Aim: To investigate the possible functions of human sperm membrane protein (hSMP-1) in the process of fertilization. Methods: A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was ...Aim: To investigate the possible functions of human sperm membrane protein (hSMP-1) in the process of fertilization. Methods: A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 (anti-rhSMP-1) antibodies. Sperm acrosome reaction and spermzona pellucida (ZP) binding assay were carried out in 10-week-old BALB/c mice. Results: Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 (P 〈 0.05). Conclusion: The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.展开更多
●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 tre...●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.展开更多
AIM: To investigate the effect of bone-marrow mesenchymal stem cells (BM MSCs) on the intestinal mucosa barrier in ischemia/reperfusion (I/R) injury. METHODS: BM MSCs were isolated from male Sprague-Dawley rats by den...AIM: To investigate the effect of bone-marrow mesenchymal stem cells (BM MSCs) on the intestinal mucosa barrier in ischemia/reperfusion (I/R) injury. METHODS: BM MSCs were isolated from male Sprague-Dawley rats by density gradient centrifugation, cultured, and analyzed by flow cytometry. I/R injury was induced by occlusion of the superior mesenteric artery for 30 min. Rats were treated with saline, BM MSCs (via intramucosal injection) or tumor necrosis factor (TNF)-α blocking antibodies (via the tail vein). I/R injury was assessed using transmission electron microscopy, hematoxylin and eosin (HE) staining, immunohistochemistry, western blotting and enzyme linked immunosorbent assay.RESULTS: Intestinal permeability increased, tight junctions (TJs) were disrupted, and zona occludens 1 (ZO-1) was downregulated after I/R injury. BM MSCs reduced intestinal mucosal barrier destruction, ZO-1 downregulation, and TJ disruption. The morphological abnormalities after intestinal I/R injury positively correlated with serum TNF-α levels. Administration of anti-TNF-α IgG or anti-TNF-α receptor 1 antibodies attenuated the intestinal ultrastructural changes, ZO-1 downregulation, and TJ disruption. CONCLUSION: Altered serum TNF-α levels play an important role in the ability of BM MSCs to protect against intestinal I/R injury.展开更多
AIM To determine whether cell division cycle(Cdc)42 is regulated by microRNA(miR)-15 a in the development of pediatric inflammatory bowel disease(IBD).METHODS We cultured 293 T cells, used plasmids and performed dual-...AIM To determine whether cell division cycle(Cdc)42 is regulated by microRNA(miR)-15 a in the development of pediatric inflammatory bowel disease(IBD).METHODS We cultured 293 T cells, used plasmids and performed dual-luciferase assay to determine whether Cdc42 is a miR-15 a target gene. We cultured Caco-2 cells, and stimulated them with tumor necrosis factor(TNF)-α. We then employed lentiviruses to alter the expression of miR-15 a and Cdc42. We performed quantitative reverse transcription polymerase chain reaction(qRTPCR) and immunofluorescence to determine whether Cdc42 is regulated by miR-15 a in Caco-2 cells. Finally, we collected ileocecal tissue by endoscopy from patients and performed qRT-PCR to examine the expression of miR-15 a and Cdc42 in pediatric IBD patients.RESULTS Target Scan and dual-luciferase assay revealed thatCdc42 was a miR-15 a target gene. MiR-15 a expression increased(P = 0.0038) and Cdc42 expression decreased(P = 0.0013) in cells stimulated with TNF-α, and the expression of the epithelial junction proteins zona occludens(ZO)-1(P < 0.05) and E-cadherin(P < 0.001) decreased. Cdc42 levels decreased in miR-15 a-mimic cells(P < 0.001) and increased in miR-15 a inhibitor cells(P < 0.05). ZO-1 and E-cadherin decreased in mi R-15 a-mimic cells(P < 0.001) but not in the miR-15 a inhibitor + TNF-α cells. In Lv-Cdc42 + TNF-α cells, ZO-1 and E-cadherin expression increased compared to the Lv-Cdc42-NC + TNF-α(P < 0.05) or miR-15 a-mimic cells(P < 0.05). Fifty-four pediatric IBD patients were included in this study, 21 in the control group, 19 in the Crohn's disease(CD) active(AC) group, seven in the CD remission(RE) group, and seven in the ulcerative colitis(UC) group. MiR-15 a increased and Cdc42 decreased in the CD AC group compared to the control group(P < 0.05). miR-15 a decreased and Cdc42 increased in the CD RE group compared to the CD AC group(P < 0.05). miR-15 a was positively correlated with the Pediatric Crohn's disease Activity Index(PCDAI)(P = 0.006), while Cdc42 was negatively correlated with PCDAI(P = 0.0008). Finally, miR-15 a expression negatively correlated with Cdc42 in pediatric IBD patients(P = 0.0045).CONCLUSION Mi R-15 a negatively regulates epithelial junctions through Cdc42 in Caco-2 cells and pediatric IBD patients.展开更多
