Proliferation Of Human Pterygium Fibroblasts Research Articles

Phytochemical Profile and Antioxidant and Antiproliferative Activity of Sedum dendroideum on Pterygium Fibroblasts.

Background Sedum dendroideum has antioxidant effects that are beneficial for different diseases. We aimed to analyze the antiproliferative activity of S. dendroideum in human pterygium fibroblasts (HPFs). Methods HPFs were treated for 24 h with 0–1000 μg/mL of S. dendroideum lyophilized to analyze its effect on cell viability using the CellTiter assay. RNA from HPF treated with 250 μg/mL of S. dendroideum lyophilized was isolated, and the expression of VEGF and CTGF genes was evaluated by qPCR. A dermal fibroblast cell line (HDFa) was used as a healthy control. The total phenolic content, antioxidant activity, and chemical profile of S. dendroideum lyophilized were determined. Results Viability of HPF decreased after 24 h treatment of S. dendroideum in a dose-dependent manner. The expression of VEGF and CTGF significantly decreased (P < 0.01) in HPF treated with 250 μg/mL of S. dendroideum when compared with untreated HPF. The total phenolic concentration in the S. dendroideum lyophilized was 33.67 mg gallic acid equivalents (GAE)/g. Antioxidant activity was 384.49 mM Trolox equivalents/mL. The main phenolic compounds identified by HPLC analysis were the kaempferol-3-O-glycoside, kaempferol-3-O-rhamnoside, kaempferol-3-O-neohesperidoside-7-O-α-rhamnopyranoside, and kaempferol-3-O-glycoside-7-O-rhamnoside. Conclusions S. dendroideum decreases the proliferation of HPF and the expression of VEGF and CTGF. The phenolic compound concentration, antioxidant activity, and phytochemical profile may play a role in these effects.

The in vitro anti-fibrotic effect of Pirfenidone on human pterygium fibroblasts is associated with down-regulation of autocrine TGF-β and MMP-1.

We aimed to investigate the in vitro effect of pirfenidone (PFD) on proliferation, migration and collagen contraction of human pterygium fibroblasts (HPFs). HPFs were obtained from tissue explants during pterygium surgery. After treatment with pirfenidone, the HPFs proliferation was measured by MTT, cell cycle progression measured by flow cytometry, cell migration measured by the scratch assay, and cell contractility evaluated in fibroblast-populated collagen gels. The expression of TGF-β1, TGF-β2, MMP-1 and TIMP-1 were also determined with quantitative PCR, western blot and immunofluorescence staining. Results showed pirfenidone markedly inhibited HPFs proliferation with an IC50 of approximately 0.2 mg/ml. After treatment with 0.2 mg/ml pirfenidone for 24 hours, HPFs were at G0/G1 cell cycle arrest, with significantly reduced cell migration capability and collagen contraction, decreased mRNA and protein expressions of TGF-β1, TGF-β2 and MMP-1, and no alterations of TIMP-1 expression. Thus, we have concluded that pirfenidone at 0.2 mg/ml inhibits proliferation, migration, and collagen contraction of HPFs, which is associated with decreased expression of TGF-β and MMP-1, and pirfenidone might represent a potentially therapeutic agent to prevent the recurrence of pterygium after surgery.

Efficacy of curcumin in inducing apoptosis and inhibiting the expression of VEGF in human pterygium fibroblasts.

Pterygium is an inflammatory and degenerative ocular surface disease, in which the conjunctiva on the cornea grows to form a fibrous tissue in the shape of a triangle. Although pterygium surgery is used broadly in clinical practice, its high recurrence rate poses a great concern for patients and doctors. In the present study, we examined the effects of curcumin on the apoptosis and proliferation of human pterygium fibroblasts (HPFs) in an in vitro culture system. HPFs were incubated with curcumin for 24 to 72 h (5, 20, 80 or 200 µmol/l). The proliferation-inhibiting effects of curcumin were assayed by MTT assay at different time points and at various concentrations. The expression of vascular endothelial growth factor (VEGF) in each group was detected by RT-qPCR and ELISA. The apoptotic effects of curcumin were detected by flow cytometry (FCM). Treatment with curcumin at 80 µmol/l for 48 h significantly inhibited HPF proliferation. Curcumin significant induced the apoptosis of HPFs. Our findings thus suggest that curcumin suppress cell proliferation in the pterygium by inducing HPF apoptosis and inhibiting VEGF expression. Thus, curcumin has potential for use as a novel agent for the treatment of pterygium in the near future.

