AIM: To study the inhibition effect of TAK-242 on the proliferation of rat eye Tenon's capsule fibroblasts via the toll-like receptor 4(TLR4) signaling pathway.METHODS: SD rat Tenon's capsule fibroblasts were ...AIM: To study the inhibition effect of TAK-242 on the proliferation of rat eye Tenon's capsule fibroblasts via the toll-like receptor 4(TLR4) signaling pathway.METHODS: SD rat Tenon's capsule fibroblasts were extracted and cultured, then the cells were divided into normal control group, lipopolysaccharide(LPS) group(10 g/m L LPS) and TAK-242 group(1 μmol/L TAK-242, and 10 μg/m L LPS after 30 min). The expressions of TLR4, transforming growth factor-β1(TGF-β1) and interleukin-6(IL-6) in each group were detected by Western blot and reverse transcriptase-polymerase chain reaction(RT-PCR). Cell proliferation was detected by cell counting kit-8(CCK-8).RESULTS: Double immunofluorescent labeling in the extracted cells showed negative keratin staining and positive vimentin staining. Western blot showed that the LPS group had the highest expression of TLR4 and TGF-β1(P<0.01). Enzyme linked immunosorbent assay(ELISA) also showed that the secretion of IL-6 was the highest in LPS group(P<0.01). But there was no significant difference in TLR4 and TGF-1, as well as IL-6 expressions between the TAK-242 group and the normal control group(P>0.05). RT-PCR showed that the IL-6 m RNA expression in LPS group was the highest in the three groups(P<0.01). CONCLUSION: TAK-242 inhibits the proliferation of LPSinduced Tenon's capsule fibroblasts and the release of inflammatory factors by regulating the TLR4 signalingpathway, providing a new idea for reducing the scarring of the filter passage after glaucoma filtration surgery.展开更多
AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-beta 1 (TGF-beta 1) in ocular Tenon capsule fibroblasts (OTFS) in v...AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-beta 1 (TGF-beta 1) in ocular Tenon capsule fibroblasts (OTFS) in vitro. METHODS: After OTFS from passages 4 to 6 47 vitro were induced by TGF-beta 1 and then treated by Y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the alpha -smooth muscular actin (alpha -SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of Y-27632, the expression levels of the alpha -SMA, CTGF and collagen I mRNA were assayed by RT-PCR. RESULTS: Y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-beta 1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-beta 1 and Y-27632 had no remarkable difference from that in control group. Y-27632 significantly inhibited the proteins expressions of both alpha -SMA and CTGF, while to some extent inhibited that of collagen I. TGF-beta 1 significantly promoted the proteins expressions of alpha -SMA, CTGF and collagen I. After OTFS treated by both TGF-beta 1 and Y-27632, of alpha -SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000<0.01). The differences of expressions of the alpha -SMA, CTGF and collagen I mRNA in 30, 150, 750 mu mol/L Y-27632 group were statistically significant, compared with those in control group, respectively (alpha -SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I,P=0.003, 0.002, 0.000). CONCLUSION: Blocking the Rho/ROCK signaling pathway by using of Y-27632 could inhibit the cellular proliferation and the expression of both CTGF and alpha -SMA whatever OTFS induced by TGF-beta 1 or not. Y-27632 suppressed the expression of collagen I mRNA without induction.展开更多
