Department of Dermatology, Zhongshan Hospital, Fudan University, Shanghai, China.
Rheumatology (Oxford). 2018 Sep 1;57(9):1675-1684. doi: 10.1093/rheumatology/key166.
To investigate the mechanism of 2-methoxyestradiol (2-ME) in inhibiting hypoxia-induced collagen synthesis of fibroblasts in SSc.
The expressions of hypoxia-inducible factor 1 alpha (HIF-1α) and connective tissue growth factor (CTGF) in skin specimens derived from SSc patients and healthy volunteers were examined by immunohistochemistry. HIF-1α was knocked down by lentiviral transduction, and SSc dermal fibroblasts cultured under normoxic (21% O2) or hypoxic (1% O2) condition were treated with PI3K inhibitor LY294002, rapamycin or 2-ME (25 μM). The protein levels of HIF-1α, CTGF, collagen I, p-Akt and p-mTOR were examined by western blotting or immunofluorescence. Apoptosis and cell cycle of fibroblasts were assessed by flow cytometry and by measuring caspase 3 activity, and cell proliferation was evaluated by Cell Counting Kit-8.
The expressions of HIF-1α and CTGF were increased in skins of SSc patients compared with healthy controls. Hypoxia up-regulated the protein levels of HIF-1α, CTGF and collagen I in SSc fibroblasts. In contrast, 2-ME inhibited PI3K/Akt/mTOR pathway and down-regulated protein levels of HIF-1α, CTGF and collagen I. Knockdown of HIF-1α reduced expressions of CTGF and collagen I, which were further down-regulated by 2-ME intervention. Moreover, 2-ME promoted the apoptosis and inhibited the proliferation of SSc fibroblasts by arresting the cell cycle at the G2/M phase.
2-ME reduced the production of CTGF and collagen I in SSc fibroblasts induced by hypoxia through PI3K/Akt/mTOR/HIF-1α signalling and inhibited the proliferation of fibroblasts. These findings suggested that 2-ME could be employed as a promising antifibrotic therapy for SSc.
研究 2-甲氧基雌二醇（2-ME）抑制 SSc 成纤维细胞缺氧诱导胶原合成的机制。
采用免疫组化法检测 SSc 患者和健康志愿者皮肤标本中缺氧诱导因子 1α（HIF-1α）和结缔组织生长因子（CTGF）的表达。通过慢病毒转导敲低 HIF-1α，在常氧（21% O2）或低氧（1% O2）条件下培养 SSc 真皮成纤维细胞，并分别用 PI3K 抑制剂 LY294002、雷帕霉素或 2-ME（25 μM）处理。通过 Western blot 或免疫荧光法检测 HIF-1α、CTGF、胶原 I、p-Akt 和 p-mTOR 的蛋白水平。通过流式细胞术和检测 caspase 3 活性评估成纤维细胞的凋亡和细胞周期，通过细胞计数试剂盒-8 评估细胞增殖。
与健康对照组相比，SSc 患者皮肤中 HIF-1α和 CTGF 的表达增加。低氧上调 SSc 成纤维细胞中 HIF-1α、CTGF 和胶原 I 的蛋白水平。相比之下，2-ME 抑制了 PI3K/Akt/mTOR 通路，并下调了 HIF-1α、CTGF 和胶原 I 的蛋白水平。HIF-1α 敲低降低了 CTGF 和胶原 I 的表达，2-ME 干预进一步下调了其表达。此外，2-ME 通过将细胞周期阻滞在 G2/M 期，促进 SSc 成纤维细胞凋亡并抑制其增殖。
2-ME 通过 PI3K/Akt/mTOR/HIF-1α 信号通路降低低氧诱导的 SSc 成纤维细胞中 CTGF 和胶原 I 的产生，并抑制成纤维细胞的增殖。这些发现表明，2-ME 可作为治疗 SSc 的一种有前途的抗纤维化疗法。
