1β-Hydroxyalantolactone from Inulae Flos alleviated the progression of pulmonary fibrosis via inhibiting JNK/FOXO1/NF-κB pathway

Highlights

• The phenotypic high-content imaging was employed for screening antifibrotic compounds in the plants.

• 1β-hydroxyalantolactone (HAL) from I. Flos had obvious antifibrotic effects.

• HAL induced apoptosis of fibroblast via reducing intracellular oxidative stress.

• HAL inhibited fibroblast activation via regulating JNK/FOXO1/NF-κB pathway.

• HAL improved bleomycin-induced lung fibrosis and inflammation in rats.

Abstract

Inulae Flos was widely distributed throughout Europe, Africa, and Asia, and was commonly used as a folk medicine in clinic for treating various respiratory diseases, including cough, asthma, bronchitis, pulmonary fibrosis, and pneumonia. However, the ingredients responsible for the pharmacology effects of I. Flos and the underlying mechanisms remain unclear. In this study, the effects of 16 known sesquiterpene lactones and flavonoids from I. Flos on TGF-β1-induced fibroblast activation were assessed by phenotypic high-content screening. Among those sixteen compounds, 1β-hydroxy alantolactone (HAL), the main characteristic sesquiterpene lactone from I. Flos, exhibited remarkable inhibitory activity. The further studies showed that HAL significantly inhibited the proliferation and induced the apoptosis of human fibroblast cell lines HELF and MRC-5 in a concentration-dependent manner. It also reduced intracellular ROS production, suppressed the mRNA expressions of E-cad, TGF-β1, Smad3, Col I, α-SMA and TNF-α, and downregulated protein expressions of α-SMA and F-actin. Furthermore, HAL significantly reduced the levels of HA, LN, PC-III and IV-C in serum, TNF-α and IL-6 in BALF, and TGF-β1, HYP and Col I in lung tissues of bleomycin (BLM)-treated rats. HAL significantly downregulated the expressions of p-JNK, FOXO1, p-p65, α-SMA, p-smad3 and Col I but upregulated p-FOXO1, which could be reversed by JNK agonist anisomycin. These results demonstrated that HAL induced the apoptosis of lung fibroblast cells activated by TGF-β1 and improved BLM-induced lung fibrosis in rats via inhibiting JNK/FOXO1/NF-κB pathway.

Introduction

Pulmonary fibrosis is a relatively rare but devastating lung disease, which is characterized by pulmonary epithelial damage, collagen deposition, and ventilation obstruction. It could be caused by multiple agents and has poor prognosis with a median survival of only 3–4 years. Since its pathogenesis is still not clear, the method to cure the disease has not been found [1]. Two antifibrotic drugs (nintedanib and pirfenidone) were conditionally approved for the treatment of patients with idiopathic pulmonary fibrosis, but they also could induce hepatic dysfunction and severe allergic reactions [2], [3]. Therefore, finding more antifibrotic therapies are in urgent demand.

Inulae Flos, prepared from the flowers of Inula japonica Thunb. or I. Britannica L. (Asteraceae family), is used as a dietary supplement and traditional medicine in many Asian, European, and African countries, including China, Korea, Japan, and France [4], [5]. In folk medicine, it was widely used to relieve cough and dyspnea and eliminate retained phlegm for the treatment of various lung diseases and digestive disorders [6]. Recent studies showed that the genus Inula mainly contains abundant sesquiterpenes, polyphenols, and flavonoids, and exerts antitumor, anti-inflammatory, gastroprotective, antitussive and expectorant properties [5], [7], [8], [9]. Up to now, more than 30 sesquiterpenoids and its glucosides had been isolated from the flowers of I. japonica, most of which could induce human cancer cell apoptosis [10], [11], [12] and inhibit nitric oxide release of RAW264.7 cells [13]. Especially, the characteristic sesquiterpenes in I. Flos, such as britanin and inulicin (1-O-acetylbritannilactone), not only inhibited IgE/Ag-induced mast cell activation and LPS-induced macrophage activation via suppressing NF-κB, JNK and MAPK pathways [14], [15], [16], but also inhibits angiogenesis and various cancer cell growth via suppressing VEGF-Src-FAK pathway [17], [18], [19]. Furthermore, the ethanol extract of I. japonica alleviated ovalbumin-induced inflammatory cell infiltration and mucus hypersecretion in mice, indicating its potential for preventing lung inflammation [20]. Most notably, I. japonica crude extract displayed the inhibition on bleomycin (BLM)-induced pulmonary fibrosis in mice via mediating soluble epoxide hydrolase activity [21]. However, the effective substances in I. Flos and their underlying molecular mechanisms on pulmonary fibrosis remain unclear. In this study, we first investigated the inhibition of 16 known sesquiterpenoids and flavonoids isolated from I. Flos on TGF-β1-induced fibroblast activation were assessed by phenotypic high-content screening, and then explored the antifibrotic mechanism of 1β-hydroxy alantolactone (HAL), which had the strongest inhibition among all the screened compounds in vitro, in TGF-β1-treated fibroblast cell model and BLM-induced pulmonary fibrotic rat model.