Purpose:To investigate the effect of apigenin on gap junctional intercellular communication (GJIC) in human Tenon's capsule fibroblasts (HTFs) and its underlying mechanism. Methods:After a 48 h treatment of cultur...Purpose:To investigate the effect of apigenin on gap junctional intercellular communication (GJIC) in human Tenon's capsule fibroblasts (HTFs) and its underlying mechanism. Methods:After a 48 h treatment of cultured HTFs with apigenin.(80 μmol/L),the GJIC was detected by a scrape-loading/dye transfer technique with Lucifer yellow dye and rhodamine (Rh) dextran. The coupling index represents a quantification of GJIC where a high coupling index is associated with a greater number of cells demonstrating cell-cell communication through gap junction channels.The changes in connexin 43 (Cx43) distribution and the expression of Cx43 at the protein and mRNA levels were statistically compared between the two groups by means of immunocytochemistry, western blotting,and real-time polymerase chain reaction (PCR). Results:The functioning of GJIC in the HTFs was significantly enhanced after 48 hours by apigenin treatment when compared with the control cells. In the apigenin group, the intercellular dye transfer grade was above 9, while this value was only grade 3-4 in the control group. The coupling index was significantly increased up to 9.205±0.3621 in the apigenin group,compared with 5.1775 ±0.3177 in the control group (F=279.581, P=0.000). The expression of Cx43 at the protein and mRNA levels was significantly up-regulated in the apigenin group compared with the control group. Conclusion:Apigenin can significantly enhance the function of GJIC in HTFs by up-regulating the expression of Cx43 at both the protein and mRNA levels,suggesting that the enhancement of GJIC in HTFs by apigenin probably acts as an important mechanism underlying the inhibitory effect of apigenin on HTF proliferation.展开更多
Purpose:To study the effect of human IFN-γon in vitro cultured human fibroblasts form Tenon's capsule.Materials and methods:The effect of different concentrations of human INF-γand mitomycin-C(MMC),5-fluorouraci...Purpose:To study the effect of human IFN-γon in vitro cultured human fibroblasts form Tenon's capsule.Materials and methods:The effect of different concentrations of human INF-γand mitomycin-C(MMC),5-fluorouracil(5-Fu）on cultured human Tenon's capsule fibroblasts(HTCF)was measured using a MTT[3-(4,5-dimethylthiazo-2-yl)]-2,5-diphenyltetrazolium bromide;Thiazolyl blue)colorimetric assay,The results were analyzed using ANOVA of the statistical package for social sciences(SPSS)9.0 version.The difference was considered to be significant if P<0.05.Results:The effects of MMC and 5-Fu on the growth of HTCF were negative,while the effects of IFN-γon the growth of HTCF were both negative(10^2-10^4units/ml in two experiments)and positive(10^6,10^5,10units/ml in two experiments).The inhibition rate of MMC ranged from5.73%to46.9%,which was similar to the inhibition rate of 5-Fu ranged from12.49%to38.92%(P=0.351),The inhibition rate of IFN-γin two experiments was smaller than MMC and 5-Fu(P<0.05).Conclusion:IFN-γhas bifunctional effect(both enhancement and inhibition)on proliferation of cultured HTCF,The antiproliferative effect of IFN-γwas weatker than MMCand 5-FU,Further study has to bd carride out to document the inhibition of scar formation of filtration bleb by IFN-γand the molecular mechanisms of its bifunctional effect on HTCF proliferation.Eye Science2000;16:43-47.展开更多
AIM： To investigate the toxicity of the E-prostanoid 2（EP2） receptor agonist, butaprost against human subconjunctival（Tenon＇s capsule） fibroblasts, and to determine the underlying mechanism. METHODS： We isolate...AIM： To investigate the toxicity of the E-prostanoid 2（EP2） receptor agonist, butaprost against human subconjunctival（Tenon＇s capsule） fibroblasts, and to determine the underlying mechanism. METHODS： We isolated Tenon＇s fibroblasts from the subconjunctival area of healthy subjects and evaluated the types of EP receptors expressed using quantitative realtime reverse transcription polymerase chain reaction（RTPCR）. The toxicity of butaprost against the fibroblasts was evaluated using methyl thiazolyl tetrazolium and lactic dehydrogenase assays. The inhibition of conjunctival fibroblast proliferation by butaprost was assessed by measuring α-actin levels. The underlying mechanism was assessed by measuring intracellular cyclic adenosine monophosphate（c AMP） levels. Intergroup differences were statistically analyzed using an independent t-test. Densitometry of the Western blot bands was performed using the Image