Abstract:

Objective To discuss whether safranal affects the proliferation， migration， and phenotypic transformation of the vascular smooth muscle cells （VSMCs） in a high-glucose environment and to clarify the function of safranal in the prevention and treatment of diabetic （DM） vascular complications.? Methods The SD rats were selected as experimental subjects； primary VSMCs were cultured from rat thoracic aortas and divided into control group， 25 mmol·L^-1 high glucose （HG） group， HG+ 20 μmol·L^-1 safranal group， HG+40 μmol·L^-1 safranal group， and HG+80 μmol·L^-1 safranal group. The cells in control group received no treatment； the cells in 25 mmol·L^-1 HG group were pretreated with 25 mmol·L^-1 HG； the cells in HG+20， 40， and 80 μmol·L^-1 safranal groups were further treated with 20， 40， and 80 μmol·L^-1 safranal respectively for 48 h on the basis of 25 mmol·L^-1 HG group. Cell counting kit-8 （CCK-8） method was used to determine the appropriate concentration of safranal and detect the viabilities of the VSMCs in various groups； cell scratch healing assay was used to detect the scratch healing rates of the VSMCs in various groups； Transwell chamber assay was used to detect the numbers of the migration VSMCs in various groups； immunofluorescence method was used to detect the fluorescence intensities of alpha-smooth muscle actin （α-SMA） and rabbit anti-osteopontin （OPN） in the VSMCs in various groups； Western blotting method was used to detect the expression levels of OPN， α-SMA， and proliferating cell nuclear antigen （PCNA） in the VSMCs in various groups. Results Under microscope， on the 4th day of in vitro culture， the spindle-shaped or triangular cells crawled out from the edge of the thoracic aorta tissue blocks， with long spindle being the most common morphology. On the 14th， the cells gradually covered the bottom of the dish； when cell density reached 80%-90%， the characteristic “hills and valleys” growth pattern appeared. Third-generation cells were taken for immunofluorescence identification； immunofluorescence staining with VSMC-specific marker α-SMA showed positive expression of α-SMA protein in the primarily cultured VSMCs. The CCK-8 assay results showed that compared with control group， the cell viability of the cells in 160 μmol·L^-1 safranal group was significantly decreased （P<0.01）， indicating toxic damage to the cells. Under the conditions of safranal concentrations at 20， 40， and 80 μmol·L^-1 respectively， after 48 h intervention on VSMCs， no significant adverse effect on cell viability was observed； considering both the effect and toxicity of safranal， these three concentrations were used in subsequent cell experiments. After 48 h intervention， compared with control group， the activity of the VSMCs in 25 mmol·L^-1 HG group was increased （P<0.001）； compared with 25 mmol·L^-1 HG group， the activities of the VSMCs in HG+20， 40， and 80 μmol·L^-1 safranal groups were gradually decreased （P<0.05）. The cell scratch healing assay and Transwell assay results showed that after 48 h intervention， the scratch healing rate of the VSMCs in 25 mmol·L^-1 HG group was significantly higher than that in control group （P<0.01）， and the number of transmembrane cells through the Transwell chamber was significantly increased （P<0.05）； compared with 25 mmol·L^-1 HG group， the scratch healing rates of the VSMCs in HG+20， 40， and 80 μmol·L^-1 safranal groups were gradually decreased （P<0.05）， and the number of transmembrane cells was decreased （P<0.05）. The immunofluorescence staining results showed that compared with control group， the fluorescence intensity of α-SMA protein in the VSMCs in 25 mmol·L^-1 HG group was significantly weakened（P<0.001）， while the fluorescence intensity of OPN protein was significantly enhanced （P<0.001）； compared with 25 mmol·L^-1 HG group， the fluorescence intensities of α-SMA protein in the VSMCs in HG+20， 40， and 80 μmol·L^-1 safranal groups were gradually increased （P<0.05）， and the fluorescence intensities of OPN were gradually weakened （P<0.05）. The Western blotting method results showed that compared with control group， the expression level of α-SMA protein in the VSMCs in 25 mmol·L^-1 HG group was decreased （P<0.05）， and the expression levels of PCNA and OPN proteins were increased （P<0.01）； compared with 25 mmol·L^-1 HG group， the expression level of α-SMA protein in the VSMCs in HG+20， 40， and 80 μmol·L^-1 safranal groups were increased （P<0.05）， and the expression levels of PCNA and OPN proteins were decreased （P<0.05）. Conclusion Safranal can inhibit the proliferation， migration， and phenotypic transformation of the VSMCs induced by high glucose.

Key words: Diabetes mellitus, Vascular diseases, Thoracic aorta smooth muscle cells, Cell migration, Cell proliferation