An average human ejaculate contains over 100 million sperm, but only a few succeed in accomplishing the journey to an egg by migration through the female reproductive tract. Among these few sperm, only one participate...An average human ejaculate contains over 100 million sperm, but only a few succeed in accomplishing the journey to an egg by migration through the female reproductive tract. Among these few sperm, only one participates in fertilization. There might be an ingenious molecular mechanism to ensure that the very best sperm fertilize an egg. However, recent gene disruption experiments in mice have revealed that many factors previously described as important for fertilization are largely dispensable. One could argue that the fertilization mechanism is made robust against gene disruptions. However, this is not likely, as there are already six different gene-disrupted mouse lines (Calmegin, Adam Ia, Adam2, Adam3, Ace and Pgapl), all of which result in male sterility. The sperm from these animals are known to have defective zona-binding ability and at the same time lose oviduct-migrating ability. Concerning spermzona binding, the widely accepted involvement of sugar moiety on zona pellucida 3 (ZP3) is indicated to be dispensable by gene disruption experiments. Thus, the landscape of the mechanism of fertilization is revolving considerably. In the sperm-egg fusion process, CD9 on egg and IZUMO1 on sperm have emerged as essential factors. This review focuses on the mechanism of fertilization elucidated by gene-manipulated animals.展开更多
Aldosterone mediates many of the physiological and pathophysiological/cardio-toxic effects of angiotensin II(Ang II). Its synthesis and secretion from the zona glomerulosa cells of the adrenal cortex, elevated in chro...Aldosterone mediates many of the physiological and pathophysiological/cardio-toxic effects of angiotensin II(Ang II). Its synthesis and secretion from the zona glomerulosa cells of the adrenal cortex, elevated in chronic heart failure(HF), is induced by Ang II type 1 receptors(AT1Rs). The AT1R is a G protein-coupled receptor, mainly coupling to Gq/11 proteins. However, it can also signal through β-arrestin-1(βarr1) or-2(βarr2), both of which mediate G protein-independent signaling. Over the past decade, a second, Gq/11 proteinindependent but βarr1-dependent signaling pathway emanating from the adrenocortical AT1R and leading to aldosterone production has become appreciated. Thus, it became apparent that AT1R antagonists that block both pathways equally well are warranted for fully effective aldosterone suppression in HF. This spurred the comparison of all of the currently marketed angiotensin receptor blockers(ARBs, AT1R antagonists or sartans) at blocking activation of the two signaling modes(G protein-, and βarr1-dependent) at the Ang IIactivated AT1R and hence, at suppression of aldosterone in vitro and in vivo. Although all agents are very potent inhibitors of G protein activation at the AT1R, candesartan and valsartan were uncovered to be the most potent ARBs at blocking βarr activation by Ang II and at suppressing aldosterone in vitro and in vivo in post-myocardial infarction HF animals. In contrast, irbesartan and losartan are virtually G protein-"biased" blockers at the human AT1R, with very low efficacy for βarr inhibition and aldosterone suppression. Therefore, candesartan and valsartan(and other, structurally similar compounds) may be the most preferred ARB agents for HF pharmacotherapy, as well as for treatment of other conditions characterized by elevated aldosterone.展开更多
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)mod...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.展开更多
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...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.展开更多
