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Objective:To study the effect and mechanism of angiotensin type 1 receptor (AGTR1) blocker olmesartan (OMS) on the apoptosis of human Tenon capsule fibroblasts (HTF).Methods:Tenon capsule tissues were obtained from patients during strabismus surgery in the Second Affiliated Hospital of Xi'an Jiaotong University.Primary HTF were cultured by explant culture.Primary cells were identified by vimentin immunofluorescence staining and flow cytometry.The fibrosis model of HTF was established using 10 ng/ml transforming growth factor-β2 (TGF-β2). The cells were divided into normal control group cultured in culture medium, TGF-β2 group in culture medium containing TGF-β2, TGF-β2+ OMS group in culture medium containing TGF-β2 and OMS, and OMS group in culture medium containing OMS, and were cultured for 48 hours.Cell apoptosis was detected by flow cytometry with annexin V/PI staining.The early apoptosis, late apoptosis, and total apoptosis rates were analyzed.The protein expression of procaspase-9, cleaved caspase-9, bax and bcl-2 in the mitochondrial apoptosis pathway was detected by Western blot.The activity of lactate dehydrogenase (LDH) and superoxide dismutase (SOD) was detected by colorimetry.The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Xi'an Jiaotong University (No.2019-014).Results:Primary HTF were successfully isolated and cultured.The cultured cells were long spindle-shaped and positive for vimentin.The expression rate of vimentin in the primary cells was greater than 99%.A statistically statistical difference was found in the early apoptosis rate, late apoptosis rate, and total apoptosis rate among the four groups ( F=24.92, 3.96, 41.82; all at P<0.05). The early and total apoptosis rates were significantly higher in TGF-β2+ OMS group than normal control group and TGF-β2 group, and the late apoptosis rate in TGF-β2+ OMS group was significantly higher than that of normal control group (all at P<0.05). There were statistically significant differences in cleaved caspase-9/procaspase-9, bax, and bax/bcl-2 among the four groups ( F=4.40, 7.98, 4.61; all at P<0.05). The bax/bcl-2 expression was significantly increased in TGF-β2+ OMS group in comparison with normal control group, and the expressions of cleaved caspase-9/procaspase-9, bax, and bax/bcl-2 were significantly elevated in TGF-β2+ OMS group compared with TGF-β2 group (all at P<0.05). LDH activity in the normal control group, TGF-β2 group, TGF-β2+ OMS group and OMS group was (783.99±79.97), (913.16±196.86), (2 529.06±240.21), and (2 134.29±138.96) μmol/(min·L), respectively, showing a statistically significant difference ( F=24.95, P<0.05). Compared with normal control group and TGF-β2 group, LDH activity in TGF-β2+ OMS group was increased, and the differences were statistically significant (both at P<0.05). SOD activity in the normal control group, TGF-β2 group, TGF-β2+ OMS group and OMS group was (50.35±0.97), (41.61±4.56), (28.88±3.26), and (37.61±4.83) μmol/(min·L), respectively, showing a statistically significant difference ( F=5.71, P<0.05). SOD activity was reduced in TGF-β2+ OMS group compared with normal control group and TGF-β2 group, reduced in OMS group compared with normal control group, and the differences were statistically significant (all at P<0.05). Conclusions:AGTR1 blocker OMS can promote the apoptosis of HTF effectively.Mitochondrial apoptosis pathway mediated by bax/bcl-2/caspase-9 and oxidative stress pathway are the potential mechanisms that OMS regulates the apoptosis of HTF.
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Objective:To investigate the effect of lithium chloride (LiCl) on the gap junctional intercellular communication (GJIC) in human Tenon capsule fibroblasts (HTFs) and its underlying mechanism.Methods:The Tenon capsule tissue of a patient who underwent strabismus surgery in Dezhou People's Hospital in April 2019 was collected and cut into tissue blocks of dimensions 1 mm×1 mm×1 mm.Primary culture and subculture were carried out, and the 4th-generation HTFs were taken for experiment.HTFs were divided into the control group and LiCl treatment group and were cultured with cell medium without or with 80 mmol/L LiCl for another 48 hours according to grouping.The cell scratch and dye labeling technique were used to label the coupling index and evaluate the GJIC function.The expression and localization of Cx43 in HTFs were detected by immunofluorescence staining.The expression levels of Cx43 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blot, respectively.The study protocol was approved by an Ethics Committee of Dezhou People's Hospital (No.2019-023). Written informed consent was obtained from the subject.Results:The cultured spindle-shaped HTFs grew adhering to the wall showing radial monolayer or vortexlike, and the cytoplasm was vimentin positive.Results of dye tracer experiment of cell scratch showed that the cell coupling index of LiCl treatment group was 9.04±0.53, which was significantly higher than 4.94±0.39 of the control group ( t=-18.79, P<0.01). Immunofluorescence staining showed that the Cx43 fluorescence was dotted in the cell membrane between adjacent cells in the control group, and Cx43 staining was obviously enhanced in the LiCl treatment group.The results of real-time fluorescence quantitative PCR showed that with relative expression level of Cx43 mRNA in the control group set to 1, the relative expression level of Cx43 in the LiCl treatment group was significantly increased to 1.97±0.23, showing a statistical significance between them ( t=-14.426, P<0.01). Western blot showed that the relative expression level of Cx43 protein was 0.871±0.057 in the LiCl treatment group, which was significantly higher than 0.446±0.028 in the control group ( t=-11.682, P<0.01). Conclusions:LiCl can enhance the GJIC function between HTFs by upregulating the expression levels of Cx43 mRNA and protein, suggesting that the enhanced GJIC function by LiCl may be one of the mechanisms of its inhibition on HTFs proliferation.
