Paeonol Prevented Ox-LDL-Induced Vascular Smooth Muscle Cells Apoptosis Via Up-regulation Of ClassⅢ PI3K/Becline-1 Signaling Pathway

Atherosclerosis is a complex chronic vascular disease,which involves the build-up of fibrous and fatty deposits called plaque inside the arteries.With the development of atherosclerotic disease,Most of the vulnerable plaques are characterized by increased apoptosis of VSMCs.Besides apoptosis,emerging evidence supports that the induction of vascular cells autophagy may play an important role in the cellular response to various important atherogenic stressors such as oxidized lipids and lipoproteins.Effects of autophagy on the role of VSMCs apoptosis are currently not repoted as well.Paeonol,isolated from mountan cortex,is an active component which has been used for its anti-inflammatory,anti-thrombotic and anti-oxidant effects.Our group research have found that paeonol can reduce the formation of AS plaques in experimental sputum,rabbits and ApoE^-/-mice.A large number of evidences have demonstrated that paeonol can inhibit apoptosis of rat endothelial cells and upregulation of autophagy activity inhibit abnormal proliferation of VSMCs.Despite these findings,the possible role of paeonol in protecting aginst VSMCs apoptosis related to autophagy has yet to be elucidated.This study is based on previous research,to establish AS model in ApoE^-/-mice.VSMCs were used as target cells,and ox-LDL was used as the injury factor to induce apoptosis of VSMCs.We discussed the effects of autopaagy on VSMCs apoptosis and the effects of paeonol,which may provide a scientific basis for revealing the autophagy involved in atherogenesis mechanism and paeonol targets with the treatment of atherosclerosis.OBJECTIVEThis study was to investigate the effect of paeonol on ox-LDL-induced apoptosis of VSMCs and the effect of paeonol on the level of autophagy induced by ox-LDL in VSMCs.And further study whether the effect of paeonol on ox-LDL-induced apoptosis of VSMCs is related to its regulation of autophagy.METHODSThe ApoE^-/-mice was induced by a high-fat diet,which was used as AS models.Effect of paeonol on LC3,P62,Caspase-3 expression in thoracic aorta of mice was examined by western blot.VSMCs of mices were cultured by pre-digested and adherent tissue method.VSMCs were identified by immunofluorescence.Autophagosomes and apoptotic body formation in VSMCs was tested by transmission electron microscopy（TEM）.The MDC-stained and DAPI-stained cells were demonstrated by fluorescent microscopy.VSMCs apoptosis was assessed by flow cytometry.ClassⅢPI3K and becline-1 gene was silenced by siRNA transfection.The expression of LC3,P62,Caspase-3 PI3K,becline-1 was detected by western blot.RESULTS1.The effect of paeonol on VSMCs apoptosis and autophagy in AS mice.Paeonol can effectively increase the expression of LC3 and decrease the level of P62and Caspase-3 in thoracic aorta of AS mice.2.Injury effect of ox-LDL on VSMCsVSMCs were treated with different concentrations of ox-LDL(50,100,150,200,250μg·mL^-1)for different periods（12,24,48h）,then,cell inhibition rate were measured using cck8 assay.Cell inhibition rate was highest when the cells were stimulated by 150μg·mL^-1 ox-LDL for 24 h,which served as optimal stimulated concentration and time in the following experiments.3.The protection effects of paeonol on injured VSMCs induced by ox-LDLWe pretreated VSMCs with paeonol at a series of concentrations（15,30,60,120,240μmol/L）for different time points（12,24,48 h）.Then,these cells were treated with 150μg·mL^-1 LPS for another 24 h.After the treatment,cell survival rate was tested with cck8 assay.An optimal condition for paeonol treatment in VSMCs was established（30,60,120μmol/L for 24 h）,which was performed in the following experiments.Flow cytometry verified this part of the results.4.Effect of paeonol on autophagy and apoptotic activity in VSMCs Paeonol concentration-dependently enhanced the fluorescence intensity of MDC-positive cells,and the number of intracellular MDC fluorescent particles increased significantly.The fluorescence intensity is proportional to the dose increase of paeonol.In most of the ox-LDL group,the nucleus was densely stained,the nucleus was white and bright,the nucleus showed irregular shape,and the apoptosis-positive cells increased significantly.Paeonol groups reduced the apoptosis-positive cells in a dose-dependent manner.In the ox-LDL group,a small number of autophagosomes were formed,and obvious apoptotic bodies were found to be absent.Paeonol groups had a certain number of autophagosomes formed,and occasionally apoptotic bodies fell off;The LC3II protein in the ox-LDL group increased slightly,and the levels of p62 and caspase-3,which was significantly increase different from the control group（P<0.01 or P<0.05）.The LC3II protein in each paeonol group increased significantly,and the p62 and caspase-3 protein levels decreased significantly（P<0.01）.5.The molecular mechanism of paeonol regulating apoptosis of VSMCsPaeonol can effectively increase the expression of LC3 and decrease the level of P62and Caspase-3 in VSMCs,but this results were reversed in 3-MA,as simiar as results of DAPI.This part of results demonstrated that paeonol upregulates autophagy to inhibit apoptosis of VSMCs.Paeonol also activate the expression of ClassⅢPI3K and becline-1,but all results were reversed in ClassⅢPI3K and becline-1 deficient cells,as simiar as the results of DAPI.This results demonstrated that regulated-autophagy of paeonol may be through ClassⅢPI3K/becline-1 Signaling Pathway.CONCLUSION1.Paeonol inhibits excessive apoptosis and up-regulates autophagy activity in thoracic aorta of ApoE-/-mice2.Paeonol prevented ox-LDL-induced VSMCs apoptosis via up-regulation of autophagy.3.Regulated-autophagy of paeonol may be through ClassⅢPI3K/becline-1 Signaling Pathway.