Triptolide inhibits TGF-β-induced matrix contraction and fibronectin production mediated by human Tenon fibroblasts

AIM: To determine if triptolide influences the contractility and fibronectin production in human Tenon fibroblasts (HTFs). METHODS: HTFs were cultured in type I collagen gels with or without transforming growth factor beta (TGF-β) and/or triptolide. The diameter of the collagen gel was used to measure contraction. Immunoblot analysis was used to quantify myosin light chain (MLC) phosphorylation and integrin expression. Laser confocal fluorescence microscopy was used to monitor the formation of actin stress fibers. Fibronectin production was measured with an enzyme immunoassay. RESULTS: Triptolide inhibition of contraction in TGF-β-induced collagen gel mediated by HTFs was dose-dependent and statistically significant at 3 nmol/L (P<0.05) and maximal at 30 nmol/L and significantly time dependent at 2d (P<0.05). Triptolide reduced TGF-β-induced expression of integrins α5 and β1, phosphorylation of MLC, and formation of stress fibers in HTFs. Furthermore, the inhibition of triptolide on the attenuated TGF-β-induced production of fibronectin by HTFs was concentration-dependent and significant at 1 nmol/L (P<0.05) and maximal at 30 nmol/L. CONCLUSION: Triptolide suppress the contractility of HTFs induced by TGF-β and the production of fibronectin by these cells. It is promising that triptolide treatment may possibly inhibit scar formation after glaucoma filtration surgery.     

Effects of 530 nm monochromatic light on basic fibroblast growth factor and transforming growth factor-β1 expression in Müller cells

AIM: To expose rat retinal Müller cells to 530 nm monochromatic light and investigate the influence of varying light illumination times on basic fibroblast growth factor (bFGF) and transforming growth factor-β1 (TGF-β1) expression.METHODS: Three groups of rat retinal Müller cells cultured in vitro under a 530 nm monochromatic light were divided into 6, 12 and 24h experimental groups , while cells incubated under dark conditions served as the control group. The bFGF and TGF-β1 mRNA expression, protein levels and fluorescence intensity of the Müller cells were analyzed.RESULTS:ThebFGF mRNA expression and protein levels were significantly upregulated in Müller cells in all three experimental groups compared with the control group (P<0.05), while that of TGF-β1 was downregulated (P<0.05). Also, bFGF expression was positively correlated, but TGF-β1 expression was negatively correlated with illumination time. The largest changes for both cytokines were seen in the 24h group. The changes in bFGF and TGF-β1 fluorescence intensity were highest in the 24h group, and significant differences were observed among the experimental groups (P<0.05).CONCLUSION:The expressions ofbFGF and TGF-β1 changed in a time-dependent manner in Müller cells exposed to 530 nm monochromatic light with 250 lx illumination intensity. Müller cells might play a role in the development of myopia by increasing bFGF expression or decreasing TGF-β1 expression. Changes in cytokine expression in retinal Müller cells may affect monochromatic light-induced myopia.     

Expression of Mesenger RNA for Transforming Growth Factor-β_1 in Bovine Trabecular Meshwork

Purpose: To investigate the relationship between transforming growth factor-β1(TGF-β1) and primary open-angle glaucoma, we have determined whether trabec-ular tissues have the expression of messenger RNA for TGF-β1.Methods: Total RNA of 24 newborn bovine trabecular tissue were extracted byGuanidine isothiocyanate method. The TGF-β33 plasmid was brought into E. col-ibacillius HB101 and amplificated. After Bam HI endolase degradation and labelwith a-32p-dATP the RNA was hybridized with the cDNA (complementary DNA)probe and examined by autoradiography.Results: The presence of mRNA for TGF-β1 in bovine trabecular meshwork wasconfirmed.Conclusions: The TGF-β1 present in normal aqueous humor must be at least partlyderived from the trabecular meshwork. It offered a basis for understanding therelationship between abnormal synthesis, activation and clearance of TGF-β1 andthe pathogenesis of primary open-angle glaucoma (POAG) in molecular biology.Eye Science 1996; 12:1-4.     

The Changes of TGF—α,TGF—β1 and Basic FGF Messenger RNA Expression i

OBJECTIVE: To study the mechanism of haze formation and investigate the expression changes of transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta 1(TGF-beta 1) and basic fibroblast growth factor (bFGF) mRNA in corneal epithelium and stroma after photorefractive keratectomy (PRK). METHODS: Sixteen white rabbits were randomly divided into 4 groups, and PRK was performed on each eye of 12 rabbits. The haze formation was examined under a slit-lamp microscope at the 1st, 2nd and 3rd month after PRK, and the expressions of TGF-alpha, TGF-beta 1 and bFGF mRNA were detected with in situ hybridization. RESULTS: The corneal haze formed at the 1st month after PRK. The most prominent haze formation was observed at the 2nd month, and declined gradually at the 3rd month after ablation. TGF-alpha mRNA expression was presented on the normal corneal epithelium and not on the corneal stroma. TGF-beta 1 and bGFG mRNA were expressed by both corneal epithelium and stroma. The capacities for cornea tissue expression of three growth factors mRNA increased after PRK, and the peaks appeared on the 1st, 2nd month. The extent for expressions of three growth factors related proportionally to the haze formation. CONCLUSION: Three growth factors took part in promoting corneal wound healing after PRK, and might contribute to corneal haze formation and development.     

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)-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-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.     

[Online First]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)-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-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 inhibite 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.     