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@#AIM: To investigate the effect of connective tissue growth factor(CTGF)on the proliferation and migration and transformation in Tenon's capsule fibroblasts(Tfb)of primary open angle glaucoma(POAG)patients.<p>METHODS: Tfb were cultured from Tenon's tissues in POAG patients <i>in vitro</i>. The free-serum DMEM-F12 containing 1.0, 10.0, 100.0ng/mL of CTGF was added into medium for 24h and 48h in different experimental group respectively, and only equal volume of free-serum DMEM-F12 was added in the negative control group. After 24h, the cell proliferation was analyzed through MTT assay, and migration was evaluated by crutch method. After 48h, Semi-quantitative RT-PCR was used to observed the mRNA expression of α-smooth muscl actin(α-SMA), and expression of α-SMA protein was examined by immunochemistry.<p>RESULTS: The proliferation values <i>A</i> of the cells in 1.0, 10.0, 100.0ng/mL of CTGF group respectively were 0.436±0.009, 0.643±0.009, 0.679±0.006, and 0.423 ±0.156 in the negative control group. The migrated cell number was 34.600±3.507, 70.400±2.074, 80.000±2.915 in different concentrations of CTGF group respectively, and 31.000±3.536 in the negative control group. And also in different experimental groups respectively, the absorbance ratio of α-SMA/β-actin was 0.873±0.161, 1.213±0.312, 1.352±0.376, and 0.851±0.158 in the negative control group, the expressing levels <i>A</i> of α-SMA protein in Tfb were 0.110±0.026, 0.141±0.017, 0.175±0.027, and 0.108±0.020 in the negative control group. The statistics of the above experimental data showed that, comparing with the negative control group, the 10.0 and 100.0ng/mL CTGF groups was statistically significant different(<i>P</i><0.05), but there was no statistical different between the 1.0ng/mL CTGF group and the negative control group(<i>P</i>>0.05). <p>CONCLUSION: The proliferation, migration, and phenotypic transformation of Tfb can be promoted in CTGF group in POAG patients. These findings suggest that CTGF may play a role in the development of filtering bleb scarring.
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PURPOSE: To investigate the role of hydrogen sulfide in the survival and collagen gel contraction of cultured human Tenon's capsule fibroblasts (HTCFs). METHODS: Primarily cultured HTCFs were exposed to 0, 100, 200, or 300 µM hydrogen sulfide (sodium hydrogen sulfide, NaHS) for 2 days. Cellular survival was assessed by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. Degree of apoptosis was assessed with flow cytometry using annexin-V/propidium iodide double staining. To evaluate the effect of NaHS on cellular transdifferentiation, HTCFs were stimulated with 5 ng/mL TGF-β1 and the level of expression of α-smooth muscle actin (SMA) mRNA was assessed using reverse-transcription polymerase chain reaction. The cells were embedded in collagen gel, and the amount of gel contraction was measured. RESULTS: NaHS at 300 µM reduced HTCF survival (p = 0.013); NaHS at both 200 and 300 µM increased apoptosis in a dose-dependent manner (p = 0.013 and p = 0.016). TGF-β1 increased the expression of α-SMA mRNA (p = 0.041); co-treatment with 100 µM NaHS decreased TGF-β1-induced α-SMA mRNA expression (p = 0.039) and inhibited collagen gel contraction. CONCLUSIONS: NaHS at high concentration reduced cellular survival and increased HTCF apoptosis. NaHS decreased TGF-β 1-induced increases in α-SMA mRNA expression and collagen gel contraction. Thus, hydrogen sulfide may suppress scar formation by inhibiting HTCF transdifferentiation and contraction of collagen gels.