J software. RESULTS： Quantitative real-time RT-PCR revealed that the fibroblast EP2 receptor levels were higher than those of the other EP receptors. Butaprost did not show toxicity against Tenon＇s tissue, but inhibited conjunctival fibroblast proliferation by reducing collagen synthesis. EP2 receptor activation enhanced the c AMP cascade, which might be an important mechanism underlying this effect.CONCLUSION： Butaprost effectively reduces the subconjunctival scarring response. Given the significanceof wound healing modulation in blebs, butaprost＇s inhibitory effect on subconjunctival Tenon＇s fibroblasts may be beneficial in managing postoperative scarring in glaucoma surgery.展开更多
基金Supported by National Natural Science Foundation Program of China (No.81770920)Hubei Health and Family Planning Commission Youth Talent Project (No. WJ2017Q037)
文摘AIM: To study the inhibition effect of TAK-242 on the proliferation of rat eye Tenon's capsule fibroblasts via the toll-like receptor 4(TLR4) signaling pathway.METHODS: SD rat Tenon's capsule fibroblasts were extracted and cultured, then the cells were divided into normal control group, lipopolysaccharide(LPS) group(10 g/m L LPS) and TAK-242 group(1 μmol/L TAK-242, and 10 μg/m L LPS after 30 min). The expressions of TLR4, transforming growth factor-β1(TGF-β1) and interleukin-6(IL-6) in each group were detected by Western blot and reverse transcriptase-polymerase chain reaction(RT-PCR). Cell proliferation was detected by cell counting kit-8(CCK-8).RESULTS: Double immunofluorescent labeling in the extracted cells showed negative keratin staining and positive vimentin staining. Western blot showed that the LPS group had the highest expression of TLR4 and TGF-β1(P<0.01). Enzyme linked immunosorbent assay(ELISA) also showed that the secretion of IL-6 was the highest in LPS group(P<0.01). But there was no significant difference in TLR4 and TGF-1, as well as IL-6 expressions between the TAK-242 group and the normal control group(P>0.05). RT-PCR showed that the IL-6 m RNA expression in LPS group was the highest in the three groups(P<0.01). CONCLUSION: TAK-242 inhibits the proliferation of LPSinduced Tenon's capsule fibroblasts and the release of inflammatory factors by regulating the TLR4 signalingpathway, providing a new idea for reducing the scarring of the filter passage after glaucoma filtration surgery.
基金Shaanxi Province Science and Technology Gongguan Program, China (No.2011-K14-02-03)
文摘AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-beta 1 (TGF-beta 1) in ocular Tenon capsule fibroblasts (OTFS) in vitro. METHODS: After OTFS from passages 4 to 6 47 vitro were induced by TGF-beta 1 and then treated by Y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the alpha -smooth muscular actin (alpha -SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of Y-27632, the expression levels of the alpha -SMA, CTGF and collagen I mRNA were assayed by RT-PCR. RESULTS: Y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-beta 1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-beta 1 and Y-27632 had no remarkable difference from that in control group. Y-27632 significantly inhibited the proteins expressions of both alpha -SMA and CTGF, while to some extent inhibited that of collagen I. TGF-beta 1 significantly promoted the proteins expressions of alpha -SMA, CTGF and collagen I. After OTFS treated by both TGF-beta 1 and Y-27632, of alpha -SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000<0.01). The differences of expressions of the alpha -SMA, CTGF and collagen I mRNA in 30, 150, 750 mu mol/L Y-27632 group were statistically significant, compared with those in control group, respectively (alpha -SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I,P=0.003, 0.002, 0.000). CONCLUSION: Blocking the Rho/ROCK signaling pathway by using of Y-27632 could inhibit the cellular proliferation and the expression of both CTGF and alpha -SMA whatever OTFS induced by TGF-beta 1 or not. Y-27632 suppressed the expression of collagen I mRNA without induction.
