Eupatilin alleviates airway remodeling via regulating phenotype plasticity of airway smooth muscle cells

Abstract

Childhood asthma is a common chronic airway disease, and its severe form remains a challenge. Eupatilin is a bioactive natural flavone that has been found to possess potential anti-asthma activity. However, the roles of eupatilin in asthma remain to be elucidated. In the present study, airway smooth muscle cells (ASMCs) were applied for the in vitro investigation since their phenotype plasticity make great contribution to airway remodeling during asthma pathogenesis. Our results showed that eupatilin suppressed the transforming growth factor β1 (TGF-β1)-induced proliferation and migration of ASMCs. Exposure of ASMCs to eupatilin increased the expressions of contractile markers smooth muscle α-actin (α-SMA) and myocardin, whereas expressions of extracellular matrix (ECM) proteins type I collagen (Coll I) and fibronectin were reduced. Furthermore, eupatilin treatment reversed the activation of nuclear factor-κ B (NF-κB), signal transducer and activator of transcription 3 (STAT3) and AKT pathways caused by TGF-β1 in ASMCs. These findings suggested that eupatilin might attenuate airway remodeling via regulating phenotype plasticity of ASMCs.

Keywords: Childhood asthma; airway remodeling; eupatilin; nuclear factor-kappa B (NF-κB); phenotype plasticity; signal transducer and activator of transcription 3 (STAT3).

Figure 1. Eupatilin significantly inhibited TGF-β1-induced ASMCs proliferation and migration

(A) Cell cytotoxicity of eupatilin on ASMCs. ASMCs were incubated with series concentrations of eupatilin (0, 10, 20, 40, 80 μM) for 48 h. MTT assay was performed to evaluate cell viability. (B) ASMCs were stimulated with different concentrations of TGF-β1 ranging from 0 to 40 ng/ml for 48 h, and cell proliferation was measured using CCK-8 assay. (C) ASMCs were pretreated with eupatilin (10, 20, or 40 μM) for 1 h, and then incubated with TGF-β1 (20 ng/ml) for 48 h. CCK-8 assay was performed to assess cell proliferation. (D) Cell migration of ASMCs. Transwell assay was conducted to evaluate cell migration. n=5. *P<0.05 vs. control cells; ^#P<0.05 vs. TGF-β1-induced ASMCs.

Figure 2. Eupatilin reversed the TGF-β1-induced inhibition of the expression of contractile phenotypic markers in ASMCs

ASMCs were pretreated with eupatilin (10, 20, or 40 μM) for 1 h, and then incubated with TGF-β1 (20 ng/ml) for 48 h. The mRNA and protein levels of contractile phenotypic markers including α-SMA and myocardin were measured using RT-PCR (A,B) and Western blot analysis (C), respectively. n=4. *P<0.05 vs. control cells; ^#P<0.05 vs. TGF-β1-induced ASMCs.

Figure 3. Eupatilin suppressed Coll I and fibronectin production in TGF-β1-stimulated ASMCs

ASMCs were pretreated with eupatilin (10, 20, or 40 μM) for 1 h, followed by incubation with TGF-β1 (20 ng/ml) for 48 h. (A–D) The mRNA levels of Col I and fibronectin in ASMCs and the secretion in cell supernatants were determined using RT-PCR and ELISA, respectively. n=4. *P<0.05 vs. control cells; ^#P<0.05 vs. TGF-β1-induced ASMCs.

Figure 4. Eupatilin prevented the activation of NF-κB and STAT3 pathways in TGF-β1-stimulated ASMCs

(A) After TGF-β1 stimulation for 2 h with or without the pretreatment of eupatilin (10, 20, or 40 μM), the expressions of p65, p-p65, STAT3, and p-STAT3 were measured using Western blot. (B) The ratio of p-p65/p65. (C) The ratio of p-STAT3/STAT3. n=3. *P<0.05 vs. control cells; ^#P<0.05 vs. TGF-β1-induced ASMCs.

Figure 5. Eupatilin prevented the activation of AKT pathway in TGF-β1-stimulated ASMCs

(A) After TGF-β1 stimulation for 30 min with or without the pretreatment of eupatilin (10, 20, or 40 μM), the expressions of p-AKT and AKT were measured using Western blot. (B) The ratio of p-AKT/AKT. n=3. *P<0.05 vs. control cells; ^#P<0.05 vs. TGF-β1-induced ASMCs.