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 transcription-polymerase 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)-1ß levels. To investigate the role of NF-κB signaling activation and IL-1ß in Sema7A's anti-barrier mechanism, we employed 0.1 µmol/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-1ß. 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-1ß. These findings suggest that Sema7A could be a potential therapeutic target for the diseases in corneal epithelium.

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

AIM: To investigate the impact of 17ß-estradiol on the collagen gels contraction (CGC) and inflammation induced by transforming growth factor (TGF)-ß in human Tenon fibroblasts (HTFs). METHODS: HTFs were three-dimensionally cultivated in type I collagen-generated gels with or without TGF-ß (5 ng/mL), 17ß-estradiol (12.5 to 100 µmol/L), or progesterone (12.5 to 100 µmol/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-ß in HTFs was significantly inhibited by 17ß-estradiol (25 to 100 µmol/L), and a statistically significant difference was observed when comparing the normal control group with 17ß-estradiol concentrations exceeding 25 µmol/L (P<0.05). The suppressive impact of 17ß-estradiol became evident 24h after administration and peaked at 72h (P<0.05), whereas progesterone had no impact. Moreover, 17ß-estradiol attenuated the formation of stress fibers, and the production of MMP-3 and MMP-1 in HTFs stimulated by TGF-ß. The expression of MCP-1, IL-6, and VEGF mRNA and protein in HTFs were suppressed by 100 µmol/L 17ß-estradiol (P<0.01). Additionally, the phosphorylation of Smad2 Smad3, p38, and extracellular signal-regulated kinase (ERK) were downregulated (P <0.01). CONCLUSION: 17ß-estradiol significantly inhibits the CGC and inflammation caused by TGF-ß in 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. 17ß-estradiol may have potential clinical benefits in preventing scar development and inflammation in the conjunctiva.

Triterpenoid saponins from Psammosilene tunicoides and their antinociceptive activities.

Herein, five undescribed oleanane-type triterpenoid saponins, namely, psammosaponins A-E, along with nine known compounds, were isolated from the roots of Psammosilene tunicoides. Moreover, part of the ethanolic extract of P. tunicoides was acid-hydrolyzed and three aglycones were isolated from the resulting hydrolysate. The structures of all compounds were established through extensive analysis involving 1D and 2D NMR experiments, HRESIMS measurements, chemical derivatization, and comparison of spectroscopic data with the values reported in the literature. In all, 10 of the isolated saponins and the three aglycones were evaluated in the acetic acid-induced writhing model for their antinociceptive activity. At a dose of 40 mg/kg, these compounds exhibited significant inhibitory effects on the mouse writhing response, with inhibitions ranging from 31.9% to 79.3%. In addition, the structure-activity relationships of the isolates were discussed. Among the isolates, quillaic acid 3-O-glucuronide and 16α-hydroxygypsogenic acid showed better antinociceptive activity with inhibitions of 79.3% and 73.7%, respectively. Both isolates also exhibited antinociceptive activities in hot plate and formalin tests on mice. Their antinociceptive mechanism was explored in lipopolysaccharide-stimulated RAW 264.7 cells. These isolates could significantly inhibit the production of nitric oxide and interleukin-6 and downregulate the expression levels of inducible NO synthase, COX-1, and COX-2.

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

AIM: To study the role of luteolin (LUT) in the expression of toll-like receptors 3 (TLR3) ligand polyI:C stimulated inflammatory factors in human corneal fibroblasts (HCFs). METHODS: HCFs cells were cultivated with or without LUT or polyI: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 enzyme-linked 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-ß (TRIF), TLR3, transforming growth factor-b-activated 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 polyI: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 polyI: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 polyI: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-ß2-induced migration and epithelial-mesenchymal transition in ARPE-19 by sulforaphane.