Proliferative Effects of Histamine on Primary Human Pterygium Fibroblasts.

Purpose. It has been confirmed that inflammatory cytokines are involved in the progression of pterygium. Histamine can enhance proliferation and migration of many cells. Therefore, we intend to investigate the proliferative and migratory effects of histamine on primary culture of human pterygium fibroblasts (HPFs). Methods. Pterygium and conjunctiva samples were obtained from surgery, and toluidine blue staining was used to identify mast cells. 3-[4, 5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was performed to evaluate the proliferative rate of HPFs and human conjunctival fibroblasts (HCFs); ki67 expression was also measured by immunofluorescence analysis. Histamine receptor-1 (H1R) antagonist (Diphenhydramine Hydrochloride) and histamine receptor-2 (H2R) antagonist (Nizatidine) were added to figure out which receptor was involved. Wound healing model was used to evaluate the migratory ability of HPFs. Results. The numbers of total mast cells and degranulated mast cells were both higher in pterygium than in conjunctiva. Histamine had a proliferative effect on both HPFs and HCFs, the effective concentration (10 μmol/L) on HPFs was lower than on HCFs (100 μmol/L), and the effect could be blocked by H1R antagonist. Histamine showed no migratory effect on HPFs. Conclusion. Histamine may play an important role in the proliferation of HPFs and act through H1R.

Antifibrotic Effect of Pirfenidone on Human Pterygium Fibroblasts

Objective: The effects of pirfenidone were investigated on cultured human pterygium fibroblasts (HPFs).Methods: HPFs were obtained from pterygium surgery and subjected to primary culture. After treatment with 0.5, 1.0 or 1.5 mg/mL pirfenidone, MTT and cell migration assays were performed, and procollagen secretion and TGF-β expression were measured by Western blotting and immunofluorescence analysis.Results: Pirfenidone had a significant inhibitory effect on HPF proliferation, migration and collagen synthesis. There were no differences between the cells treated with 0.5, 1.0 and 1.5 mg/mL pirfenidone and the controls in the MTT assay. After 48 h of treatment with 1.0 or 1.5 mg/mL pirfenidone, TGF-β expression was significantly decreased.Conclusions: These findings demonstrate that pirfenidone inhibits the proliferation, migration and procollagen secretion of HPFs at nontoxic concentrations by decreasing TGF-β expression. Thus, pirfenidone may be considered as a safe adjuvant for pterygium surgery to prevent recurrence.

Effects of mitomycin C on membrane permeability and ultrastructure changes in human pterygium fibroblasts

Background Mitomycin C (MMC) has an inhibitory effect on the growth and proliferation of human pterygium fibroblasts,however,there is little literature about its influence on plasma membrane. Objective This study was to investigate the influence of MMC on the physical and chemical features and ultrastructures of plasma membrane. Methods Human pterygium tissues were obtained during the surgery.Human pterygium fibroblasts were primarily cultured and passaged using explant cultured method and identified by Vimentin staining.The third generation of cells were incubated to 96 well plate at a density of 5 × 103 cells/well,and 0,50,100,200,300 and 400 mg/L MMC was added in the culture well respectively to act for 12 hours.Cell viability was assayed using cell counting kit-8 ( CCK-8 ),and cellular apoptosis was detected using annexin V-FICT/PI.The changes of cell membrane structure were examined under an atomic force microscope.Malondialdehyde( MDA ) content in cell supernatant and level of lactate dehydrogenase ( LDH ) in extracellular fluid were detected to assess the lipid peroxidation level and permeability of cell membrane.Intracellular Ca2+ changes and membrane surface topography were assessed by Fluo-3/AM mark and flow cytometry( FCM ).This study was approved by Ethic Commission of Affiliated First Hospital of Jinan University.Informed consent was obtained from each patient. Results A lot of cells grew with the shape of spindle 1-2 weeks after culture.Positive response was seen in cultured cells for Vimentin.Growth and proliferation of the cells reduced 12 hours after action of MMC with the increase of MMC concentrations.The apoptosis rate of human pterygium fibroblasts was 4.2％,4.2％,5.4％,19.3％ and 25.8％ in 0,50,100,200 and 300 mg/L MMC groups respectively.Different degrees of abnormalities of cells membrane were found in various concentrations of MMC groups.The elevated contents of LDH and MDA in extracellular fluid were detected in various concentrations of MMC groups compared with the control group,and the LDH and MDA levels were gradually ascended as the increase of MMC concentrations,with a significant difference between any two groups(P＜0.05).The disturbance of intracellular Ca2+ homeostasis was also been seen after MMC acted. Conclusions MMC leads to the changes of physical and chemical features in human pterygium fibroblasts at a dose-dependent manner.Cell membrane may be the acting target of MMC. Key words: Mitomycin C ; Pterygium; Cytotoxicity ; Ultrastructure

