Engeletin ameliorates pulmonary fibrosis through endoplasmic reticulum stress depending on lnc949-mediated TGF-β1-Smad2/3 and JNK signalling pathways

摘要

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?×?10sup6/sup/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 ICsub50/sub 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.