Vascular endothelial growth factor-165b protects the blood-retinal barrier from damage after acute high intraocular pressure in rats

AIM:To elucidate the role of vascular endothelial growth factor-165b(VEGF-165b)in blood-retinal barrier(BRB)injury in the rat acute glaucoma model.METHODS:In this study,the rat acute high intraocular pressure(HIOP)model was established before and after intravitreous injection of anti-VEGF-165b antibody.The expression of VEGF-165b and zonula occludens-1(ZO-1)in rat retina was detected by double immunofluorescence staining and Western blotting,and the breakdown of BRB was detected by Evans blue(EB)dye.RESULTS:The intact retina of rats expressed VEGF-165b and ZO-1 protein,which were mainly located in the retinal ganglion cell layer and the inner nuclear layer and were both co-expressed with vascular endothelial cell markers CD31.After acute HIOP,the expression of VEGF-165b was up-regulated;the expression of ZO-1 was down-regulated at 12h and then recovered at 3d;EB leakage increased,peaking at 12h.After intravitreous injection of anti-VEGF-165b antibody,the expression of VEGF-165b protein was no significantly changed;and the down-regulation of the expression of ZO-1 was more obvious;EB leakage became more serious,peaking at 3d.EB analysis also showed that EB leakage in the peripheral retina was greater than that in the central retina.CONCLUSION:The endogenous VEGF-165b protein may protect the BRB from acute HIOP by regulating the expression of ZO-1.The differential destruction of BRB after acute HIOP may be related to the selective loss of retinal ganglion cells.

Semaphorin 7A impairs barrier function in cultured human corneal epithelial cells in a manner dependent on nuclear factor-kappa B

●AIM:To evaluate the role of semaphorin 7A(Sema7A)and its associated regulatory mechanisms in modulating the barrier function of cultured human corneal epithelial cells(HCEs).●METHODS:Barrier models of HCEs were treated with recombinant human Sema7A at concentrations of 0,125,250,or 500 ng/mL for 24,48,or 72h in vitro.Transepithelial electrical resistance(TEER)as well as Dextran-fluorescein isothiocyanate(FITC)permeability assays were conducted to assess barrier function.To quantify tight junctions(TJs)such as occludin and zonula occludens-1(ZO-1)at the mRNA level,reverse transcriptionpolymerase chain reaction(RT-PCR)analysis was performed.Immunoblotting was used to examine the activity of the nuclear factor-kappa B(NF-κB)signaling pathway and the production of TJs proteins.Immunofluorescence analyses were employed to localize the TJs.Enzyme-linked immunosorbent assay(ELISA)and RT-PCR were utilized to observe changes in interleukin(IL)-1βlevels.To investigate the role of NF-κB signaling activation and IL^(-1)βin Sema7A’s anti-barrier mechanism,we employed 0.1μmol/L IκB kinase 2(IKK2)inhibitor IV or 500 ng/mL IL^(-1)receptor(IL-1R)antagonist.●RESULTS:Treatment with Sema7A resulted in decreased TEER and increased permeability of Dextran-FITC in HCEs through down-regulating mRNA and protein levels of TJs in a time-and dose-dependent manner,as well as altering the localization of TJs.Furthermore,Sema7A stimulated the activation of inhibitor of kappa B alpha(IκBα)and expression of IL-1β.The anti-barrier function of Sema7A was significantly suppressed by treatment with IKK2 inhibitor IV or IL-1R antagonists.●CONCLUSION:Sema7A disrupts barrier function through its influence on NF-κB-mediated expression of TJ proteins,as well as the expression of IL-1β.These findings suggest that Sema7A could be a potential therapeutic target for the diseases in corneal epithelium.

Research on the Relationship between Thick Greasy Tongue Fur Formation and Vascular Endothelial Cell Permeability with the Protein Expression of Zonula Occludens-1

Objective：To determine the relationship of thick greasy tongue fur formation and permeability of vascular endothelial cells（ECs）with the protein expression of zonula occludens-1（ZO-1）.Methods：Sprague Dawley rats were randomly divided into a model group of severe acute pancreatitis（SAP）and a sham-operated （SO）group.The SAP rats were further divided into two subgroups on the basis of tongue-coating status：a thick greasy tongue fur group（SAP-TGF）and a normal tongue fur group（SAP-NF）.Six rats were chosen randomly from every group mentioned above for an Evans blue assay 5 days after model establishment.For the histomorphology analysis,the expressions of ZO-1 protein and mRNA were studied by hematoxylin-eosin （HE）staining,transmission electron microscope,Western blot,and Q-PCR using blood and tongue tissues, which were collected from 8 rats randomly chosen from each group.Results：The papillae density of the rat tongue surface and the caryocinesis frequency of the basal layer were significantly increased in the SAP-TGF group compared with the SO group（P0.05）.Evans blue levels in the tongue tissue of the SAP-TGF group were significantly higher than that of the SO and SAP-NF groups（P0.05）.Vascular ECs were wider and obviously fissured in the SAP-TGF group under transmission electron microscope observation.The protein and mRNA expression of ZO-1 in the SAP-TGF group were lower than those in the SAP-NF（P0.05）.Conclusions： Reproductive activity enhancement of glossal epithelial cells was one of the main characteristics of thick greasy tongue fur formation.An increase in vasopermeability was closely associated with thick greasy tongue fur formation.Tight junction structural variation of vascular ECs might play an important role in the pathological and physiological process of thick greasy tongue fur formation.

