sham group and XNJ group was significantly higher than that in model group and solvent group (P<0.05).CONCLU-SION:At 48 h and 72 h after global ischemia-reperfusion, Xingnaojing injection play a protective role in blood-brain barri-er and this role may be associated with the increase in ZO-1 protein expression by Xingnaojing injection.
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PURPOSE: The aim of this study was to investigate whether pathologic changes in zonula occludens-1 (ZO-1) are induced by interleukin-13 (IL-13) in the experimental minimal-change nephrotic syndrome (MCNS) model and to determine whether montelukast, a leukotriene receptor antagonist, has an effect on ZO-1 restoration in cultured human podocytes. MATERIALS AND METHODS: Human podocytes cultured on bovine serum albumin-coated plates were treated with different doses of IL-13 and montelukast and then examined for distribution using confocal microscopy and for ZO-1 protein levels using Western blotting. RESULTS: ZO-1 was internalized and shown to accumulate in the cytoplasm of human podocytes in an IL-13 dose-dependent manner. High doses (50 and 100 ng/mL) of IL-13 decreased the levels of ZO-1 protein at 12 and 24 h (both p<0.01; n=3), which were significantly reversed by a high dose (0.5 microM) montelukast treatment (p<0.01; n=3). CONCLUSION: Our results suggest that IL-13 alters the expression of ZO-1, and such alterations in the content and distribution of ZO-1 may be relevant in the pathogenesis of proteinuria in the MCNS model.
Subject(s)
Humans , Acetates/pharmacology , Blotting, Western , Dose-Response Relationship, Drug , Interleukin-13/pharmacology , Leukotriene Antagonists/pharmacology , Microscopy, Confocal , Podocytes/drug effects , Proteinuria/pathology , Quinolines/pharmacology , Tight Junctions , Zonula Occludens-1 Protein/metabolismABSTRACT
PURPOSE: The aim of this study was to investigate whether pathologic changes in zonula occludens-1 (ZO-1) are induced by interleukin-13 (IL-13) in the experimental minimal-change nephrotic syndrome (MCNS) model and to determine whether montelukast, a leukotriene receptor antagonist, has an effect on ZO-1 restoration in cultured human podocytes. MATERIALS AND METHODS: Human podocytes cultured on bovine serum albumin-coated plates were treated with different doses of IL-13 and montelukast and then examined for distribution using confocal microscopy and for ZO-1 protein levels using Western blotting. RESULTS: ZO-1 was internalized and shown to accumulate in the cytoplasm of human podocytes in an IL-13 dose-dependent manner. High doses (50 and 100 ng/mL) of IL-13 decreased the levels of ZO-1 protein at 12 and 24 h (both p<0.01; n=3), which were significantly reversed by a high dose (0.5 microM) montelukast treatment (p<0.01; n=3). CONCLUSION: Our results suggest that IL-13 alters the expression of ZO-1, and such alterations in the content and distribution of ZO-1 may be relevant in the pathogenesis of proteinuria in the MCNS model.
Subject(s)
Humans , Acetates/pharmacology , Blotting, Western , Dose-Response Relationship, Drug , Interleukin-13/pharmacology , Leukotriene Antagonists/pharmacology , Microscopy, Confocal , Podocytes/drug effects , Proteinuria/pathology , Quinolines/pharmacology , Tight Junctions , Zonula Occludens-1 Protein/metabolismABSTRACT
The severe local thermal trauma activates a number of systemic inflammatory mediators, such as TNF-α, NF-κB, resulting in a disruption of gut barrier. The gastrointestinal tight junction (TJ) is highly regulated by membrane-associated proteins including zonula occludens protein-1 (ZO-1) and occludin, which can be modulated by inflammatory cytokines. As splenectomy has been shown to reduce secretion of cytokines, we hypothesized that (1) severe scald injury up-regulates TNF-α and NF-κB, meanwhile down-regulates expression of ZO-1 and occludin, leading to the increased intestinal permeability, and (2) splenectomy can prevent the burn-induced decrease in ZO-1 and occludin expression, resulting in improved intestinal barrier. Wistar rats undergoing a 30% total body surface area (TBSA) thermal trauma were randomized to receive an accessorial splenectomy meanwhile or not. Intestinal injury was assessed by histological morphological analysis, and serum endotoxin levels, TNF-α, NF-κB, ZO-1 and occludin levels were detected by Western blotting in the terminal ileum mucosal tissue. 30% TBSA burn caused a significant increase in serum endotoxin levels, but NF-κB, and TNF-α, and the average intestinal villus height and mucosal thickness were decreased significantly. Burn injury could also markedly decrease the levels of ZO-1 and occludin in terminal ileum mucosal tissue (all P<0.01). Splenectomy at 7th day after burn significantly reversed the burn-induced breakdown of ZO-1 and occludin (all P<0.01). The results of this study suggest that severe thermal injury damages the intestinal mucosal barrier. Splenectomy may provide a therapeutic benefit in restoring burn-induced intestinal barrier by decreasing the release of inflammatory cytokines and recovering TJ proteins.