AIM: To investigate the effects of sulforaphane (SFN) on transforming growth factor (TGF)-ß2 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-ß2. SFN toxicity was assessed by performing a lactate dehydrogenase assay (LDH) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-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-ß2-induced appearance of actin stress fibers as well as fibronectin and α-smooth muscle actin expressions in these cells. SFN also hindered the TGF-ß2-stimulated phosphorylation of Smad2, Smad3, and Akt. SFN showed no cytotoxicity towards ARPE-19 cells. CONCLUSION: SFN inhibits TGF-ß2-stimulated migration and EMT in ARPE-19 cells, probably by preventing the establishment of actin stress fibers and Akt and Smad2/3 signaling.

Alarmins from conjunctival fibroblasts up-regulate matrix metalloproteinases in corneal fibroblasts.

AIM: To explore the effects of alarmins produced by necrotic human conjunctival fibroblasts on the release of matrix metalloproteinases (MMPs) by human corneal fibroblasts (HCFs). METHODS: A necrotic cell supernatant (NHCS) was prepared by subjecting human conjunctival fibroblasts to three cycles of freezing and thawing. The amounts of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α in NHCS were determined by enzyme-linked immunosorbent assays. HCFs exposed to NHCS or other agents in culture were assayed for the release of MMPs as well as for intracellular signaling by immunoblot analysis. The abundance of MMP mRNAs in HCFs was examined by reverse transcription and real-time polymerase chain reaction analysis. RESULTS: NHCS increased the release of MMP-1 and MMP-3 by HCFs as well as the amounts of the corresponding mRNAs in the cells. NHCS also induced activation of mitogen-activated protein kinase (MAPK) signaling pathways mediated by extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK) as well as elicited that of the nuclear factor (NF)-κB signaling pathway by promoting phosphorylation of the endogenous NF-κB inhibitor IκB-α. Inhibitors of MAPK and NF-κB signaling as well as IL-1 and TNF-α receptor antagonists attenuated the NHCS-induced release of MMP-1 and MMP-3 by HCFs. Furthermore, IL-1ß and TNF-α were both detected in NHCS, and treatment of HCFs with these cytokines induced the release of MMP-1 and MMP-3 in a concentration-dependent manner. CONCLUSION: Alarmins, including IL-1ß and TNF-α, produced by necrotic human conjunctival fibroblasts triggered MMP release in HCFs through activation of MAPK and NF-κB signaling. IL-1ß and TNF-α are therefore potential therapeutic targets for the amelioration of corneal stromal degradation in severe ocular burns.

Transforming growth factor-ß2 induces morphological alteration of human corneal endothelial cells in vitro.

AIM: To investigate the morphological altering effect of transforming growth factor-ß2 (TGF-ß2) on untransfected human corneal endothelial cells (HCECs) in vitro. METHODS: After untransfected HCECs were treated with TGF-ß2 at different concentrations, the morphology, cytoskeleton distribution, and type IV collagen expression of the cells were examined with inverted contrast light microscopy, fluorescence microscopy, immunofluorescence or Western Blot. RESULTS: TGF-ß2 at the concentration of 3-15 µg/L had obviously alterative effects on HCECs morphology in dose and time-dependent manner, and 9 µg/L was the peak concentration. TGF-ß2 (9 µg/L) altered HCE cell morphology after treatment for 36h, increased the mean optical density (P<0.01) and the length of F-actin, reduced the mean optical density (P<0.01) of the collagen type IV in extracellular matrix (ECM) and induced the rearrangement of F-actin, microtubule in cytoplasm and collagen type IV in ECM after treatment for 72h. CONCLUTION: TGF-ß2 has obviously alterative effect on the morphology of HCECs from polygonal phenotype to enlarged spindle-shaped phenotype, in dose and time-dependence manner by inducing more, elongation and alignment of F-actin, rearrangement of microtubule and larger spread area of collagen type IV.

Comparison of Combined Phacoemulsification, Intraocular Lens Implantation, and Goniosynechialysis With Phacotrabeculectomy in the Treatment of Primary Angle-Closure Glaucoma and Cataract.