Role of zonula occludens in gastrointestinal and liver cancers

A growing body of evidence suggests that tight junction(TJ)proteins play a crucial role in the pathogenesis of various diseases,including gastrointestinal(GI)cancer and hepatocellular carcinoma(HCC).TJ proteins primarily maintain the epithelial and endothelial cells intact together through integral proteins however,recent reports suggest that they also regulate gene expression necessary for cell proliferation,angiogenesis,and metastasis through adapter proteins such as zonula occludens(ZO).ZO proteins are membrane-associated cytosolic scaffolding proteins that modulate cell proliferation by interacting with several transcription factors.Reduced ZO proteins in GI cancer and HCC are correlated with tumor development and poor prognosis.Pubmed has searched for using the keyword ZO and gastric cancer,ZO and cancer,and ZO and HCC for the last ten years to date.This review summarized the role of ZO proteins in cell proliferation and their expression in GI cancer and HCC.Furthermore,therapeutic interventions targeting ZO in GI and liver cancers are reviewed.

黄体酮对脑缺血再灌注大鼠紧密连接蛋白ZO－1和occludin表达的影响

目的探讨黄体酮对大鼠局灶性脑缺血再灌注后血脑屏障紧密连接蛋白ZO—1、occludin表达及血脑屏障通透性的影响。方法将42只健康雄性SD大鼠按随机数字表法分为假手术组（6只）和缺血再灌注组，后者再按再灌注时间分为缺血2h再灌注3h、6h、12h、24h、48h及72h组（各6只）。缺血再灌注组用线栓法制备成大鼠大脑中动脉缺血再灌注模型。采用荧光分光光度法测定缺血侧脑组织中伊文氏蓝（EB）含虽来评价血脑屏障的通透性，Westernblotting法检测脑组织ZO－1和occludin的表达。取EB漏出最多组的时间点，增设黄体酮干预组和溶剂对照组（各6只1．与相同时间点的缺血再灌注组比较，观察黄体酮对ZO－1、occludin表达及fffL脑屏障通透性的影响。结果缺血2h冉灌注3h时脯组织EB含量丌始增加，再灌注24h时达高峰；ZO—1、occludin的表达在缺血2h再灌注3h时开始下降，再灌注24h时达最低。黄体酮干预组EB含量明显低于缺血2h再灌注24h组，差异有统计学意义（P〈0．05）。黄体酮干预组ZO—1和occludin的表达水平均明显高于缺血2h再灌注24h组，差异有统计学意义（P〈0．05）。结论黄体酮可抑制缺出再灌注大鼠紧密连接蛋白ZO－1和occludin表达的降低，从而起到保护舡脑屏障的作用。

Progesterone is neuroprotective by inhibiting cerebral edema after ischemia

Ischemic edema can alter the structure and permeability of the blood-brain barrier. Recent studies have reported that progesterone reduces cerebral edema after cerebral ischemia. However, the underlying mechanism of this effect has not yet been elucidated. In the present study, progesterone effectively reduced Evans blue extravasation in the ischemic penumbra, but not in the ischemic core, 48 hours after cerebral ischemia in rats. Progesterone also inhibited the down-regulation of gene and protein levels of occludin and zonula occludens-1 in the penumbra. These results indicate that progesterone may effectively inhibit the down-regulation of tight junctions, thereby maintaining the integrity of the blood-brain barrier and reducing cerebral edema.

Efficacy of Qifu Lizhong enema prescription(芪附理中灌肠方)on intestinal mucosal tight junction function modulation of ulcerative colitis rat model

OBJECTIVE:To investigate the efficacy and mechanism of Qifu Lizhong enema prescription(芪附理中灌肠方,QFLZ)on intervening ulcerative colitis(UC)rat model with TCM spleen and kidney Yang insufficiency syndrome METHODS:Seventy-two male Sprague-Dawley rats were randomly assigned to six groups:normal model,mesalazine,and QFLZ high,medium,and low dose groups,each with 12 rats.After 3 d of adaptation feeding,all groups except the normal group were induced using rhubarb decoction in combination with trinitrobenzene sulfonic acid(TNBS)/55%ethanol to establish a UC rat model.Following successful modeling,the normal and model groups received daily saline enema,while the Chinese medicine and Western medicine groups received daily QFLZ and Mesalazine enema for 2 weeks respectively.The disease activity index score,hematoxylin and eosin staining,immunohistochemistry,and Western blotting were used to determine the expression of claudin 1,claudin 2,zonula occludens-1protein(ZO-1),and F-actin proteins in each rat colon tissue following treatment.RESULTS:QFLZ significantly alleviated the structural disorganization in the form of epithelial glands in the intestinal mucosa of rats with UC and retarded the progression of the disease.The intestinal mucosal epithelial cells of UC rats showed decreased expression of claudin 1,ZO-1,F-actin(P<0.05),claudin 2 appeared elevated(P<0.05),which resulted in impaired TJ.Treatment with QFLZ resulted in elevated expression of claudin 1(P<0.05),ZO-1(P<0.05)and F-actin(P<0.05)and decreased expression of claudin 2(P<0.05),which allowed for repair of the intestinal mucosal TJ,which in turn served as a treatment for UC.CONCLUSIONS:The mechanism of repairing TJ function and repairing the intestinal mucosal barrier by QFLZ may be associated with up-regulation of claudin 1,ZO-1,and F-actin levels,and down-regulation of claudin 2 expression level.