AIM:To explore the protective effect of bone marrow mesenchymal stem cells(BM MSCs)in the small intestinal mucosal barrier following heterotopic intestinal transplantation(HIT)in a rat model.METHODS:BM MSCs were isola...AIM:To explore the protective effect of bone marrow mesenchymal stem cells(BM MSCs)in the small intestinal mucosal barrier following heterotopic intestinal transplantation(HIT)in a rat model.METHODS:BM MSCs were isolated from male Lewis rats by density gradient centrifugation,cultured,and analyzed by flow cytometry.The HIT models were divided into a non-rejection group,saline-treated rejection group(via penile vein),and BM MSC–treated group(via penile vein).Intestinal mucosal barrier injury was estimated by diamine oxidase(DAO)and D-lactic acid(D-LA)expression levels.Tumor necrosis factor-α(TNF-α),interferon-γ(INF-γ),interleukin-10(IL-10),and transforming growth factor-β(TGF-β)were detected by enzyme-linked immunosorbent assay.Ultrastructural change of tight junctions(TJs)was observed under transmission electron microscope.Expression levels of the TJ proteins occludin and zona occludens(ZO)-1,affected by the inflammatory factors,were measured using real-time polymerase chain reaction and Western blotting.RESULTS:The pathological score at each time point after surgery indicated significantly less serious injury in the BM MSCs-treated group than in the rejection group(P<0.05).In the former,graft levels of DAO and D-LA were reduced,and TNF-αand INF-γproduction was inhibited(at day 7:10.6473±0.0710vs 17.2128±0.4991,P<0.05;545.1506±31.9416vs 810.2637±25.1175,P<0.05).IL-10 and TGF-βproduction was increased greatly(at day 7:125.7773±4.7719 vs 80.3756±2.5866,P<0.05;234.5273±9.3980 vs 545.1506±31.9416,P<0.05).There was increased expression of occludin and ZO-1 protein(at day 7:0.2674±0.0128 vs 0.1352±0.0142,P<0.05;at day 5:0.7189±0.0289 vs 0.4556±0.0242,P<0.05)and mRNA(at day 7:0.3860±0.0254 vs 0.1673±0.0369,P<0.05;at day 5:0.5727±0.0419 vs0.3598±0.0242,P<0.05).CONCLUSION:BM MSCs can improve intestinal barrier permeability,repair TJs,and increase occludin and ZO-1 protein expression.With altered cytokine levels,they can protect the intestinal mucosa after transplantation.展开更多
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 t...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.展开更多
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 primar...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.展开更多
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:...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.展开更多
文摘Aim: To investigate the possible functions of human sperm membrane protein (hSMP-1) in the process of fertilization. Methods: A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 (anti-rhSMP-1) antibodies. Sperm acrosome reaction and spermzona pellucida (ZP) binding assay were carried out in 10-week-old BALB/c mice. Results: Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 (P 〈 0.05). Conclusion: The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.
基金Supported by the National Natural Science Foundation of China(No.81770889)Zhuhai Science and Technology Program(No.ZH22036201210134PWC).
文摘●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.
基金Supported by Natural Science Foundation of China, No.81270528the Natural Science Foundation of Tianjin, No. 08JCYBJC08400, No. 11JCZDJC27800 and No. 12JCZDJC25200the Technology Foundation of Health Bureau in Tianjin, No.2011KY11
文摘AIM: To investigate the effect of bone-marrow mesenchymal stem cells (BM MSCs) on the intestinal mucosa barrier in ischemia/reperfusion (I/R) injury. METHODS: BM MSCs were isolated from male Sprague-Dawley rats by density gradient centrifugation, cultured, and analyzed by flow cytometry. I/R injury was induced by occlusion of the superior mesenteric artery for 30 min. Rats were treated with saline, BM MSCs (via intramucosal injection) or tumor necrosis factor (TNF)-α blocking antibodies (via the tail vein). I/R injury was assessed using transmission electron microscopy, hematoxylin and eosin (HE) staining, immunohistochemistry, western blotting and enzyme linked immunosorbent assay.RESULTS: Intestinal permeability increased, tight junctions (TJs) were disrupted, and zona occludens 1 (ZO-1) was downregulated after I/R injury. BM MSCs reduced intestinal mucosal barrier destruction, ZO-1 downregulation, and TJ disruption. The morphological abnormalities after intestinal I/R injury positively correlated with serum TNF-α levels. Administration of anti-TNF-α IgG or anti-TNF-α receptor 1 antibodies attenuated the intestinal ultrastructural changes, ZO-1 downregulation, and TJ disruption. CONCLUSION: Altered serum TNF-α levels play an important role in the ability of BM MSCs to protect against intestinal I/R injury.
