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.

17β-estradiol inhibits TGF-β-induced collagen gel contraction mediated by human Tenon fibroblasts via Smads and MAPK signaling pathways

AIM:To investigate the impact of 17β-estradiol on the collagen gels contraction(CGC)and inflammation induced by transforming growth factor(TGF)-βin human Tenon fibroblasts(HTFs).METHODS:HTFs were three-dimensionally cultivated in type I collagen-generated gels with or without TGF-β(5 ng/mL),17β-estradiol(12.5 to 100μmol/L),or progesterone(12.5 to 100μmol/L).Then,the collagen gel diameter was determined to assess the contraction,and the development of stress fibers was analyzed using immunofluorescence staining.Immunoblot and gelatin zymography assays were used to analyze matrix metalloproteinases(MMPs)and tissue inhibitors of metalloproteinases(TIMPs)being released into culture supernatants.Enzyme-linked immunosorbent assay(ELISA)and reverse transcription-quantitative polymerase chain reaction(RT-PCR)were used to detect interleukin(IL)-6,monocyte chemoattractant proteins(MCP)-1,and vascular endothelial growth factor(VEGF)in HTFs at the translational and transcriptional levels.The phosphorylation levels of Sma-and Mad-related proteins(Smads),mitogen-activated protein kinases(MAPKs),and protein kinase B(AKT)were measured by immunoblotting.Statistical analysis was performed using either the Tukey-Kramer test or Student’s unpaired t-test to compare the various treatments.RESULTS:The CGC caused by TGF-βin HTFs was significantly inhibited by 17β-estradiol(25 to 100μmol/L),and a statistically significant difference was observed when comparing the normal control group with 17β-estradiol concentrations exceeding 25μmol/L(P<0.05).The suppressive impact of 17β-estradiol became evident 24h after administration and peaked at 72h(P<0.05),whereas progesterone had no impact.Moreover,17β-estradiol attenuated the formation of stress fibers,and the production of MMP-3 and MMP-1 in HTFs stimulated by TGF-β.The expression of MCP-1,IL-6,and VEGF mRNA and protein in HTFs were suppressed by 100μmol/L 17β-estradiol(P<0.01).Additionally,the phosphorylation of Smad2 Smad3,p38,and extracellular signal-regulated kinase(ERK)were downregulated(P<0.01).CONCLUSION:17β-estradiol significantly inhibits the CGC and inflammation caused by TGF-βin HTFs.This inhibition is likely related to the suppression of stress fibers,inhibition of MMPs,and attenuation of Smads and MAPK(ERK and p38)signaling.17β-estradiol may have potential clinical benefits in preventing scar development and inflammation in the conjunctiva.

Inhibitory effects of luteolin on TLR3-mediated inflammation caused by TAK/NF-κB signaling in human corneal flbroblasts

AIM: To study the role of luteolin(LUT) in the expression of toll-like receptors 3(TLR3) ligand poly I:C stimulated inflammatory factors in human corneal fibroblasts(HCFs).METHODS: HCFs cells were cultivated with or without LUT or poly I:C.The expression levels of interleukin(IL)-6, IL-8, monocyte chemotactic protein-1(MCP-1), vascular cell adhesion molecule(VCAM)-1, as well as intercellular adhesion molecule(ICAM)-1 were measured using enzymelinked immunosorbent assay(ELISA), immunoblotting or reverse transcription-quantitative polymerase chain reaction(PCR) analyses.Immunoblotting was used to assess toll-interleukin-1 receptor-domain-containing adapter-inducing interferon-β(TRIF), TLR3, transforming growth factor-bactivated kinase 1(TAK1), tumor necrosis factor receptor-associated factor 6(TRAF6), the transcription factor AP-1, as well as transcription factor nuclear factor(NF-κB)–inhibitory protein IκB-α degradation and phosphorylation.Immunofluorescence assays were used to localize the cellular location of the p65 subunit of NF-κB.RESULTS: Corneal fibroblasts exposed to poly I:C demonstrated decreased VCAM-1, ICAM-1, MCP-1, IL-6, and IL-8 expression levels upon exposure to LUT in a time-dependent and concentration-dependent manner.LUT was observed to suppress poly I:C-triggered expression of TLR3, the translocation of NF-κB p65 into cell nuclei, as well as the phosphorylation of TAK, c-Jun, and IκB-α, while no impact on the expression levels of TRIF and TRAF6 were observed.CONCLUSION: LUT suppress the expression of proinflammatory adhesion molecules, chemokines, and cytokines in poly I:C exposed HCFs.These effects are likely mediated through TAK/NF-κB signal attenuation.Therefore, LUT is a candidate molecule that can prevent the TLR3-mediated inflammation response associated with corneal viral infection.