基金supported by Shandong Provincial Natural Science Foundation Project (No.ZR2010HM015)
文摘Purpose:To investigate the effect of apigenin on gap junctional intercellular communication (GJIC) in human Tenon's capsule fibroblasts (HTFs) and its underlying mechanism. Methods:After a 48 h treatment of cultured HTFs with apigenin.(80 μmol/L),the GJIC was detected by a scrape-loading/dye transfer technique with Lucifer yellow dye and rhodamine (Rh) dextran. The coupling index represents a quantification of GJIC where a high coupling index is associated with a greater number of cells demonstrating cell-cell communication through gap junction channels.The changes in connexin 43 (Cx43) distribution and the expression of Cx43 at the protein and mRNA levels were statistically compared between the two groups by means of immunocytochemistry, western blotting,and real-time polymerase chain reaction (PCR). Results:The functioning of GJIC in the HTFs was significantly enhanced after 48 hours by apigenin treatment when compared with the control cells. In the apigenin group, the intercellular dye transfer grade was above 9, while this value was only grade 3-4 in the control group. The coupling index was significantly increased up to 9.205±0.3621 in the apigenin group,compared with 5.1775 ±0.3177 in the control group (F=279.581, P=0.000). The expression of Cx43 at the protein and mRNA levels was significantly up-regulated in the apigenin group compared with the control group. Conclusion:Apigenin can significantly enhance the function of GJIC in HTFs by up-regulating the expression of Cx43 at both the protein and mRNA levels,suggesting that the enhancement of GJIC in HTFs by apigenin probably acts as an important mechanism underlying the inhibitory effect of apigenin on HTF proliferation.
基金This paper is granted by National Natural Sciences Foundation of China (No.39700153) Nutural Sciences Foundation of Guangdong Province (No.970082)
文摘Purpose:To study the effect of human IFN-γon in vitro cultured human fibroblasts form Tenon's capsule.Materials and methods:The effect of different concentrations of human INF-γand mitomycin-C(MMC),5-fluorouracil(5-Fu）on cultured human Tenon's capsule fibroblasts(HTCF)was measured using a MTT[3-(4,5-dimethylthiazo-2-yl)]-2,5-diphenyltetrazolium bromide;Thiazolyl blue)colorimetric assay,The results were analyzed using ANOVA of the statistical package for social sciences(SPSS)9.0 version.The difference was considered to be significant if P<0.05.Results:The effects of MMC and 5-Fu on the growth of HTCF were negative,while the effects of IFN-γon the growth of HTCF were both negative(10^2-10^4units/ml in two experiments)and positive(10^6,10^5,10units/ml in two experiments).The inhibition rate of MMC ranged from5.73%to46.9%,which was similar to the inhibition rate of 5-Fu ranged from12.49%to38.92%(P=0.351),The inhibition rate of IFN-γin two experiments was smaller than MMC and 5-Fu(P<0.05).Conclusion:IFN-γhas bifunctional effect(both enhancement and inhibition)on proliferation of cultured HTCF,The antiproliferative effect of IFN-γwas weatker than MMCand 5-FU,Further study has to bd carride out to document the inhibition of scar formation of filtration bleb by IFN-γand the molecular mechanisms of its bifunctional effect on HTCF proliferation.Eye Science2000;16:43-47.
基金Supported by the Research Institute for Convergence of Biomedical Science and Technology,Pusan National University Yangsan Hospital,Korea(No.30-2013-009)
文摘AIM： To investigate the toxicity of the E-prostanoid 2（EP2） receptor agonist, butaprost against human subconjunctival（Tenon＇s capsule） fibroblasts, and to determine the underlying mechanism. METHODS： We isolated Tenon＇s fibroblasts from the subconjunctival area of healthy subjects and evaluated the types of EP receptors expressed using quantitative realtime reverse transcription polymerase chain reaction（RTPCR）. The toxicity of butaprost against the fibroblasts was evaluated using methyl thiazolyl tetrazolium and lactic dehydrogenase assays. The inhibition of conjunctival fibroblast proliferation by butaprost was assessed by measuring α-actin levels. The underlying mechanism was assessed by measuring intracellular cyclic adenosine monophosphate（c AMP） levels. Intergroup differences were statistically analyzed using an independent t-test. Densitometry of the Western blot bands was performed using the Image J software. RESULTS： Quantitative real-time RT-PCR revealed that the fibroblast EP2 receptor levels were higher than those of the other EP receptors. Butaprost did not show toxicity against Tenon＇s tissue, but inhibited conjunctival fibroblast proliferation by reducing collagen synthesis. EP2 receptor activation enhanced the c AMP cascade, which might be an important mechanism underlying this effect.CONCLUSION： Butaprost effectively reduces the subconjunctival scarring response. Given the significanceof wound healing modulation in blebs, butaprost＇s inhibitory effect on subconjunctival Tenon＇s fibroblasts may be beneficial in managing postoperative scarring in glaucoma surgery.
