Atractylenolide III Ameliorated Autophagy Dysfunction via EpidermalGrowth Factor Receptor-Mammalian Target of Rapamycin Signals and Alleviated Silicosis Fibrosis in Mice

Atractylenolide III (ATL-III) is a major active constituent of the natural plant Atractylodes rhizome. Our previous study has shown that ATL-III may alleviate alveolar macrophage apoptosis via the inhibition of the mammalian target of rapamycin (mTOR)-mediated autophagy of human silicosis. Therefore, we aimed to further explore the function of ATL-III in autophagy, apoptosis, and pulmonary fibrosis by establishing the ATL-III-intervened silicosis mouse model in this study. Meanwhile, we sought and then verified potential autophagy-related signaling pathways by matching differentially expressed genes (attained by RNA sequencing) and the autophagy database. In this study, RNA-sequencing results implied that the epidermal growth factor receptor, the crucial upstream activator of mTOR, was seen as a potential autophagy-regulatory molecule in the ATL-IIIeintervened silicosis mouse model. The finding of this study was that ATL-III might improve the disorder of autophagic degradation via the activation of epidermal growth factor receptor-mTOR signals in the pulmonary tissue of the silicosis mouse model. ATL-III also alleviated cell apoptosis and silicotic fibrosis. Overall, we supposed that ATL-III might be a potential protective medicine, which had a regulatory effect on autophagy, for the intervention of silicotic fibrosis. In the future, the therapeutic drugs for silicosis should be further focused on the development and application of such natural autophagy agents. Male C57BL/6J mice (6 to 8 weeks old, 20 to 25g), were housed in an air-conditioned room under light-controlled conditions (twelve-hour light/twelve-hour dark cycle) with standard laboratory food and water ad libitum. A total of 60 mice were randomly divided into 3 groups as follows (n = 20 for every group): the Ctrl group, which was instilled intratracheally with 50 mL of saline; the Silica group, which was instilled intratracheally with 50 mL of saline containing 5 mg of silica; and the Silica þ ATL-III group, which was injected intraperitoneally with 0.2 mL of saline containing 30 mg/kg/d of ATL-III on the basis of the Silica group.. Mice were executed with pentobarbital sodium anesthesia on the 28th day after instillation, and lung tissues were extracted for RNA sequencing.

苍术内酯III（Atractylenolide III, ATL-III）是天然植物苍术根茎的主要活性成分。本课题组此前的研究表明，ATL-III可通过抑制哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）介导的人硅肺细胞自噬，缓解肺泡巨噬细胞凋亡。因此，本研究通过构建ATL-III干预的硅肺小鼠模型，进一步探究ATL-III在自噬、细胞凋亡及肺纤维化中的作用。
同时，本研究通过匹配经RNA测序（RNA-sequencing）获得的差异表达基因与自噬数据库，筛选并验证了潜在的自噬相关信号通路。本研究的RNA测序结果显示，作为mTOR关键上游激活因子的表皮生长因子受体（epidermal growth factor receptor, EGFR），在ATL-III干预的硅肺小鼠模型中可作为潜在的自噬调控分子。
本研究发现，在硅肺小鼠模型的肺组织中，ATL-III可通过激活表皮生长因子受体-mTOR信号通路，改善自噬降解紊乱；同时ATL-III能够缓解细胞凋亡与硅肺纤维化。综上，我们推测ATL-III可能是一种潜在的保护性药物，可通过调控自噬实现硅肺纤维化的干预。未来，硅肺治疗药物的研发应进一步聚焦于此类天然自噬调节剂的开发与应用。
本研究选取6~8周龄、体重20~25g的雄性C57BL/6J小鼠，在光照可控（12小时光照/12小时黑暗循环）的空调房内饲养，自由采食标准实验饲料并自由饮水。将60只小鼠随机分为3组（每组n=20）：对照组（Ctrl组），经气管滴注50mL生理盐水；硅肺模型组（Silica组），经气管滴注50mL含5mg二氧化硅的生理盐水；ATL-III干预组（Silica+ATL-III组），在硅肺模型组基础上，每日腹腔注射0.2mL含30mg/kg ATL-III的生理盐水。
于滴注后第28天，采用戊巴比妥钠麻醉并处死小鼠，提取肺组织进行RNA测序。