Betulinic Acid Promotes Autophagy and Improves Vascular Smooth Muscle Cell Calcification through RRAGD/mTOR/ULK1
Objective To discuss the mechanism of betulinic acid(BA)on calcification of vascular smooth muscle cells(VSMCs)induced by high phosphorus.Methods In vitro model of high phosphorus induced VSMCs calcification model.CCK8 detected the cell viability on betulinic acid(BA).BA with low,medium and high dose(5 μmol·L-1,10 μmol·L-1,20μmol·L-1)and 3-MA(5 μmol·g-1)were added into VSMCs.Slow virus overexpression of Ras related GTP binding protein D(RRAGD)was transfected into VSMCs,and the transfection efficiency was verified by fluorescence microscopy and RT-PCR.Alizarin red staining,intracellular calcium content and alkaline phosphatase activity were used to detect the degree of cell calcifi-cation.Western blot was used to detect the expressions of VSMCs calcification indicators RUNT related transcription factor 2(RUNX2)bone morphogenetic protein 2(BMP2)and smooth muscle protein 22α(SM22),α-smooth muscle action(α-SMA),the expressions of UNC-51 like kinase 1(ULK1),autophagy protein microtubule associated protein 1 light chain 3(LC3Ⅱ/LC3Ⅰ),ubiquitin binding protein P62(P62),mammalian target of rapamycin(mTOR)and RRAGD.Results According to the results of CCK8,BA doses of 5,10 and 20 μmol·L-1 were selected for subsequent experiments.Calcium level,ALP activ-ity and alizarin red staining showed that BA improved high phosphorus induced VSMCs calcification.The protein expression lev-els of RUNX2,BMP2,RRAGD,p-mTOR and P62 were significantly reduced in BA groups while the protein levels of SM22α,α-SMA,LC3Ⅱ/Ⅰ and p-ULK1 were increased significantly(P＜0.05).The effect of BA on hyperphosphorus-induced VSMCs calcification was blocked by the autophagy inhibitor 3-MA and overexpression lentivirus of RRAGD.Conclusion BA may activate autophagy and inhibit vascular smooth muscle cell calcification through RRAGD/mTOR/ULK1 signaling pathway.
万方数据
维普
