Inhibition Effect Of Pirfenidone On Stress Fibers And Focal Adhesions Remodeling And Cell Function Of Orbital Fibroblasts

PartⅠ Primary culture, identification and myofibroblast differentiation of orbital fibroblastsObjective: Primary culture of the orbital fibroblasts derived from TAO and control group was applied to evaluate the growth and differentiation state of the cells under the stimulation effect of TGF-β1.Methods: Orbital fibroblasts were collected by tissue explant culture technique and identified by morphological and immunocytochemistry analysis. MTT was used to assess the proliferation of orbital fibroblasts in the inducing effect of different concentrations of TGF-β1(1μg/L, 5μg/L, 10μg/L). The α-SMA expression levels induced by TGF-β1 with different dose and time effects were detected by immunocytochemistry and Western blot.Results: ⑴The orbital fibroblasts were cultured and passaged successfully. The morphological features of fibroblast were long spindle appearance, plump cytoplasm, round or oval nuclei; the immunocytochemistry analysis showed that Vimentin staining was positive, Desmin, Keratin and S-100 staining were all negative, indicating that the cells were mesoderm derived fibroblasts. ⑵ Fibroblasts proliferation in MTT assay were induced by TGF-β1 in a dose-effect. After 72 h, the highest rates of cell proliferation, 107.35%, was found in the 10μg/L TGF-β1 group(P﹤0.05). MTT optical density values in TAO and control groups were compared, and showed no statistically significant difference(P﹥0.05). ⑶The cytoplasm and area were enlarged by the stimulation of 10μg/L TGF-β1 for 24h; Immunostaining displayed α-SMA expression in fibril form parallel to the long axis of cell, after stimulation with 10μg/L TGF-β1 for 72 h. ⑷ Western-blot detection of TGF-β1-induced α-SMA protein expression was in a dose and time effect, which was found to reached a plateau at the concentration of 10μg/L TGF for 48h(P﹤0.05). No statistically significant difference of α-SMA expression was found between TAO and control groups(P﹥0.05).Conclusions: The orbital fibroblasts show morphological and immunologicalcharacteristics significantly by tissue explant culture. TGF-β1 has promotion effect in fibroblast proliferation, differentiation to myofibroblast, expression of α-SMA in a concentration and time dependent manner within a certain range.PartⅡ Inhibition effects of pirfenidone on proliferation, differentiation, migration, and collagen contraction of orbital fibroblasts in vitroObjective: To investigate the effect of pirfenidone on proliferation, differentiation, migration, and collagen contraction of orbital fibroblasts.Methods: After stimulating by different concentration of TGF-β1, pirfenidone(PFD), dexamethasone(DXM), fibroblast proliferation was measured by MTT assay; cell viability was detected by trypan blue exclusion assay. In the 10μg/L TGF-β1 group, 1.0mg/ml PFD group, 1000 n M DXM group, α-SMA and fibronectin expression were estimated in immunocytochemistry assay; cell migration was evaluated by scratch assay; fibroblast contractility was investigated by fibroblast-mediated collagen gels contraction assay.Results: ⑴ In MTT assay, pirfenidone inhibited fibroblasts proliferation in a dose-effect, 72 h cell proliferation rate in 0.5mg/ml PFD group and 1.0mg/ml PFD group were-24.82%,-41.84%; the difference, compared with control group, had statistically significant(P﹤0.05). Pirfenidone displayed more powerful effect than dexamethasone in the inhibition of TGF-β1-induced cell proliferation(P﹤0.05). ⑵In trypan blue exclusion test, after treating with different concentrations of pirfenidone for 72 h, the cell viability was good, no statistically significant difference was found between drag treated and control groups. ⑶Compared with control group, TGF-β1 has the effect of promoting cell migration and scratch healing, while pirfenidone significantly reduced cell motility in the scratch test(P﹤ 0.05). ⑷Immunocytochemistry assay showed that the expression of α-SMA in fibroblasts was inhibited in PFD group, but normal in DXM group. In control group, TGF-β1 group, DXM group, fibronectin expression was found in the cytoplasm and outside thefibroblast; while pirfenidone inhibited fibronectin expression with or without TGF-β1 treatment. ⑸Exposure to TGF-β1 for 6 days, contractile index in TGF-β1 group was 59.38%, which indicated TGF-β1 induced more powerful promotion in fibroblast mediated collagen contraction than conrol(P﹤0.05). Pirfenidone induced significant inhibition of collagen contraction with the index of 6.70% and 8.02% in the absence or presence of TGF-β1(P﹤0.05). In DXM group, the index was 25.48%; there was no statistical significance(P﹥0.05).Conclusions: Pirfenidone inhibits proliferation, differentiation, migration, and collagen contraction of orbital fibroblasts and down regulates their fibronectin expression. These results provide a better effectiveness of pirfenidone than dexamethasone in the suppression of fibrosis inducing by TGF-β1.PartⅡ Inhibition effect of pirfenidone on stress fiber remodeling and focal adhesions maturation in orbital fibroblasts in vitroObjective: To research inhibition role of pirfenidone on TGF-β1 induced actin stress fibers remodeling and focal adhesions maturation, and their signaling pathways. Methods: After stimulation by TGF-β1 or pirfenidone(PFD), the morphology and subcellular localization of actin stress fibers, focal adhesion protein and collagenⅠwere evaluated by immunofluorescence assay under confocal laser scanning microscopy; the expression of Akt, Phospho-Akt, FAK, and Phospho-FAK protein were detected by Western-blot; cell adhesion to the Max GelTM was investigated by fluorescein stain.Results: ⑴ Observed by the confocal microscope, pirfenidone inhibited TGF-β1-induced fibroblast to myofibroblast differentiation, reduced α-SMA-positive stress fibers building; it diminished the expression of paxillin and tensin, prohibited differentiation and maturation of focal adhesions by decreasing their size and number(P ﹤ 0.05). ⑵ Western Blot results showed that pirfenidone inhibited TGF-β1-induced expression levels of Akt, Phospho-Akt, FAK, and Phospho-FAK protein(P﹤0.05). ⑶Cell adhesion experiments showed that few cell adhered to theextracellular matrix, after treated with pirfenidone for 24 h, which indicated pirfenidone prevented fibroblast adhesion to extracellular matrix(P﹤0.05).Conclusions: By down regulation of TGF-β1-mediated Akt/FAK signaling pathway activity, pirfenidone could inhibit α-SMA stress fibers assembly and focal adhesions maturation, which may serve as significant regulation role to reduce fibroblasts adhesion to the extracellular matrix, and further suppress their activity in the fibrosis process. So, pirfenidone can be used against TAO fibrosis development and improvement of clinical symptoms.