PURPOSE: This study aimed to compare the efficacy and safety of combined phacoemulsification, intraocular lens implantation, and goniosynechialysis with phacotrabeculectomy in the treatment of primary angle-closure glaucoma (PACG) and cataract. DESIGN: A comparative case series. METHODS: Sixty-five patients (65 eyes) with PACG and cataract from the Fifth Affiliated Hospital of Sun Yat-Sen University were enrolled for this study between October 2009 and July 2011. Of these, 33 underwent combined phacoemulsification, intraocular lens implantation, and goniosynechialysis (treatment group), and 32 underwent phacotrabeculectomy (control group). The effects on intraocular pressure, best-corrected visual acuity, anterior chamber angle, number of antiglaucoma medications, and complications were evaluated. RESULTS: Both the treatment group and the control group had lowered intraocular pressure, reduced the use of antiglaucoma medications, and improved vision in patients with PACG and cataract. Complications were 8 (24.2%) of 33 in the treatment group and 12 (37.5%) of 32 in the control group. CONCLUSIONS: Combined phacoemulsification, intraocular lens implantation, and goniosynechialysis appears to be a preferred method for the treatment of PACG and cataract because it seems to have the same efficacy as phacotrabeculectomy and has much less surgical complications.

A comparing study of quantitative staining techniques for retinal neovascularization in a mouse model of oxygen-induced retinopathy.

AIM: To explore an efficient, practical and objective quantitative method to evaluate the retinal neovascularization in mouse model of oxygen induced retinopathy (OIR). METHODS: Thirty C57BL/6J mice were explored in OIR model procedure. Eyes were removed for different staining methods including: (1) HE staining; (2) immunohistochemistry with Griffonia Simplicifolia Lectin(GSL); (3) Immunofluorescence with FITC labeled CD31 antibody; (4) Two-step immunofluorescence with purified-CD31 antibody; (5) FITC-Dextran perfusion combined with two-step purified-CD31immunofluorescence. Images of the retinal vasculature were analyzed by imaging software. RESULTS: GSL immunohistochemistry could clearly demonstrate the deep and superficial capillary beds. FITC labeled CD31 Immunofluorescence was blurring with high fluorescence background which was hard to distinguish retinal neovascularization in some area. Excellent detail of neovascularization and preexistent retinal vessels was provided in two-step Purified-CD31 immunofluorescence group. CONCLUSION: GSL immunohistochemistry can clearly demonstrate neovascularization tufts in deep and superficial capillary beds. Immunofluorescence of specific antigen CD31 on vascular endothelium can selectively label the neovascularization of mouse retina. When combined with computer analysis software, it is an effective and objective quantitative method to evaluate the retinal neovascularization in OIR mouse model.

[Studies on the purification of water-soluble holothurian glycosides from Apostichopus japonicus and their tumor suppressing activity].

To obtain water-soluble holothurian glycosides with high tumor suppressing activities from Apostichopus japonicus, macroporous resin, silica gel and gel-filtration column chromatograghy were used to purify the water-soluble holothurian glycosides, and their tumor suppressing activity and inducing apoptosis of tumor cells were examined in this study. The 70% ethanol fraction of macroporous resin column, the pSC-2 and pSC-3 fractions from silica gel column showed very strong tumor suppressing activity towards HeLa cells, A-549 lung cancer cells, SGC-7901 stomach cancer cells and Bel-7402 liver cancer cells. SC-2 and SC-3 fraction purified from Sephadex LH-20 gel-filtration column chromatography, with a purity above 99.6%, all had the properties of triterpenoid glycosides. Purified SC-2 fraction had remarkable tumor suppressing activity on HeLa cells in a dose- and time-dependent manner, and had prominent tumor suppressing activity to mouse S180 solid tumors in a dose-dependent manner. Besides, the SC-2 fraction also had remarkable ability in elevating mouse thymus index and spleen index. The purified SC-2 fraction could induce apoptosis of HeLa cells in a dose-dependent manner, and DNA fragmentation of HeLa cells occurred after treated 12 h with 10 mg x L(-1) and 50 mg x L(-1) of SC-2 fractions. From the results, it can be concluded that the purified SC-2 fraction of water-soluble holothurian glycosides has extremely strong tumor suppressing activity, and the suppression is realized by inducing tumor cells to undergo apoptosis. This study lays solid foundation for development of highly effective new natural anticancer agents from sea cucumbers.