目的:探讨ZD6474(一种新型的VEGF和EGF抑制剂)对TGF-β1刺激的人Tenon囊成纤维细胞(HTFs)的抗纤维化作用及其在HUVECs中的抗血管生成作用。与丝裂霉素C(MMC)相比。
方法:测定ZD6474对TGF-β1刺激的HTFs和HUVECs细胞增殖或迁移的影响,使用CCK8或伤口愈合试验,分别。检测TGF-β1刺激的HTFs中典型的纤维化标志物,通过免疫荧光波形蛋白,α-SMA和蜗牛的蛋白质印迹。在ZD6474处理后,应用管形成以验证HUVEC中的抗血管生成作用。此外,磷酸化AKT和mTOR(p-AKT和p-mTOR)进行评估,与标准化的总AKT和mTOR相比,使用蛋白质印迹。
结果:在ZD6474(≤1μM/mL)处理后,HTFs的细胞活力几乎没有降低,但是MMC(>50μg/mL)显著损害细胞活力。ZD6474显著抑制TGF-β1刺激的HTFs增殖和迁移,与对照组比较(**P<0.01)。ZD6474还显著减弱了TGF-β1刺激的波形蛋白表达,HTF中的α-SMA和蜗牛。ZD6474处理后HUVEC中的管形成明显中断(**P<0.01)。在TGF-β1-诱导的HTFs和HUVECs中,响应于ZD6474处理,P-AKT和p-mTOR显著降低。
结论:ZD6474可能通过调节AKT-mTOR信号通路在TGF-β1刺激的HTFs中发挥抗增殖和抗纤维化作用。ZD6474也抑制增殖,通过相同的信号通路在HUVECs中迁移和管形成。我们得出结论,ZD6474可能是预防青光眼滤过手术(GFS)后气泡功能障碍的新型药物。
OBJECTIVE: To investigate the anti-fibrotic effect of ZD6474 (a novel inhibitor of VEGF and EGF) in TGF-β1 stimulated human Tenon\'s capsule fibroblasts (HTFs) and the anti-angiogenetic role in HUVECs, compared to that of mitomycin C (MMC).
METHODS: The effects of ZD6474 on cell proliferation or migration in TGF-β1-stimulated HTFs and HUVECs were determined, using CCK8 or wound healing assay, respectively. The typical markers of fibrosis in TGF-β1-stimuated HTFs were detected, vimentin by immunofluorescence, α-SMA and snail by western blot. Tube formation was applied to validate the anti-angiogenesis effect in HUVECs following ZD6474 treatment. Furthermore, phosphorylated AKT and mTOR (p-AKT and p-mTOR) were evaluated, compared to the standardized total AKT and mTOR, using western blot.
RESULTS: There was almost no decreased cell viability in HTFs following ZD6474 (≤ 1 μM/mL) treatment, but MMC (> 50 μg/mL) significantly impaired cell viability. ZD6474 significantly inhibited TGF-β1-stimulated proliferation and migration in HTFs, compared to control group (**P < 0.01). ZD6474 also significantly attenuated the TGF-β1-stimulated expression of vimentin, α-SMA and snail in HTFs. Tube formation was notably interrupted in HUVECs following ZD6474 treatment (**P < 0.01). P-AKT and p-mTOR were significantly decreased in response to ZD6474 treatment in TGF-β1- induced HTFs and HUVECs.
CONCLUSIONS: ZD6474 exerts anti-proliferation and anti-fibrotic effects in TGF-β1-stimulated HTFs perhaps via regulating AKT-mTOR signaling pathway. ZD6474 also inhibited proliferation, migration and tube formation in HUVECs via the same signaling pathway. We concluded that ZD6474 may be potentially a novel agent in preventing bleb dysfunction following glaucoma filtration surgery (GFS).