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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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OBJECTIVE: To investigate the effect of transforming growth factor β1( TGF-β1) type Ⅰ receptor kinase inhibitor SB431542 on epithelial-mesenchymal transition( EMT) induced by paraquat in type Ⅱ alveolar epithelial cells A549. METHODS: A549 cells were randomly divided into control group,paraquat group and TGF-β1 blockade group,with3 samples in each group. The cells in the control group were cultured without any treatment,cells in paraquat group were stimulated by paraquat of a final concentration of 20 μmol/L,cells in TGF-β1 blockade group were treated with paraquat with a final concentration of 20 μmol/L and SB431542 with a final concentration of 20 μg/L. After 5 days,cultured cells were harvested and observed for morphologic and phenotypic characteristics using inverted phase contrast microscope and scanning electron microscope. Cell migration was assayed by Transwell chamber. The expression of target protein was detected by Western blot. The enzyme-linked immunosorbent assay was used to detect the TGF-β1 levels. RESULTS: Under inverted phase contrast microscope and scanning electron microscope,A549 cells in control group grew normally,cells in paraquat group changed from epithelial morphology to mesenchymal morphology,cells in TGF-β1 blockade group reversed to epithelial cell morphology. The results of cell migration showed that the number of cells in the paraquat group passed through the membrane was higher than that in the control group and TGF-β1 blockade group( P < 0. 05). The relative expression of E-cadherin and zonula occluden-1 in paraquat group was decreased( P < 0. 05),while the relative expression of vimentin,α-smooth muscle actin,type I collagen,the phosphorylation Smad and Mad related protein( p-Smad) 2 and p-Smad3 was elevated( P < 0. 05),compared to control group and TGF-β1 blockade group. The levels of total TGF-β1 and active TGF-β1 increased in paraquat group than that in control group( P < 0. 05). CONCLUSION: Paraquat induced EMT in A549 cells by activating TGF-β1/Smads signaling pathway. Early treatment with SB431542 can inhibit paraquatinduced EMT by blocking TGF-β1/Smads signaling pathway.
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Objective To observe the effects of perfusion of rosiglitazone (RSG) in lesion areas on the expression levels of the perihematomal tight junction-associated proteins occludin and zonula occluden-1 (ZO-1) mRNA,the permeability of blood-brain-barrier (BBB),and neurological function score in a rabbit model of cerebral hemorrhage (ICH).Methods A total of 45 healthy male rabbits were selected (a body mass of 2.0 to 2.5 kg).They were divided into 3 groups,a control group,a ICH model group,and a RSG treatment group (n =15,5 of them for BBB determination) according to the random number table.The control group was use to simulate the process of making intracranial hematoma.After successful puncture,the target was iujected with isotonic saline 0.3 ml and isotonic saline 0.1 ml was injected again after 6 h;after successful puncture,the ICH model group was injected with 0.3 ml of autologous non-anticoagulant arterial blood,and the target was injected into isotonic saline 0.1 ml after 6 h;RSG 0.5 mg was infused into the hematoma area (dissolved in 0.1 ml isotonic saline) in the RSG treatment group at 6 h after the ICH model was successfully induced.All rabbits in each group were sacrificed on day 7 after the neurological deficit scale score (Purdy score).Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression levels of perihematomal oecludin and ZO-1 mRNA.The formamide method was used to measure the Evans blue (EB) content in the perihematomal tissue in order to evaluate the permeability of BBB.Results (1) Neurological function scores:Purdy scores of the control group,ICH model group,and RSG treatment group were 2.53 ± 0.05,8.13 ± 0.06),and 6.67 ± 0.08,respectively.There were significant differences among the groups (F =459.116,P ＜ 0.01).Compared with the control group,Purdy scores of the ICH model group and RSG treatment group were increased significantly (all P ＜ 0.01).Compared with the ICH model group,Purdy scores of the RSG treatment group were decreased (P ＜ 0.05).(2) The expression levels of occludin and ZO-1 mRNA:The differences were statistically significant in occludin and ZO-1 mRNA in the control group,ICH model group,and RSG treatment group (1.013 ±0.051,1.001 ± 0.045;0.221 ± 0.017,0.247 ± 0.019;0.498 ± 0.041,and 0.613 ± 0.045,respectively in each group;F =443.924 and 381.929 respectively,all P ＜ 0.01).Compared with the control group,the expression levels of occludin and ZO-1 mRNA were significantly decreased in the ICH model group and RSG treatment group (all P ＜ 0.01).Compared with the ICH model group,the expression levels of occludin and ZO-1 mRNA were increased in the RSG treatment group (all P ＜ 0.05).(3) The permeability of BBB:The EB content in the control group,ICH model group,and RSG treatment group were 12.0 ± 1.0,51.6 ± 0.9,and 36.4 ± 1.0 μg/g,respectively.The differences were statistically significant among the groups (F =223.516,P ＜ 0.01).Compared with the control group,the EB content was significantly increased in the ICH model group and RSG treatment group (all P ＜ 0.01).Compared with the model group,the EB content was significantly decreased in the RSG treatment group (P ＜ 0.01).Conclusion The perfusion of RSG in the lesion area can significantly improve the neurological function of rabbits after ICH,increase the expression levels of occludin and ZO-1 mRNA in the perihematomal tissue,and decrease the permeability of BBB.
