This study aimed to evaluate the effect of sanguinarine on biomechanical properties of rat airway smooth muscle cells (rASMCs) including stiffness, traction force and cytoskeletal stress fiber organization. To do so, rASMCs cultured in vitro were treated with sanguinarine solution at different concentrations (0.005~5 μmol/L) for 12 h, 24 h, 36 h, and 48 h, respectively. Subsequently, the cells were tested for their viability, stiffness, traction force, migration and microfilament distribution by using methylthiazolyldiphenyl-tetrazolium bromide assay, optical magnetic twisting cytometry, Fourier transform traction microscopy, scratch wound healing method, and immunofluorescence microscopy, respectively. The results showed that at concentration below 0.5 μmol/L sanguinarine had no effect on cell viability, but caused dose and time dependent effect on cell biomechanics. Specifically, rASMCs treated with sanguinarine at 0.05 μmol/L and 0.5 μmol/L for 12 and 24 h exhibited significant reduction in stiffness, traction force and migration speed, together with disorganization of the cytoskeletal stress fibers. Considering the essential role of airway smooth muscle cells (ASMCs) biomechanics in the airway hyperresponsiveness (AHR) of asthma, these findings suggest that sanguinarine may ameliorate AHR via alteration of ASMCs biomechanical properties, thus providing a novel approach for asthma drug development.
Objective To explore the effect of salbutamol combined with Rho associated coiled-coil forming protein kinase (ROCK) inhibitor Y-27632 on airway smooth muscle and to find a new way for drug treatment of asthma. Methods Pig tracheal smooth muscle tissue strips were prepared, and after treatment they were divided into an electrical stimulation group (Fmax, 50%Fmax) and a blank group. The smooth muscle tissue strips were quickly frozen to determine the expression level of Rock-Ⅱ and the phosphorylation level of MLC20. The Fmax and 50%Fmax electrical stimulation groups were divided into a blank group, a salbutamol group, a Y-27632 group, and a salbutamol combined with Y-27632 group according to different intervention drugs. The relaxation of smooth muscle strips was observed. Results In the blank group, 50%Fmax group and Fmax group, the expression level of Rock-Ⅱ and the phosphorylation of MLC20 in smooth muscle tissue showed an increasing trend, with statistically significant differences (P<0.05). In the 4 subgroups of the 50%Fmax group intervention with different drugs (blank group, salbutamol group, Y-27632 group, salbutamol plus Y-27632 group), the diastolic ratio smooth muscle tissue strips showed an increasing trend. When the time reaches 10 min, the diastolic ratios were 0.7%, 2.5%, 6.0%, and 15.0%. the diastolic ratios were 1.8%, 4.5%, 7.5%, and 21.0% at the time of 20 min. the diastolic ratios were 1.9%, 7.5%, 7.9% and 22.0% at the time of 40 min. the diastolic ratios were 2.0%, 8.0%, 8.8%, and 22.5% at the time of 60 min. In the four subgroups of the Fmax electrical stimulation group, the relaxation ratio of smooth muscle tissue strips also showed an increasing trend. When the time reaches 10 min, the diastolic ratios were 1.0%, 3.0%, 7.0%, and 17.0%. the diastolic ratios were 2.6%, 5.5%, 9.0%, and 24.0% at the time of 20 min. the diastolic ratios were 2.8%, 9.0%, 9.5%, and 27.5% at the time of 40 min. diastolic ratios were 2.9%, 10.5%, 10.5%, and 28.0% at the time of 60 min. The analysis of difference between groups showed that at the same time, the diastolic ratio of smooth muscle in salbutamol combined with Y-27632 group was significantly higher than that in salbutamol alone group and Y-27632 group (P<0.05). In addition, the smooth muscle diastolic ratio of combined intervention was also better than the the mathematical sum effect of both single drug intervention (P<0.05). Conclusions The contractility and intensity of smooth muscle are positively correlated with the expression level of ROCK and the phosphorylation level of MLC20. Salbutamol combined with Y-27632 can enhance the relaxation of porcine airway smooth muscle, which may have a synergistic effect.