基金Supported by the National Natural Science Foundation of Shanghai,No.201540068
文摘AIM To determine whether cell division cycle(Cdc)42 is regulated by microRNA(miR)-15 a in the development of pediatric inflammatory bowel disease(IBD).METHODS We cultured 293 T cells, used plasmids and performed dual-luciferase assay to determine whether Cdc42 is a miR-15 a target gene. We cultured Caco-2 cells, and stimulated them with tumor necrosis factor(TNF)-α. We then employed lentiviruses to alter the expression of miR-15 a and Cdc42. We performed quantitative reverse transcription polymerase chain reaction(qRTPCR) and immunofluorescence to determine whether Cdc42 is regulated by miR-15 a in Caco-2 cells. Finally, we collected ileocecal tissue by endoscopy from patients and performed qRT-PCR to examine the expression of miR-15 a and Cdc42 in pediatric IBD patients.RESULTS Target Scan and dual-luciferase assay revealed thatCdc42 was a miR-15 a target gene. MiR-15 a expression increased(P = 0.0038) and Cdc42 expression decreased(P = 0.0013) in cells stimulated with TNF-α, and the expression of the epithelial junction proteins zona occludens(ZO)-1(P < 0.05) and E-cadherin(P < 0.001) decreased. Cdc42 levels decreased in miR-15 a-mimic cells(P < 0.001) and increased in miR-15 a inhibitor cells(P < 0.05). ZO-1 and E-cadherin decreased in mi R-15 a-mimic cells(P < 0.001) but not in the miR-15 a inhibitor + TNF-α cells. In Lv-Cdc42 + TNF-α cells, ZO-1 and E-cadherin expression increased compared to the Lv-Cdc42-NC + TNF-α(P < 0.05) or miR-15 a-mimic cells(P < 0.05). Fifty-four pediatric IBD patients were included in this study, 21 in the control group, 19 in the Crohn's disease(CD) active(AC) group, seven in the CD remission(RE) group, and seven in the ulcerative colitis(UC) group. MiR-15 a increased and Cdc42 decreased in the CD AC group compared to the control group(P < 0.05). miR-15 a decreased and Cdc42 increased in the CD RE group compared to the CD AC group(P < 0.05). miR-15 a was positively correlated with the Pediatric Crohn's disease Activity Index(PCDAI)(P = 0.006), while Cdc42 was negatively correlated with PCDAI(P = 0.0008). Finally, miR-15 a expression negatively correlated with Cdc42 in pediatric IBD patients(P = 0.0045).CONCLUSION Mi R-15 a negatively regulates epithelial junctions through Cdc42 in Caco-2 cells and pediatric IBD patients.
文摘An average human ejaculate contains over 100 million sperm, but only a few succeed in accomplishing the journey to an egg by migration through the female reproductive tract. Among these few sperm, only one participates in fertilization. There might be an ingenious molecular mechanism to ensure that the very best sperm fertilize an egg. However, recent gene disruption experiments in mice have revealed that many factors previously described as important for fertilization are largely dispensable. One could argue that the fertilization mechanism is made robust against gene disruptions. However, this is not likely, as there are already six different gene-disrupted mouse lines (Calmegin, Adam Ia, Adam2, Adam3, Ace and Pgapl), all of which result in male sterility. The sperm from these animals are known to have defective zona-binding ability and at the same time lose oviduct-migrating ability. Concerning spermzona binding, the widely accepted involvement of sugar moiety on zona pellucida 3 (ZP3) is indicated to be dispensable by gene disruption experiments. Thus, the landscape of the mechanism of fertilization is revolving considerably. In the sperm-egg fusion process, CD9 on egg and IZUMO1 on sperm have emerged as essential factors. This review focuses on the mechanism of fertilization elucidated by gene-manipulated animals.
