Pulmonary fibrosis may be partially the result of deregulated tissue repair in response to chronic hypoxia. In this study we explored the effects of hypoxia on lung fibroblasts and the effects of neotuberostemonine (NTS), a natural alkaloid isolated from Stemona tuberosa, on activation of fibroblasts in vitro and in vivo. PLFs (primary mouse lung fibroblasts) were activated and differentiated after exposure to 1% O₂ or treatment with CoCl₂ (100 μmol/L), evidenced by markedly increased protein or mRNA expression of HIF-1α, TGF-β, FGF2, α-SMA and Col-1α/3α, which was blocked after silencing HIF-1α, suggesting that the activation of fibroblasts was HIF-1α-dependent. NTS (0.1-10 μmol/L) dose-dependently suppressed hypoxia-induced activation and differentiation of PLFs, whereas the inhibitory effect of NTS was abolished by co-treatment with MG132, a proteasome inhibitor. Since prolyl hydroxylation is a critical step in initiation of HIF-1α degradation, we further showed that NTS treatment reversed hypoxia- or CoCl₂-induced reduction in expression of prolyl hydroxylated-HIF-1α. With hypoxyprobe immunofiuorescence staining, we showed that NTS treatment directly reversed the lower oxygen tension in hypoxia-exposed PLFs. In a mouse model of lung fibrosis, oral administration of NTS (30 mg·kg^-1·d^-1, for 1 or 2 weeks) effectively attenuated bleomycin-induced pulmonary fibrosis by inhibiting the levels of HIF-1α and its downstream profibrotic factors (TGF-β, FGF2 and α-SMA). Taken together, these results demonstrate that NTS inhibits the protein expression of HIF-1α and its downstream factors TGF-β, FGF2 and α-SMA both in hypoxia-exposed fibroblasts and in lung tissues of BLM-treated mice. NTS with anti-HIF-1α activity may be a promising pharmacological agent for the treatment of pulmonary fibrosis.

中文翻译：

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Neotuberostemine 通过调节 HIF-1α 信号传导抑制小鼠肺成纤维细胞向肌成纤维细胞的分化。

肺纤维化可能部分是由于慢性缺氧而导致的组织修复失调的结果。在这项研究中，我们探讨了缺氧对肺成纤维细胞的影响以及新块百部碱 (NTS)（一种从百部中分离出来的天然生物碱）对体外和体内成纤维细胞活化的影响。_{PLFs（原代小鼠肺成纤维细胞）在1% O 2}暴露或CoCl ₂ (100 μmol/L)处理后被激活和分化，HIF-1α、TGF-β、FGF2、α蛋白或mRNA表达显着增加。 -SMA和Col-1α/3α，在沉默HIF-1α后被阻断，表明成纤维细胞的激活是HIF-1α依赖性的。NTS (0.1-10 μmol/L) 剂量依赖性地抑制缺氧诱导的 PLF 活化和分化，而 NTS 的抑制作用通过与蛋白酶体抑制剂 MG132 共同处理而被消除。由于脯氨酰羟基化是HIF-1α降解起始的关键步骤，我们进一步表明NTS处理逆转了缺氧或CoCl ₂诱导的脯氨酰羟基化-HIF-1α表达的减少。通过低氧探针免疫荧光染色，我们发现 NTS 处理直接逆转了缺氧暴露的 PLF 中较低的氧张力。在肺纤维化小鼠模型中，口服NTS（30 mg·kg ^-1 ·d ^-1，持续1或2周）通过抑制HIF-1α及其下游促纤维化因子的水平，有效减轻博莱霉素诱导的肺纤维化（TGF-β、FGF2 和 α-SMA）。总而言之，这些结果表明，NTS 抑制缺氧暴露的成纤维细胞和 BLM 治疗小鼠肺组织中 HIF-1α 及其下游因子 TGF-β、FGF2 和 α-SMA 的蛋白表达。具有抗HIF-1α活性的NTS可能是一种有前景的治疗肺纤维化的药物。