Qingfei xieding prescription ameliorates mitochondrial DNA-initiated inflammation in bleomycin-induced pulmonary fibrosis through activating autophagy

Ethnopharmacological relevance: Qingfei Xieding prescription was gradually refined and produced by Hangzhou Red Cross Hospital. The raw material includes Ephedra sinica Stapf, Morus alba L., Bombyx Batryticatus, Gypsum Fibrosum, Prunus armeniaca L. var. ansu Maxim., Houttuynia cordata Thunb. , Pueraria edulis Pamp. Paeonia L., Scutellaria baicalensis Georgi and Anemarrhena asphodeloides Bge. It is effective in clinical adjuvant treatment of patients with pulmonary diseases. Aim of the study: To explore the efficacy and underlying mechanism of Qingfei Xieding (QF) in the treatment of bleomycin-induced mouse model. Materials and methods: TGF-ll induced fibrotic phenotype in vitro. Bleomycin injection induced lung tissue fibrosis mouse model in vivo. Flow cytometry was used to detect apoptosis, cellular ROS and lipid oxidation. Mitochondria substructure was observed by transmission electron microscopy. Autophagolysosome and nuclear entry of P65 were monitored by immunofluorescence. Quantitative real-time PCR was performed to detect the transcription of genes associated with mtDNA-cGAS-STING pathway and subsequent inflammatory signaling activation. Results: TGF-ll induced the expression of a-SMA and Collagen I, inhibited cell viability in lung epithelial MLE-12 cells that was reversed by QF-containing serum. TGF-ll-mediated downregulation in autophagy, upregulation in lipid oxidation and ROS contents, and mitochondrial damage were rescued by QF-containing serum treatment, but CQ exposure, an autophagy inhibitor, prevented the protective role of QF. In addition to that, the decreased autophagolysosome in TGF-ll-exposed MLE-12 cells was reversed by QF and restored to low level in the combination treatment of QF and CQ. Mechanistically, QF-containing serum treatment significantly inhibited mtDNA-cGAS-STING pathway and subsequent inflammatory signaling in TGF-ll-challenged cells, which were abolished by CQ-mediated autophagy inhibition. In bleomycin-induced mouse model, QF ameliorated pulmonary fibrosis, reduced mortality, re -activated autophagy in lung tissues and restrained mtDNA-cGAS-STING inflammation pathway. However, the protective effects of QF in bleomycin-induced model mice were also abrogated by CQ. Conclusion: QF alleviated bleomycin-induced pulmonary fibrosis by activating autophagy, inhibiting mtDNAcGAS-STING pathway -mediated inflammation. This research recognizes the protection role of QF on bleomycin-induced mouse model, and offers evidence for the potentiality of QF in clinical application for pulmonary fibrosis treatment.