Inhibition of TGF-β2-induced migration and epithelial-mesenchymal transition in ARPE-19 by sulforaphane

AIM: To investigate the effects of sulforaphane(SFN) on transforming growth factor(TGF)-β2 stimulated migration and epithelial-mesenchymal transition(EMT) in ARPE-19 cells.METHODS: ARPE-19 cells were cultured in the presence or absence of SFN or TGF-β2. SFN toxicity was assessed by performing a lactate dehydrogenase assay(LDH) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium(MTS) assays, and cell migration was evaluated by Transwell migration assay. Actin stress fiber formation in ARPE-19 cells was determined using immunofluorescence analysis. Immunoblotting analysis was used to determine fibronectin and α-smooth muscle actin expressions along with the degree of Smad and Akt phosphorylation.RESULTS: SFN inhibited ARPE-19 migration. Additionally, SFN attenuated TGF-β2-induced appearance of actin stress fibers as well as fibronectin and α-smooth muscle actin expressions in these cells. SFN also hindered the TGF-β2-stimulated phosphorylation of Smad2, Smad3, and Akt. SFN showed no cytotoxicity towards ARPE-19 cells. CONCLUSION: SFN inhibits TGF-β2-stimulated migration and EMT in ARPE-19 cells, probably by preventing the establishment of actin stress fibers and Akt and Smad2/3 signaling.

A randomized placebo-controlled trial of Chinese medicine acupoint application on gastrointestinal dysfunction after appendectomy

Background:Gastrointestinal dysfunction is one of the common complications of appendectomy,which seriously affects the postoperative recovery and clinical prognosis.Through traditional Chinese medicine acupoint application is suggested for managing postoperative gastrointestinal dysfunction,supporting evidence is weak.Here,the prospective randomized placebo-controlled study was designed to provide high-level evidence regarding whether traditional Chinese medicine acupoint application is effective on the gastrointestinal dysfunction after appendectomy.Methods:A total of 60 patients who underwent appendectomy in Dongfang Hospital Beijing University of Chinese Medicine(Beijing,China)from November 2016 to December 2017 were selected as study objects and randomly divided into control group(n=30)and observation group(n=30).Based on routine postoperative care,the acupoints Zusanli(ST36)and Yongquan(KI1)were selected.The control group was given acupoint application of traditional Chinese medicine placebo and the observation group was given acupoint application of clinical empirical Chinese medicine called Wentongliqi prescription.The course of treatment was performed on the 1st,2nd,and 3rd days after appendectomy,once a day and 4 hours each time.The primary outcome includes the time until the recovery time of bowel sounds(h),the first postoperative flatus(h)and first bowel movement time(h)on the 1st,2nd,and 3rd days after appendectomy.The secondary outcome includes clinical symptom score,life ability score and adverse reactions were observed and recorded on the 1st,2nd,and 3rd days after appendectomy.Results:After treatment,the recovery time of intestinal sound in the observation group was earlier than that in the control group(P<0.05).However,there were no significant difference between the two groups in first anal exhaust time and first bowel movement time,clinical symptom scores and life ability scores between the two groups before and after treatment(P>0.05).Conclusion:Acupoint application therapy has limited effect on the recovery of gastrointestinal dysfunction after appendectomy.Further study with large sample size is needed to confirm its therapeutic effects.

ATRP-Template Dispersion Polymerization of Methacrylic Acid/PVP

ATRP-template dispersion polymerization of methacrylic acid （MAA） on the template of polyvinyl pyrrolidone （PVP K-30） was carried out in the aqueous solution by using methyl 2-bromopropionate （MBP）/CuC1/2,2＇-bipyridine （bpy） as the initiation system. The scanning electron microscopy （SEM）, dynamic light scattering （DLS） and gel permeation chromatography （GPC） were employed for evaluating the results of polymerization. As a result, the minimonomer droplets formed due to the H-bond interaction of PVP-MAA. The stability of droplets was dependent on pH and the concentrations of both PVP and MAA. When pH 〈 2, the coagulum of PVP-MAA formed, whereas when pH 〉 4.5, the droplets were not observable by DLS. In order to prepare the stable latex, the concentration of PVP should be lower than 9 wt%, whilst the concentration of MAA should be lower than 5.5 wt%. The optimum condition was pH 2.4, PVP 4.76 wt% and MAA 5 wt%, by which the stable latex of ca. 50 nm nanoparticles of PMAA/PVP was prepared by ATRP polymerization and simultaneously the molar mass of PVP was duplicated by PMAA according to GPC diagrams. In contrast, by using AIBN, KPS and KPS-Na2SO3 redox initiation system, the coagulum accompanying with the larger molar mass of PMAA was obtained, irrespective of pH and concentrations of PVP and MAA.