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Humans , Actins , Apoptosis , Cicatrix , Collagen , Fibroblasts , Flow Cytometry , Gels , Hydrogen Sulfide , Hydrogen , Polymerase Chain Reaction , RNA, Messenger , Tenon CapsuleABSTRACT
Objective To investigate the effects of artesunate (Art) on cell proliferation and apoptosis of human Tenon's capsule fibroblasts (HTFs),and discuss the countermeasures of bleb scarfing in glaucoma.Methods In vitro,HTFs were cultivated and applicated by different concentrations (50 μg · mL-1,100 μg · mL-1,150 μg ·mL-1,200 μg · mL-1) of Art for 48 hours.The effect of Art on cell proliferation was assessed by MTT method.The rate of apoptosis induced by Art was determined by flow cytometry.Western Blot was performed to detect the relative expression levels of Bax and Bcl-2 after Art was treated.Results After treated with Art for 48 hours,compared with blank control group,Art (50 μg · mL-1,100 μg · mL-1,150 μg · mL-1,200 μg · mL-1) group exhibited notable anti-proliferative effect on HTFs with concentration-dependence (all P ＜ 0.05).The results of flow cytometry showed that the apoptosis rates (8.80％ ±0.88％,11.60％ ±0.56％,16.30％ ±1.03％,23.40％ ±1.62％) of HTFs were significantly enhanced with the increase of Art concentration (all P ＜ 0.05).The relative expression levels of Bax were obviously high with the increase of Art concentration,while Bcl-2 levels were significantly low with the increase of Art concentration (all P ＜ 0.05).Conclusion Art can inhibit the proliferation and induce cell apoptosis of HTFs possibly by enhancing the expression of Bax and reducing the expression of Bcl2.Art may be a potential drug in preventing fibrous scar formation after glaucoma filtration surgery.
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Objective To investigate the effects of colchicine on the proliferation and apoptosis of human Tenon's capsule fibroblasts (HTCFs) in vitro.Methods HTCFs were cultured in vitro,and the subcultured cells were identified.MTS assay was used to observe the inhibitory actions of colchicine on HTCFs at different concentrations.The apoptotic rates of the cells were detected by flow cytometry.The apoptosisrelated proteins,including PARP,Caspase-9,Caspase-3 and active-Caspase-3,were detected by Western blot.Results MTS assay showed that colchicine inhibited the proliferation of HTCFs in a dose-and time-dependent manner.Flow cytometry showed that colchicine induced a dose-dependent apoptosis of HTCFs for 48 hours,the apoptotic rates of 5 μmol · L-1,10 μmol · L-1 and 20 μmol · L-1 were (18.37 ±4.26)％,(33.80 ±5.02)％ and (52.00 ± 2.00)％,respectively,there were significant differences compared with the control (F =91.59,P ＜ 0.001).Western blot showed the activation of Caspase-3 and PARP followed by colchicine treatment.Conclusion Colchicine significantly inhibits the proliferation of HTCFs in vitro,and induced apoptosis,which may be associated with the activation of mitochondrial apoptotic pathways.
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Abstract? AlM: To investigate the inhibition effect of bevacizumab on human Tenon capsule fibroblasts ( HTFs ) and discuss the countermeasures of bleb scarringscarring in glaucoma surgery countermeasures.? METHODS: Adopted cell recovery method and followed the aseptic principles, we performed the culture of HTFs which came from the Central Laboratory of Shaanxi People's Hospital cell library. Wound Healing assay:We scraped a cell-free zone on the cell surface when the cells reached confluence at 80%. The control group was added to serum-free DMEM medium. The HTFs of bevacizumab group were stimulated with 1mg/mL concentrations without DEME for 0, 24, 48, and 72h. The scratch width was observed and measured.? RESULTS: HTFs were long fusiform shape under microscope, the nucleus is in the center of the cell with larger nucleus, abundant cytoplasm, were arranged in a whorled growth out of shape, strong ability to proliferate, conform to general forms of fibroblast. The morphological and biological characteristics of cells after cryopreservation and resuscitation remain unchanged. Wound healing assay: 0h, equal to the initial width of the two groups, 24h when the migration distance of the two groups of cells are basically the same, 48h when the control group cell migration distance is greater than that in the bevacizumab processing group, 72h when the control group scratches basichealing, bevacizumab treated cells migrate closer than 48h no significant change, and a lot of cells died.?CONCLUSlON:Cell recovery method can successfully cultured HTFs, which was stability on morphology and biological properties, laying the cellular basis for experimental research. Fibroblast itself has a strong ability to migrate, outside - derived bevacizumab can inhibit HTFs migration evidently and it will cause excessive cell death when. Bevacizumab has certain extent inhibitory effect on HTFs migration, and it is likely to become one of the important drugs for creating bleb scarring after glaucoma surgery in the future.