Cordyceps Sinensis Alleviates ?-Glycerophosphate-induced Vascular Smooth Muscle Cell Calcification By Promoting Autophagy

Objective: To investigate the effects of cordyceps sinensis on ?-glycerophosphateinduced vascular smooth muscle cell(VSMC)calcification,and the role of AMPK/m TOR-promoted autophagy in this process.Methods: The rat VSMCs were treated with ?-glycerophosphate(?-GP)to induce calcification.Cordyceps sinensis was also given to the cells to investigate their effect on calcification.Cells were pre-treated with autophagy inhibitor 3-methyladenine and AMPK inhibitor Compound C to explore the potential effect of autophagy and the involvement of AMPK/m TOR signaling pathway,respectively.The effect of cordyceps sinensis on VSMC cell viability was detected by using CCK-8.The calcium deposition was examined by Alizarin red S staining and the O-cresolphthalein complexone method.To evaluate autophagy,the autophagosomes in the cells were observed by using transmission electron microscope(TEM),and the GFP-LC3 puncta were examined by using fluorescent microscope.In addition,the expression levels of ?-SMA,Runx2,autophagy-associated genes LC3?/?,beclin1,and AMPK,m TOR were determined by using Western blotting.Results: Cordyceps sinensis further increased the number of autophagosomes,GFP-LC3 puncta,and the expression levels of LC3?/?,Beclin1,?-SMA,and p-AMPK in VSMCs treated with ?-GP;whereas,the calcium deposition and the expression levels of Runx2 and p-m TOR were decreased.When we pre-treated the cells with 3-MA to repress autophagy,the increase in the number of autophagosomes,GFP-LC3 puncta,and the expression levels of LC3?/?,Beclin1,?-SMA,as well as the decrease in Runx2 and calcium deposition were reversed.When we further pre-treated the cells with Compound C to inhibit the AMPK/m TOR signaling pathway,the number of autophagosomes,GFP-LC3 puncta,and the expression levels of LC3?/?,Beclin1,?-SMA,and p-AMPK were reduced;however,the calcium deposition and the expression levels of Runx2 and pm TOR were raised.Conclusion: Cordyceps sinensis effectively alleviated ?-glycerophosphate-induced vascular smooth muscle cell(VSMC)calcification,which might possibly be accomplished by promoting the autophagic activity of the cells dependent on the activation of AMPK/m TOR signaling pathway.