Subject(s)
Animals , Female , Male , Rats , Blotting, Western , Endotoxins , Blood , Hot Temperature , Intestinal Mucosa , NF-kappa B , Blood , Occludin , Metabolism , Rats, Wistar , Splenectomy , Tumor Necrosis Factor-alpha , Blood , Zonula Occludens-1 Protein , MetabolismABSTRACT
Objective To observe the effect of astragaloside IV (A) and SB203580 antagonize cadmium (Cd) toxicity on expression of associated protein and blood-testis barrier(BTB) in rats and to study the protective mechanism of A on it.Methods Totally 21 SD male rats were randomly divided into 7 groups, 3 rats per group:Cd [ intraperitoneally injected with 0.1%CdCl2,1mg/(kg?d)],Cd+A [at the above dose of CdCl2,at the same time with A,10mg/(kg?d)], and Cd +SB203580 [at the above dose of CdCl2,at the same time with SB203580,100μg/(kg?d)], each of the above groups was further divided into continuous five and ten days treatment groups .The control group was intraperitoneally injected with equal dosage of normal saline .The testes were studied by light , electron microscopy , immunohistochemistry and Western blotting .Results In the control group ,irregular and lightly stained nuclei of Sertoli cell ( Sc) in seminiferous tubules were observed by HE staining .A continuous electron density line of tight junction ( TJ) and normal ultrastructure of BTB were observed .After Cd treatment ,the vesicular formation in the Sc was observed .The ultrastructural damage of Sc and TJ was observed .Compared with the corresponding time point of Cd group ,these were weakened in morphology of testis and ultrastructure of TJ after Cd +A or Cd +SB203580 treatment .The positive products of zonula occludens-1 ( ZO-1 ) and claudin-11 were localized mainly in the base of the seminiferous tubule .After Cd treatment , the average absorbance (AA) of ZO-1 and Claudin-11 was decreased significantly compared with that of the control group (P<0.05).After Cd +A or Cd +SB203580 treatment,AA of ZO-1 and Claudin-11 were increased significantly compared with that of the Cd group(P<0.05),though lower than that of the control group .The result of Western blotting showed that phosphorylation-p38MAPK in Cd group was increased significantly compared with that of the control group (P<0.05).After Cd +A or Cd+SB203580 treatment, it was decreased significantly compared with that of the Cd group (P<0.05).Conclusion Cd decreases ZO-1 and Claudin-11 expression and damages ultrastructure of TJ in BTB , asⅣhas protective effect on it , and is related to inhibiting activation of p 38 MAPK pathway .
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AIM:To investigate the effect of hydrogen sulfide ( H2 S) on high glucose ( HG)-induced injury of the mouse podocyte cell line MPC5.METHODS: The cultured MPC5 cells were randomly divided into 4 groups: HG group, normal glucose (NG) group, NG+DL-propargylglycine (PPG) group, and HG+NaHS group.After treated for a certain time, the cells were collected for further detection .The expression of zonula occludens-2 (ZO-2), nephrin,β-cate-nin and cystathionine γ-lyase ( CSE) was determined by Western blotting .RESULTS:High glucose significantly reduced the expression of nephrin, ZO-2 and CSE (P<0.05), while the level of β-catenin was elevated obviously (P<0.05), all in a time-dependent manner.NG+PPG inhibited the levels of ZO-2 and nephrin significantly (P<0.05), and increased the level of β-catenin (P<0.05), all in a PPG concentration-dependent manner.HG+NaHS induced a more significant increase in the levels of ZO-2 and nephrin as compared with HG group (P<0.01), whereas a severe reduction of β-cate-nin in HG+NaHS group was observed as compared with HG group .Compared with NG group , the expression of ZO-2 and nephrin was decreased obviously , and the level of β-catenin was increased in HG +NaHS group.CONCLUSION:Down-regulation of CSE contributes to hyperglycemia-induced podocyte injury .Exogenous H 2 S protects against hyperglycemia-in-duced podocyte injury , possibly through up-regulation of ZO-2 and subsequent suppression of Wnt/β-catenin pathway .