Bioactive Molecule Icariin Regulated Rat Airway Smooth Muscle Cells Behaviors

Objective: Icariin is the main active compound extracted from Epimedium Flavonoids(EFs)and involved in regulation of cell behaviors(proliferation,apoptosis,and autophagy etc.)for many cell types,but the effect of Icariin on airway smooth muscle cells(ASMCs)has not been studied.The aim of the present study is to examine the role of Icariin on rat ASMCs proliferation,apoptosis and autophagy.Methods : Anesthetized the SD rats and Isolated the airway,extracted and purified ASMCs from rats by tissue patch method and differential adherence method,and identified them by fluorescence of smooth muscle ?-actin(SM ?-actin).The Cell Counting Kit8(CCK-8)and Edu proliferation kit assay were used to detect the effect of ICA on the proliferation of rat ASMCs at different concentrations and at different times.The cell cycle was analyzed by flow cytometry.Apoptosis was detected by flow cytometry and TdT-UTP nick end labeling(TUNEL)assay.The expression of CylinE1,CylinA1,and Caspase-3 proteins in cells was detected by Western blotting.The mRNA expression of CyclinE1,CyclinA1,Atg3,Atg5 and Beclin1 were detected by reverse transcription-polymerase chain reaction(RT-PCR).Results:Rat ASMCs were successfully extracted by tissue patch method,and the puritywas over 95% by fluorescence.The Cell Counting Kit8(CCK-8)and Edu proliferation kit assay showed that Icariin significantly inhibited the proliferation of ASMCs in rat starting from 75 ?M in a time-dose-dependent manner(P<0.01).The Flow Cytometry assay showed that the Icariin interfered with ASMCs cell cycle,when treated with Icariin,S phase shortened while G2 phase prolonged,cyclinE1 and cyclinA1 gene and protein expressions decreased.It was shown that icariin could down-regulate the expression of cyclin E1 and cyclinA1 to affect cell cycle and inhibit cell proliferation.Flow Cytometey and TUNEL showed that icariin promoted apoptosis of rat ASMCs,with the optimal concentration of 200 ?M(P<0.01).Western blot analysis revealed that icariin could activate the expression of the apoptosis protein caspase-3.It was suggested that icariin might induce apoptosis by activating caspase-3-related pathways.Finally,RT-PCR and Western blot assay showed that icariin up-regulated the expression of Atg3,Atg5 and Beclin1 autophagy-related factors,and promoted the conversion of LC3 I to LC3 II.The findings showed that icariin induced autophagy in rat ASMCs by affecting autophagy-related factors.Conclusions: Icariin inhibited the expression of CyclinA1 and CyclinE1 to restrained the proliferation and interfered with cell cycle of ASMCs,it can induced apoptosis and activated casepase apoptosis-related pathways;in addition,it can regulated the expression of autophagy-related factors to induce autophagy in rat ASMCs.Experiments showed that icariin can affect the cell behavior of rat ASMCs.