文摘Aldosterone mediates many of the physiological and pathophysiological/cardio-toxic effects of angiotensin II(Ang II). Its synthesis and secretion from the zona glomerulosa cells of the adrenal cortex, elevated in chronic heart failure(HF), is induced by Ang II type 1 receptors(AT1Rs). The AT1R is a G protein-coupled receptor, mainly coupling to Gq/11 proteins. However, it can also signal through β-arrestin-1(βarr1) or-2(βarr2), both of which mediate G protein-independent signaling. Over the past decade, a second, Gq/11 proteinindependent but βarr1-dependent signaling pathway emanating from the adrenocortical AT1R and leading to aldosterone production has become appreciated. Thus, it became apparent that AT1R antagonists that block both pathways equally well are warranted for fully effective aldosterone suppression in HF. This spurred the comparison of all of the currently marketed angiotensin receptor blockers(ARBs, AT1R antagonists or sartans) at blocking activation of the two signaling modes(G protein-, and βarr1-dependent) at the Ang IIactivated AT1R and hence, at suppression of aldosterone in vitro and in vivo. Although all agents are very potent inhibitors of G protein activation at the AT1R, candesartan and valsartan were uncovered to be the most potent ARBs at blocking βarr activation by Ang II and at suppressing aldosterone in vitro and in vivo in post-myocardial infarction HF animals. In contrast, irbesartan and losartan are virtually G protein-"biased" blockers at the human AT1R, with very low efficacy for βarr inhibition and aldosterone suppression. Therefore, candesartan and valsartan(and other, structurally similar compounds) may be the most preferred ARB agents for HF pharmacotherapy, as well as for treatment of other conditions characterized by elevated aldosterone.
基金Supported by the National Natural Science Foundation of China(No.81660217)Youth Foundation of the First Affiliated Hospital of Hainan Medical University(No.HYYFYPY201922)。
文摘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.
基金Supported by a Research Grant from Beijing Administration of Traditional Chinese Medicine(No.JJ 2008-015)
文摘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.
基金Supported by The Natural Science Foundation of China,No.81270528the Natural Science Foundation of Tianjin,China,No.08JCYBJC08400,No.11JCZDJC27800 and No.12JCZDJC25200the Technology Foundation of Health Bureau of Tianjin,China,No.2011KY11
文摘AIM:To explore the protective effect of bone marrow mesenchymal stem cells(BM MSCs)in the small intestinal mucosal barrier following heterotopic intestinal transplantation(HIT)in a rat model.METHODS:BM MSCs were isolated from male Lewis rats by density gradient centrifugation,cultured,and analyzed by flow cytometry.The HIT models were divided into a non-rejection group,saline-treated rejection group(via penile vein),and BM MSC–treated group(via penile vein).Intestinal mucosal barrier injury was estimated by diamine oxidase(DAO)and D-lactic acid(D-LA)expression levels.Tumor necrosis factor-α(TNF-α),interferon-γ(INF-γ),interleukin-10(IL-10),and transforming growth factor-β(TGF-β)were detected by enzyme-linked immunosorbent assay.Ultrastructural change of tight junctions(TJs)was observed under transmission electron microscope.Expression levels of the TJ proteins occludin and zona occludens(ZO)-1,affected by the inflammatory factors,were measured using real-time polymerase chain reaction and Western blotting.RESULTS:The pathological score at each time point after surgery indicated significantly less serious injury in the BM MSCs-treated group than in the rejection group(P<0.05).In the former,graft levels of DAO and D-LA were reduced,and TNF-αand INF-γproduction was inhibited(at day 7:10.6473±0.0710vs 17.2128±0.4991,P<0.05;545.1506±31.9416vs 810.2637±25.1175,P<0.05).IL-10 and TGF-βproduction was increased greatly(at day 7:125.7773±4.7719 vs 80.3756±2.5866,P<0.05;234.5273±9.3980 vs 545.1506±31.9416,P<0.05).There was increased expression of occludin and ZO-1 protein(at day 7:0.2674±0.0128 vs 0.1352±0.0142,P<0.05;at day 5:0.7189±0.0289 vs 0.4556±0.0242,P<0.05)and mRNA(at day 7:0.3860±0.0254 vs 0.1673±0.0369,P<0.05;at day 5:0.5727±0.0419 vs0.3598±0.0242,P<0.05).CONCLUSION:BM MSCs can improve intestinal barrier permeability,repair TJs,and increase occludin and ZO-1 protein expression.With altered cytokine levels,they can protect the intestinal mucosa after transplantation.
基金financially supported by the National Natural Science Foundation of China,No.81301006a grant from Henan Provincial Scientific and Technological Research Projects of China,No.132102310092
文摘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.
基金Supported by JIPMER intramural research grant,Indian Council of Medical Research (ICMR),New Delhi,India,No. 3/1/3 J.R.F.-2016/LS/HRD
文摘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.
基金Supported by National Natural Science Foundation of China:Research on the Biological Mechanism of Nourishing Fire and Nourishing Soil Building Muscle Regeneration in Repairing Intestinal Mucosal Barrier Damage in UC Based on MLCK/Rho/PKC-Crosstalk(No.81973821)。
文摘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.