The apoptosis and expression of TGF-β1 and NF-κB of conjunctival epithelium cell in diabetes mellitus patients

Objective To apprehend the ocular surface condition of type 2 diabetes mellitus patients by detecting the apoptosis index and the inflammation condition of conjunctival epithelial cell. To analyze the reason of the type 2 DM' s dry eye. Methods Thirty-five type 2 DM patients and 25 healthy adults were se lected randomly to detect the break-up time (BUT ), Schirmer I test (SIt) and Fluorescein staining (FL). Con junctival epithelial cells were collected by impression cytology from the type 2 DM patients and healthy adults. Flow cytometry was taken to measure apoptosis. Immunohistochemical staining was performed to detect trans forming TGF-P ,and NF-K B. Results The value of SIt and BUT in type 2 DM patients obviously increased man those in control group (P <0.01). The FL in type 2 DM patients positively expressed than that in control group (P<0.01). The apoptotic index of conjunctival epithelium in type 2 DM patients was significantly higher than that in control group (P <0.01). The expression of TGF-β1 ,and NF-k B in DM patients was obviously stronger than those in control group (P <0.01). The apoptotic index showed positive correlation with the ex pression of TGF-* 1 (P<0.01). Conclusions The increase of apoptotic and inflammation of conjunctival ep ithelium may have close relationship with the type 2 DM' s dry eye.     

The apoptosis and expression of TGF-β1 and NF-κB of conjunctival epithelium cell in diabetes mellitus patients

Objective To apprehend the ocular surface condition of type 2 diabetes mellitus patients by detecting the apoptosis index and the inflammation condition of conjunctival epithelial cell. To analyze the reason of the type 2 DM' s dry eye. Methods Thirty-five type 2 DM patients and 25 healthy adults were se lected randomly to detect the break-up time (BUT ), Schirmer I test (SIt) and Fluorescein staining (FL). Con junctival epithelial cells were collected by impression cytology from the type 2 DM patients and healthy adults. Flow cytometry was taken to measure apoptosis. Immunohistochemical staining was performed to detect trans forming TGF-P ,and NF-K B. Results The value of SIt and BUT in type 2 DM patients obviously increased man those in control group (P <0.01). The FL in type 2 DM patients positively expressed than that in control group (P<0.01). The apoptotic index of conjunctival epithelium in type 2 DM patients was significantly higher than that in control group (P <0.01). The expression of TGF-β1 ,and NF-k B in DM patients was obviously stronger than those in control group (P <0.01). The apoptotic index showed positive correlation with the ex pression of TGF-* 1 (P<0.01). Conclusions The increase of apoptotic and inflammation of conjunctival ep ithelium may have close relationship with the type 2 DM' s dry eye.     

Inhibition of β-elemene on the expressions of HIF-lα, VEGF and iNOS in diabetic rats model

AIM: To evaluate the effect of β-elemene on the expressions of hypoxia-inducible factor (HIF)-lα, vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS) in a streptozotocin (STZ) induced diabetic Sprague-Dawley (SD) rat model. METHODS: SD rats were administered an abdominal injection of STZ and induced to a diabetic model. After 6wk course of diabetes, the treatment groups were given β-elemene through periocular and intravitreous injection separately and the control groups were given blank emulsion injection. HE staining was used to observe the morphology of retina. The mRNA expressions of HIF-1α, VEGF and iNOS was assayed by real-time polymerase chain reaction (PCR) and the protein expression was measured by Western blot and immunocytochemistry methods. RESULTS: The results indicated that the protein and mRNA expressions of HIF-1α, VEGF and iNOS after treated by β-elemene periocularly and intravitreally injections were all found to be reduced compared with the levels in the diabetic rats group (P<0.05). The inhibitory effect of intravitreal injection was more remarkable. CONCLUSION: The results show β-elemene protect the retina of diabetic rats from high glucose damage by downregulating the expression of HIF-1α, VEGF and iNOS.     

Involvement of Rho-associated coiled-coil kinase signaling inhibition in TGF-β1/Smad2, 3 signal transduction in vitro

AIM: To research the effect of Y-27632, a selective Rho-associated coiled-coil kinase (ROCK) inhibitor, on TGF-β1/Smad2, 3 signal transduction in ocular Tenon’s capsule fibroblasts (OTFs). METHODS: Primary ocular Tenon’s capsule fibroblasts had been cultured in vitro. The effect of Y27632 on proliferation of OTF stimulated by lysophosphatidic acid (LPA) was evaluated by MTT colorimetric assay so as to sift out the proper concentrations range of Y-27632 for the next experiment. Real time-polymerase chain reactor (RT-PCR) was to analyze the changes of Smad2 and Smad3 genes of cells affected by Y-27632, though unaffected by transforming growth factor-beta1 (TGF-β1). Proteins of Smad2, Smad3, phosphorylated Smad2 (Ser245/250/255), and phosphorylated Smad3 (Ser423/425/203) were respectively quantified by Western blot after OTFs were successively incubated by TGF-β1 and Y-27632. Meanwhile, α-smooth muscular actin (α-SMA) protein was also quantified after the small intervening gene fragments of human Smad2 and Smad3 were designed, synthesized, and then transfected to OTFs. RESULTS: Y-27632 significantly inhibited OTFs proliferation stimulated by LPA. Also Y-27632 significantly suppressed the expressions of Smad2 mRNA, Smad2, 3 proteins expressions, Smad3 phosphorylation at the carboxylic terminals of Ser423/425/203 which had been radically promoted by TGF-β1. SiRNA-Smad2, 3 suppressed α-SMA expressions, but less effectively than Y-27632. CONCLUSION: The inhibition of ROCK signaling may be a potential therapeutic candidate for the treatment of the filtration channel fibrosis.