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Background:With the development of capsule endoscopy,small intestinal injury induced by non-steroidal anti-inflammatory drugs( NSAIDs)has become an issue of growing concern. Although there are a variety of drugs used for NSAIDs-induced gastric mucosal injury,small intestinal injury caused by NSAIDs is lack of effective prevention and treatment modalities. Aims:To investigate the protective effect and possible mechanism of rebamipide on human colon cancer cell line Caco-2 injury induced by aspirin. Methods:In aspirin group,Caco-2 cells were treated with aspirin 10 mmol/L;in rebamipide group,Caco-2 cells were treated with aspirin and different concentrations of rebamipide(0. 1, 0. 5,1. 0 mmol/L),and a negative control group was established. Cell proliferation inhibition was measured by MTT assay. Cell apoptosis was determined by flow cytometry. Morphological changes of cells were observed under inverted phase contrast microscope. Permeability of cells was assessed by Transwell assay. Expressions of tight junction proteins occludin and zonula occluden-1(ZO-1),as well as mitogen activated protein kinase(MAPK)signaling pathway-associated proteins including extracellular signal-regulated kinase(ERK)1/2,phosphorylated ERK1/2(p-ERK1/2),p38,p-p38,c-Jun N-terminal kinase( JNK),and p-JNK,were determined by Western blotting. Results:Proliferation inhibition rate,apoptosis rate and permeability of Caco-2 cells in rebamipide 0. 1,0. 5,1. 0 mmol/L groups were significantly lower than those in aspirin group in a dose-dependent manner(P<0. 05). Injuries of Caco-2 cells were seen in aspirin group by inverted phase contrast microscope and rebamipide could reduce these injuries. Expressions of occludin,ZO-1 and p-JNK were significantly higher and expressions of p-p38 and p-ERK1/2 were significantly lower in rebamipide 0. 1,0. 5,1. 0 mmol/L groups than those in aspirin group in a dose-dependent manner(P<0. 05). Conclusions:Rebamipide have a protective effect against aspirin-induced Caco-2 cell injury,probably through regulating MAPK signaling pathway( inhibiting p38 and ERK1/2 phosphorylation,stimulating JNK phosphorylation),and subsequently up-regulating the expressions of tight junction proteins and decreasing the permeability of cells.
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Objective To discuss the effect of hyperthermia on tight junctions of the endothelial cells in the blood-brain barrier and explore the molecular mechanism. Methods An in vitro blood-brain barrier model was established by coculture of ECV304 and astrocytes. Transendothelial resistance (TER) of in vitro blood-brain barrier was determined by Millicell-ERS system. The morphological change of tight junctions of the endothelial cells in the in vitro blood-brain barrier was determined by the method of silver staining. The expression levels of zonula occluden 1 (ZO-1) and occludin were analyzed by means of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western bloting. Results After two hours at 43℃, the mean value of TER was decreased from (321.30? 58.59) ??cm2 to (65.67?6.02) ??cm2. The integrity of tight junctions was destroyed and the expressions of ZO-1 and occludin decreased significantly. Conclusions Hyperthermia can destroy the tight junctions of the endothelial cells in the in vitro blood-brain barrier. The expression decrease of ZO-1 and occludin induced by hyperthermia is one of the most important molecular mechanisms.