Notch1 promotes the pericyte-myofibroblast transition in idiopathic pulmonary fibrosis through the PDGFR/ROCK1 signal pathway.

The goals of this study were to investigate the role of the Notch1/PDGFRß/ROCK1 signaling pathway in the pathogenesis of pulmonary fibrosis and to explore the possibility of treating fibrosis by targeting Notch1. Lung tissues from patients with pulmonary fibrosis were examined for the expression of Notch1/PDGFRß/ROCK1 using RT-qPCR, western blotting, and immunostaining. Cultured mouse lung pericytes were transfected with Notch1-overexpressed vectors or shRNA targeting PDGFRß/ROCK1 to examine cell behaviors, including proliferation, cell cycle arrest, and differentiation toward myofibroblasts. Finally, a mouse pulmonary fibrosis model was prepared, and a Notch1 inhibitor was administered to observe tissue morphology and pericyte cell behaviors. Human pulmonary fibrotic tissues presented with overexpression of Notch1, PDGFRß, and ROCK1, in addition to a prominent transition of pericytes into myofibroblasts. In cultured mouse lung pericytes, overexpression of Notch1 led to the accelerated proliferation and differentiation of cells, and it also increased the expression of the PDGFRß and ROCK1 proteins. The knockdown of PDGFRß/ROCK1 in pericytes remarkably suppressed pericyte proliferation and differentiation. As further substantiation, the administration of a Notch1 inhibitor in a mouse model of lung fibrosis inhibited the PDGFRß/ROCK1 pathway, suppressed pericyte proliferation and differentiation, and alleviated the severity of fibrosis. Our results showed that the Notch1 signaling pathway was aberrantly activated in pulmonary fibrosis, and this pathway may facilitate disease progression via mediating pericyte proliferation and differentiation. The inhibition of the Notch1 pathway may provide one promising treatment strategy for pulmonary fibrosis.

Amelioration of bleomycin-induced pulmonary fibrosis by chlorogenic acid through endoplasmic reticulum stress inhibition.

To investigate the inhibitory effects of chlorogenic acid on pulmonary fibrosis and the internal mechanisms in vivo and in vitro. 30 male BALB/C mice were randomized into 5 groups: control group, pulmonary fibrosis model group, low, middle and high dose of chlorogenic acid groups. Mice in pulmonary fibrosis model group were administered 5.0 mg/kg bleomycin with intracheal instillation and mice in 3 chlorogenic acid groups were treated with chlorogenic acid every day for 28 days after bleomycin administration. Lung tissue histology was observed using HE staining. Primary pulmonary fibroblasts were isolated and cultured. The expressions of fibrosis related factors (α-SMA and collagen I), as well as ER stress markers (CHOP and GRP78) were determined by both real-time PCR assay and Western blotting, while the expressions of other ER stress signaling pathway factors PERK, IRE-1, ATF-6 and protein levels of caspase-12, caspase-9, caspase-3, PARP were determined by Western blotting. RLE-6TN cell line induced by TGF-ß1 was also used to verify the amelioration effects in vitro study. In both in vivo and in vitro studies, TUNEL staining was used to evaluate cell apoptosis. Expressions of collagen I, α-SMA, GRP78, and CHOP were significantly inhibited by chlorogenic acid in dose-dependent manner. Similarly, decreasing levels of cleaved caspase-12, caspase-9, caspase-3 and increasing level of uncleaved PARP were observed in chlorogenic acid groups compared with those in the fibrosis group both in vivo and in vitro. Chlorogenic acid could also significantly down-regulate the level of phosphorylation of PERK and cleaved ATF-6 in vivo study. Moreover, MTT assay demonstrated chlorogenic acid could enhance proliferation of RLE-6TN cells induced by TGFß1 in vitro. And the apoptosis assays indicated that chlorogenic acid could significantly inhibit cell apoptosis both in vivo and in vitro studies. Chlorogenic acid could inhibit the pulmonary fibrosis through endoplasmic reticulum stress inhibition in vivo and in vitro.