Sinomenine Relieves Airway Remodeling By Inhibiting Epithelial-Mesenchymal Transition Through Downregulating TGF-β1 and Smad3 Expression In Vitro and In Vivo

Abstract

Airway remodeling is associated with dysregulation of epithelial-mesenchymal transition (EMT) in patients with asthma. Sinomenine (Sin) is an effective, biologically active alkaloid that has been reported to suppress airway remodeling in mice with asthma. However, the molecular mechanisms behind this effect remain unclear. We aimed to explore the potential relationship between Sin and EMT in respiratory epithelial cells in vitro and in vivo. First, 16HBE cells were exposed to 100 μg/mL LPS and treated with 200 μg/mL Sin. Cell proliferation, migration, and wound healing assays were performed to evaluate EMT, and EMT-related markers were detected using Western blotting. Mice with OVA-induced asthma were administered 35 mg/kg or 75 mg/kg Sin. Airway inflammation and remodeling detection experiments were performed, and EMT-related factors and proteins in the TGF-β1 pathway were detected using IHC and Western blotting. We found that Sin suppressed cell migration but not proliferation in LPS-exposed 16HBE cells. Sin also inhibited MMP7, MMP9, and vimentin expression in 16HBE cells and respiratory epithelial cells from mice with asthma. Furthermore, it decreased OVA-specific IgE and IL-4 levels in serum, relieved airway remodeling, attenuated subepithelial collagen deposition, and downregulating TGF-β1and Smad3 expression in mice with asthma. Our results suggest that Sin suppresses EMT by inhibiting IL-4 and downregulating TGF-β1 and Smad3 expression.

Keywords: EMT; Sinomenine; TGF-β1/Smad3 expression; airway remodeling; asthma.

Figure 1

Sin inhibited cell migration but not proliferation in LPS-exposed 16HBE cells. (A) A total of 2×10⁴ 16HBE cells were seeded in 96-well plates and stimulated with Sin at concentrations of 0, 2, 10, 20, 100, 200, 500, 1000, 2000 μg/mL for 24 h. Cell viability was analyzed using a CCK-8 assay. The OD450 values of the cells stimulated at different doses are shown in the Y-axis. (B) A total of 1×10⁴ 16HBE cells were seeded in 96-well plates and divided into three groups: 100 μg/mL LPS, 100 μg/mL LPS and 200 μg/mL Sin, control (untreated) group. Cell proliferation ability was analyzed using a CCK-8 assay. The OD450 values of the cells at 0, 24, 48, and 72 h were detected and shown by the ordinate. (C) The cell layers were horizontally scraped using a sterile 10 μL pipette tip upon reaching 90% confluence in six-well plates. Images of the wound areas were captured at 0 and 24 h after stimulation. (D) Migration area was measured to analyze cell migration. (E) A total of 6×10⁴ HBE16 cells were seeded onto the upper wells with no more than 200 μL serum‐free medium. The bottom chamber was filled with 800 μL 10% FBS medium with 100 μg/mL LPS, 100 μg/mL LPS with 200 μg/mL Sin, 200 μg/mL Sin, or 10% FBS medium. The migrated cells were observed and captured at 100× magnification after 48 h. (F) Numbers of migration cells were counted from 6 randomly chosen fields. Scale bar indicates 200 μm. The data are presented as mean ± SEM from three independent experiments. The one-way analysis of variance followed by Dunnett’s Multiple Comparison Test was used. *p < 0.05. **p < 0.01. ***p < 0.001.

Figure 2

Sin inhibited EMT in LPS-exposed human airway epithelial cells. (A) The 16HBE cells were seeded in a 6-well plate and stimulated with LPS at concentrations of 0, 10, 20, 50, 100 μg/mL for 72 h. Protein levels of MMP9, MMP7, and vimentin were determined using Western blotting. (B) The 16HBE cells were seeded in a 6-well plate and divided into four groups: control (untreated), LPS (100 μg/mL), LPS with Sin (100 μg/mL and 200 μg/mL, respectively), and sin (200 μg/mL) groups. The cells were incubated for 72 h to perform Western blotting. (C, D) Statistical analysis of the gray values of each protein normalized to GAPDH. The data are presented as mean ± SEM from three independent experiments. The one-way analysis of variance followed by Dunnett’s Multiple Comparison Test was used. *p < 0.05. **p < 0.01. ***p < 0.001.

Figure 3

Sin relieved Th2 airway inflammation in mice with OVA-induced asthma. (A–C). IL-4, IL-6, and IL-10 expressions in serum were detected using ELISA on mice from control, model, and Sin-treated (35 mg/kg or 75 mg/kg) groups. OD450 absorbances are shown in the Y-axis. (D) OVA-specific IgE in the serum was detected using ELISA in the abovementioned group. The data are presented as mean ± SEM (n=5-6/group). The one-way analysis of variance followed by Dunnett’s Multiple Comparison Test was used. *p < 0.05. **p < 0.01. ***p < 0.001. ns, no significant difference between the two groups.

Figure 4

Sin relieved airway remodeling and reduced collagen deposition in the lungs of mice with OVA-induced asthma. Scale bar indicates 50 μm. (A) Hematoxylin & eosin (H&E)-stained lung tissue specimen sections. The total area of the airway wall and the perimeter of the basement membrane (Wat/Pbm) were used to evaluate the airway remodeling. The Wat/Pbm values (μm²/μm) are shown in Y-the axis. The non-parametric multiple comparison followed by Dunnett’s Multiple Comparison Test was used. (B) Masson staining was conducted on sections of the lung bronchi tissue, in which collagen fibers are shown as blue. The subepithelial collagen density was quantified. The one-way analysis of variance followed by Dunnett’s Multiple Comparison Test was used. The data are presented as mean ± SEM (n=5-6/group). *p < 0.05. ***p < 0.001.

Figure 5

Sin reduced the EMT of the airway epithelial cells in the lungs of the mice with OVA-induced asthma. (A) The protein levels of MMP9, MMP7, and vimentin were detected via immunohistochemistry. Scale bar indicates 50 μm. (B) The expressions of each protein were determined using Western blotting. (C) Statistical analysis of the gray values of each band normalized to GAPDH. The data are presented as mean ± SEM from three independent experiments. The one-way analysis of variance followed by Dunnett’s Multiple Comparison Test was used. *p < 0.05. **p < 0.01. ns, no significant difference between the two groups.

Figure 6

Sin inhibited the TGF-β1/Smad3 expression in the lungs of mice with OVA-induced mice asthma. (A) Protein levels of TGF-β1, Smad3, p-Smad3 and Smad2 were detected using immunohistochemistry. Scale bar indicates 50 μm. (B) Protein levels of TGF-β1 and Smad3 in the lung tissue were determined by Western blotting. (C), Statistical data of the gray values of each protein normalized to GAPDH. The data are presented as mean ± SEM from three independent experiments. The one-way analysis of variance followed by Dunnett’s Multiple Comparison Test was used. **p < 0.01. ***p < 0.001. ns, no significant difference between the two groups.