Inhibitory effect of PPARγ agonist on the proliferation of human pterygium fibroblasts

The effects of DK2, a peroxisome proliferator-activated receptor γ agonist, on cultured human pterygium fibroblasts (HPFs) in virto were studied. The HPFs were incubated with 0-200 μmol/L DK2 for 12-72 h. The MTT method was used to assay the bio-activity of DK2 at different doses and time. The cytotoxic effect of DK2 was measured by LDH release assay. The cell cycle distribution and apoptosis were flow cytometrically detected. The expression of proliferating cell nuclear antigen (PCNA) in each group was detected by real-time PCR (RT-PCR) and Western blotting. The results showed that administration of 1-75 μmol/L DK2 for 12-72 h could significantly inhibit HPF proliferation in a dose- and time-dependent manner. DK2-treated cells did not release significant amount of LDH as compared with rosiglitazone-treated cells. After treatment with DK2 at concentrations of 15, 25 μmol/L for 24 h, the number of HPFs in G(0)/G(1) phase was significantly increased while that in S phase was significantly decreased (P<0.05), leading to arrest at G(0)/G(1) phase. The apoptosis rates of HPF cells in drug-treated groups were significantly higher than the rate of control group (P<0.05). At the dosage range between 15-25 μmol/L, DK2 could inhibit the expression of PCNA mRNA and protein in HPFs in a dose-dependent fashion (P<0.05). It was concluded that PPARγ agonist can significantly inhibit HPF proliferation, resulting in the arrest at G(0)/G(1) phase, induce the apoptosis of HPFs, and suppress the synthesis of PCNA, in dose- and time-dependent manners.

Atomic Force Microscope-Related Study Membrane-Associated Cytotoxicity in Human Pterygium Fibroblasts Induced by Mitomycin C

Mitomycin C (MMC) has been shown to have a therapeutic effect against human pterygium fibroblasts (HPFs) by inducing apoptosis. However, there is little data about the effect of it on plasma membrane. In the present study, the cytotoxicity of MMC to HPFs including inhibiting cell growth, inducing apoptosis and bringing about membrane toxicity was investigated. It was found that MMC could significantly suppress the proliferation of HPFs in a dose-dependent manner by CCK-8 assay. Flow cytometric analysis also revealed that treatment with MMC resulted in increased percentages of apoptotic cells in a dose-dependent manner. Membrane lipid peroxidation level, lactate dehydrogenase (LDH) leakage, membrane surface topography, and membrane rigidity alterations were investigated to assess the membrane toxicity induced by MMC. Treatment with MMC at different concentrations accelerated membrane lipid peroxidation and potentiated LDH leakage, which was consistent with disturbance of membrane surface and decrease of membrane elasticity detected by atomic force microscopy. All the above changes led to the disturbed intracellular Ca(2+) homeostasis, which was an important signal triggering apoptosis. Hence, the membrane toxicity induced by MMC might play an important role in the process of apoptotic induction and the calcium channel may be one of the apoptosis mechanisms.

