Sanguinarine Decreases Cell Stiffness and Traction Force and Inhibits the Reactivity of Airway Smooth Muscle Cells in Culture

Abstract

Airway hyperresponsiveness (AHR) is the cardinal character of asthma, which involves the biomechanical properties such as cell stiffness and traction force of airway smooth muscle cells (ASMCs). Therefore, these biomechanical properties comprise logical targets of therapy. β2-adrenergic agonist is currently the mainstream drug to target ASMCs in clinical practice for treating asthma. However, this drug is known for side effects such as desensitization and non-responsiveness in some patients. Therefore, it is desirable to search for new drug agents to be alternative of β2-adrenergic agonist. In this context, sanguinarine, a natural product derived from plants such as bloodroots, that has been reported to relax gut smooth muscle emerges as a potential candidate. So far, it is unknown whether sanguinarine can regulate the biomechanical properties of ASMCs and reactivity of ASMCs to irritants. Thus, we tested the hypothesis that sanguinarine reduce the contractile potentials of ASMCs in culture. To do so, the primary cultured rat ASMCs were first treated with different concentration of sanguinarine. Then, cell stiffness, traction force, fiber distribution, and calcium signaling of the ASMCs were evaluated by optical magnetic twisting cytometry, Fourier transform traction microscopy, atomic force microscopy, and Fluo-4/AM based fluorescence confocal scanning microscopy, respectively. The results indicated that sanguinarine (0.05 and 0.5 µmol/L) significantly decreased cell stiffness and traction force, inhibited reactivity of ASMCs to histamine, and disrupted the fiber structures in ASMCs in dose-dependent manner. These findings establish that sanguinarine can indeed change the biomechanical properties of ASMCs and may be used to treat AHR in asthma.

Sensing Traction Force on the Matrix Induces Cell–Cell Distant Mechanical Communications for Self-Assembly

“…The hydrogel stiffness was measured using a Nanowizard II atomic force microscope (AFM, JPK Instruments AG, Berlin, Germany) on an Olympus IX 81 inverted light microscope, of which procedures have been described recently. 61 Briefly, a local region (∼10 × 10 μm) on the hydrogel surface was selected for force−distance measurement by using the force spectrum mode of AFM. During measurement, the cantilever was pressed onto the hydrogel surfaces followed by standard retraction of the cantilever to generate a force−indentation curve, and the surface stiffness was determined by fitting the curve to the Hertz model.…”

“…The hydrogel stiffness was measured using a Nanowizard II atomic force microscope (AFM, JPK Instruments AG, Berlin, Germany) on an Olympus IX 81 inverted light microscope, of which procedures have been described recently . Briefly, a local region (∼10 × 10 μm) on the hydrogel surface was selected for force–distance measurement by using the force spectrum mode of AFM.…”

Chemical Activation of Piezo1 Alters Biomechanical Behaviors toward Relaxation of Cultured Airway Smooth Muscle Cells

“…The dynamic response of [Ca 2+ ] i in ASMCs was visualized using Fluo-4/AM as described previously. 33) Briefly, cultured ASMCs were seeded into confocal Petri dishes (1 × 10 5 cells per dish) and cultured for 24 h, after which the cells were incubated with 5 µM Fluo-4/AM for 30 min at 37 °C. Then the cells were incubated for 20 min in Tyrode solution to allow de-esterification of Fluo-4/AM.…”

“…Assessment of Cell Stiffness of ASMCs Cell stiffness of cultured ASMCs was measured by optical magnetic twisting cytometry (OMTC) as described previously. 33) In brief, ASMCs cultured on type I collagen-coated dishes (96-well plate, Immunon II) were bathed in serum-free culture media containing 1 µM Yoda1 or vehicle (0.1% DMSO) for 24 h. Then the cells were first labeled with Arg-Gly-Asp (RGD)coated ferrimagnetic microbeads for 20 min and washed with DMEM to remove unbound beads. Then the microbeads were magnetized horizontally with a brief 1000-Gauss pulse and twisted in a vertically aligned homogenous magnetic field (20 Gauss) oscillating at 0.75 Hz, resulting in rotation and displacement of the bead.…”

“…Assessment of Cell Traction Force of ASMCs Traction force generated by ASMCs was measured by using Fourier transformation traction force microscopy (FTTFM) as described previously. 32,33) Briefly, ASMCs were cultured in serum-free DMEM containing 1 µM Yoda1 or vehicle (0.1% DMSO) for 24 h and then seeded onto type I collagen (5000 cells/dish)-coated polyacrylamide gel in DMEM without FBS for 6 h. A single cell and the fluorescent microbeads (Molecular Probes, Eugene, OR, U.S.A.) were then imaged by phase-contrast and fluorescence microscopy, respectively. Then NaOH was added to cell cultures and the cell-free bead positions were recorded as a reference point (traction-free) for bead displacement.…”