Abstract Context Pulmonary fibrosis (PF) is a highly heterogeneous and lethal pathological process having no effective drug. Engeletin exerts multiple biological activities including anti-inflammatory and lung repair. Whether engeletin has therapeutic effects on PF remains unclear. Objective Examining effect and mechanism of engeletin on PF in vivo and in vitro. Materials and methods L929 cells (1 × 106/well) were treated with TGF-β1 (5 ng/mL). Sixty male C57BL/6 mice were divided into three groups and given saline or single intratracheal instillation bleomycin (5 mg/kg) or both bleomycin and intraperitoneally injected engeletin (25 mg/kg). Results Histological staining showed engeletin inhibited myofibrobasts activation and improved alveolar structure. Engeletin elevated forced vital capacity from 12 induced by bleomycin to 17. CCK-8 assay reported IC50 value of engeletin was 270 μg/mL. Real-time cellular analysis showed engeletin reduced proliferation and migration of myofibroblasts by 2.5- and 2-fold. Engeletin blocked α-SMA, vimentin, and collagen expression. RNA sequencing revealed PERK-ATF4 signalling pathway relating to ER stress involved in anti-fibrotic function of engeletin. Engeletin reduced ATF4, CHOP and BIP expression. Chemical inhibitors of smad2/3- (SB431542) and JNK- (SP600125) signalling pathways blocked expression of long noncoding RNA (lncRNA) – lnc949. Engeletin inhibited phosphorylation of smad2/3 and JNK leading to lower level of lnc949. Knockdown lnc949 inhibited ATF4, CHOP and BIP expression. Conclusions We reported gene expression profiling of engeletin through RNA-seq; and identified lnc949-mediated TGF-β1-Smad2/3 and JNK were upstream signalling pathways of ER stress induced by engeletin. Our results showed engeletin remedies pulmonary fibrogenesis and may be a new drug candidate.

中文翻译：

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Engeletin 通过内质网应激改善肺纤维化，这取决于 lnc949 介导的 TGF-β1-Smad2/3 和 JNK 信号通路

摘要 背景肺纤维化（PF）是一种高度异质性和致命的病理过程，没有有效的药物。Engeletin 发挥多种生物活性，包括抗炎和肺修复。恩格列汀是否对 PF 有治疗作用尚不清楚。目的考察engeletin对PF的体内外作用及机制。材料和方法 L929 细胞（1 × 106/孔）用 TGF-β1（5 ng/mL）处理。将 60 只雄性 C57BL/6 小鼠分为三组，给予生理盐水或单次气管内滴注博来霉素（5mg/kg）或博来霉素和腹腔注射恩格列汀（25mg/kg）。结果组织学染色显示恩格列汀抑制肌成纤维细胞活化并改善肺泡结构。Engeletin 将博来霉素诱导的用力肺活量从 12 提高到 17。CCK-8 测定报告恩格列汀的 IC50 值为 270 μg/mL。实时细胞分析显示 engeletin 将肌成纤维细胞的增殖和迁移降低了 2.5 倍和 2 倍。Engeletin 阻断了 α-SMA、波形蛋白和胶原蛋白的表达。RNA测序揭示了与内质网应激相关的PERK-ATF4信号通路参与engeletin的抗纤维化功能。Engeletin 降低了 ATF4、CHOP 和 BIP 的表达。smad2/3- (SB431542) 和 JNK- (SP600125) 信号通路的化学抑制剂阻断了长链非编码 RNA (lncRNA) – lnc949 的表达。Engeletin 抑制 smad2/3 和 JNK 的磷酸化，导致 lnc949 水平降低。敲低 lnc949 抑制 ATF4、CHOP 和 BIP 表达。结论 我们通过 RNA-seq 报告了 engeletin 的基因表达谱；并确定 lnc949 介导的 TGF-β1-Smad2/3 和 JNK 是 engeletin 诱导的 ER 应激的上游信号通路。我们的结果表明 engeletin 可以治疗肺纤维化，并且可能是一种新的候选药物。