Inhibitory Effect of PPARγ Agonist on the Proliferation of Human Pterygium Fibroblasts

Summary: The effects of DK2, a peroxisome proliferator-activated receptor γ agonist, on cultured human pterygium fibroblasts (HPFs) in virto were studied. The HPFs were incubated with 0–200 μmol/L DK2 for 12–72 h. The MTT method was used to assay the bio-activity of DK2 at different doses and time. The cytotoxic effect of DK2 was measured by LDH release assay. The cell cycle distribution and apoptosis were flow cytometrically detected. The expression of proliferating cell nuclear antigen (PCNA) in each group was detected by real-time PCR (RT-PCR) and Western blotting. The results showed that administration of 1–75 μmol/L DK2 for 12–72 h could significantly inhibit HPF proliferation in a dose- and time-dependent manner. DK2-treated cells did not release significant amount of LDH as compared with rosiglitazone-treated cells. After treatment with DK2 at concentrations of 15, 25 μmol/L for 24 h, the number of HPFs in G0/G1 phase was significantly increased while that in S phase was significantly decreased (P<0.05), leading to arrest at G0/G1 phase. The apoptosis rates of HPF cells in drug-treated groups were significantly higher than the rate of control group (P<0.05). At the dosage range between 15–25 μmol/L, DK2 could inhibit the expression of PCNA mRNA and protein in HPFs in a dose-dependent fashion (P<0.05). It was concluded that PPARγ agonist can significantly inhibit HPF proliferation, resulting in the arrest at G0/G1 phase, induce the apoptosis of HPFs, and suppress the synthesis of PCNA, in dose- and time-dependent manners.

The effects of β-NGF on proliferation of human pterygium fibroblasts

目的 观察神经生长因子β(β-NGF)对体外培养的人翼状胬肉成纤维细胞(HPF) 增生的影响,探讨翼状胬肉的发病机制.方法 对翼状胬肉组织标本行组织块培养.待细胞生长汇合至80% 后以0.25%胰蛋白酶+ 0.02%EDTA 1:1 混合消化细胞,取3～5代细胞用于实验.通过细胞中波形蛋白、角蛋白及α平滑肌肌动蛋白的表达鉴定培养的细胞,免疫荧光法检测HPF中β-NGF受体trkA、p75的表达,MTT法检测不同质量浓度β-NGF对HPF的作用,Western blot及RT-PCR法半定量检测细胞核抗原(PCNA)来评估HPF的增生.结果 波形蛋白、α平滑肌肌动蛋白、trkA和p75在HPF中呈阳性表达;MTT结果显示,在β-NGF 作用12、24、48、72和96h 后,48h为β-NGF促增生的高峰期;在不同质量浓度β-NGF(5～50ng/mL)作用下,HPF中PCNA蛋白及mRNA的表达与0和1ng/mL β-NGF作用组之间比较差异均有统计学意义(Fprotein=24.980,P=0.000;FmRNA=64.490,P=0.000).结论 NGF可能通过结合两受体trkA、p75促进了HPF的增生。

Inhibitory effect of curcumin on proliferation of human pterygium fibroblasts

In order to investigate the effect of curcumin on proliferation and apoptosis of human pterygium fibroblasts (HPF) in culture and search for a new method to prevent the recurrence after pterygium surgery, HPF was incubated with 0-160 micromol/L curcumin for 24-96 h. The MTT method was used to assay the biologic activities of curcumin at different time points and different doses. The expression of proliferating cell nuclear antigen (PCNA) in each group was detected by immunohistochemistry. The cell cycle distribution was detected by flow cytometry (FCM). Administration of 20-80 micromol/L curcumin for 24-72 h could significantly inhibit HPF proliferation in a dose-and time-dependent manner (P<0.05). After treatment with curcumin at different concentrations of 20, 40, 80 and 160 micromol/L for 24 h, FCM revealed there was a significant sub-G1 peak at each concentration. The number of HPF in G0/G1 phase was increased, while in S phase, it was decreased (P<0.05). At the concentration of 20-80 micromol/L, curcumin, in a dose-dependent manner (P<0.05), could inhibit the expression of PCNA in HPF. It was suggested that curcumin could significantly inhibit the proliferation of HPF, make HPF arrest in G0/G1 phase and induce the apoptosis of HPF in a dose-and time-dependent manner.