Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, China; Jiangsu Key Laboratory of Pediatric Respiratory Disease, Institute of Pediatrics, Nanjing University of Chinese Medicine, Nanjing, China.
Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, China; Jiangsu Key Laboratory of Pediatric Respiratory Disease, Institute of Pediatrics, Nanjing University of Chinese Medicine, Nanjing, China; Department of Respiratory and Critical Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, China.
Phytomedicine. 2021 Aug;89:153599. doi: 10.1016/j.phymed.2021.153599. Epub 2021 May 21.
Idiopathic pulmonary fibrosis is a chronic, progressive, fibrotic disease. Although the pathogenesis remains unclear, the effect of endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AEC IIs) is increasingly thought to be a critical mechanism.
We investigated the effects of citrus alkaline extracts (CAE) on AEC IIs and elucidated the underlying mechanism for their possible use in ameliorating pulmonary fibrosis (PF).
A bleomycin-induced mouse model of PF, and an in vitro tunicamycin (TM) -induced ER stress model in A549 cells were successfully established. Accumulation of collagen in lung tissues in vivo was assessed using histological analysis and western blotting. The expression levels of the ER-stress marker BiP and other related proteins were assessed by western blotting and immunofluorescence staining. Mitochondrial membrane potential was assessed to evaluate mitochondrial homeostasis.
CAE mitigated collagen deposition to ameliorate PF in vivo. CAE suppressed the bleomycin or TM-induced increases in ER-stress biomarker, BiP, and PERK pathway proteins, resulting in a decrease in ER stress in mouse lung tissues and A549 cells, respectively. Additionally, CAE treatment suppressed the bleomycin or TM-induced increase in the ER-stress downstream proteins, activating ATF3 and increased the levels of PINK1 in AEC IIs, both in vivo and in vitro. The reduced mitochondrial homeostasis induced by TM was restored by CAE-treatment in A549 cells. Furthermore, conditioned media from TM-treated A549 cells increased collagen deposition in MRC5 cells mainly via TGF-β1. The increased collagen deposition was not seen using conditioned media from CAE-treated A549 cells.
These results provide novel insights into the potential mechanism of CAE in inhibiting ER stress in AEC IIs, and suggests that it has great potential to ameliorate PF via the ATF3/PINK1 pathway.
特发性肺纤维化是一种慢性、进行性、纤维化疾病。尽管其发病机制仍不清楚，但内质网（ER）应激在 II 型肺泡上皮细胞（AEC II）中的作用越来越被认为是一个关键机制。
我们研究了柑橘碱提取物（CAE）对 AEC II 的影响，并阐明了其在改善肺纤维化（PF）中的潜在机制。
成功建立了博来霉素诱导的 PF 小鼠模型和 A549 细胞中的衣霉素（TM）诱导的 ER 应激模型。通过组织学分析和 Western blot 评估体内肺组织中胶原蛋白的积累。通过 Western blot 和免疫荧光染色评估 ER 应激标志物 BiP 和其他相关蛋白的表达水平。评估线粒体膜电位以评估线粒体稳态。
CAE 减轻胶原蛋白沉积，改善体内 PF。CAE 抑制博来霉素或 TM 诱导的 ER 应激生物标志物 BiP 和 PERK 通路蛋白的增加，导致小鼠肺组织和 A549 细胞中的 ER 应激分别降低。此外，CAE 处理抑制了博来霉素或 TM 诱导的 ER 应激下游蛋白的增加，激活了 ATF3 并增加了 AEC II 中的 PINK1 水平，无论是在体内还是体外。TM 诱导的线粒体稳态降低在 A549 细胞中通过 CAE 处理得到恢复。此外，来自 TM 处理的 A549 细胞的条件培养基主要通过 TGF-β1 增加 MRC5 细胞中的胶原蛋白沉积。使用来自 CAE 处理的 A549 细胞的条件培养基没有观察到增加的胶原蛋白沉积。
这些结果为 CAE 抑制 AEC II 中的 ER 应激的潜在机制提供了新的见解，并表明它通过 ATF3/PINK1 途径具有改善 PF 的巨大潜力。
