Based On PI3K/AKT/mTOR Autophagy Pathway To Study The Effect Of Gualou-xiebai On Inhibiting The Proliferation Of Vascular Smooth Muscle Cells Against Atherosclerosis

Atherosclerosis（Atherosclerosis,AS）is a type of blood vessels that has a typical characteristic of pathological hyperplasia and is a series of cardiovascular diseases.The common pathological basis of the disease.Vascular smooth muscle cells（VSMCs）,as vascular skeleton granulocytes,constitute the media layer of blood vessels throughout the body.The abnormal spread of VSMCs is the pathological process and key complication of AS plaque formation.The process in the inner double-layer membrane vesicles,the contents of the double-layer membrane vesicles are transported to the lysosome for fusion and degradation.This process is closely related to cell proliferation,survival and the maintenance of the cell’s internal environment.Similar to the stage of AS plaque formation,insufficient autophagy of VSMCs leads to abnormal proliferation of VSMCs.Gualou-Xiebai medicine pair was first published in the“Jin Kui Yao Lue”for the treatment of“heart arterial obstruction”.The combination of the two medicines can relieve the evil turbidity accumulated and relieve the cold accumulation in the chest,and now widely used in clinical practice.Traditional Chinese medicine believes that AS is caused by lung stasis and obstruction of the collaterals,and is mostly related to the“chest obstruction”of Chinese medicine.Therefore,proved by clinical practice,modern physicians extend the Gualou-Xiebai formula for AS.In this thesis,the starting point of this Compound medicine pair of Gualou-Xiebai drug pair is to investigate the Regulation mechanism of Gualou-Xiebai on the aortic plaque formation,aortic VSMCs proliferation and autophagy level of AS mice.Gualou-Xiebai inhibits the autophagy mechanism of aortic plaque formation.OBJECTIVEIn order to study the inhibition of autophagy on the pathological formation and abnormal proliferation of VSMCs in AS mice,and to explore the pathway mechanism of Gualou-Xiebai regulating VSMCs autophagy,and to provide the basis for the treatment of AS.METHODSApoE^-/-mice fed with high-fat diet were used to establish AS animal pathological model.Applicating oil red O staining and He staining to observe the pathological changes of aorta.The serum levels of TC,TG,HDL-C and LDL-C were detected by enzyme labeling instrument.The actinα-SMA and autophagy characteristic protein of smooth muscle cells of active VSMCs were detected by laser co localization microscope The distribution of LC3,the formation of autophagosome was detected by transmission electron microscope,and the expressions of PCNA,LC3,p62 were detected by Western blot.The primary culture of VSMCs by pre digestion and adherence,then identification of VSMCs was detected by immunocytochemistry SP,the detection of cell proliferation would be detected by CCK8.Finally,the detection of PCNA,LC3,p62,PI3K and p-PI3K,Akt and p-AKT,m TOR and p-m TOR was checked by Western blot to explore the expression of the protein.RESULTS1.The effect of Gualou-Xiebai on aortic pathological plaque,VSMCs proliferation and autophagy activity in AS mice.Western diet ApoE^-/-mice reproduced as model,GLXB could significantly reduce the lipid level in vivo,down regulate the lipid in peripheral blood,and reduce the lipid level of accumulation,reduce the accumulation of TC,TG and LDL-C in vivo.GLXB could significantly reduce the number of VSMCs in aortic lumen,reduce the expression of PCNA andα-SMA in VSMCs;GLXB could improve the LC3Ⅱ/Ⅰ,reduce the degradation of p62,promote the autophagy.2.Autophagy mechanism of Gualou-Xiebai inhibiting VSMCs abnormal proliferation.GLXB significantly induced the formation of autophagosomes,increased the level of LC3Ⅱ/Ⅰ,accelerated the degradation of accumulated p62,and effectively increased the autophagy activity of VSMCs.The results showed that the accumulation of p62protein in VSMCs induced by CQ was blocked at the later stage of autophagy,LC3Ⅱprotein increased but failed to accumulate,and autophagy activity decreased;the addition of CQ alone had no effect on the proliferation rate of VSMCs,while the addition of CQ and GLXB abolished the inhibitory effect of GLXB on proliferation and reversed the increase of proliferation rate.3.Mechanism of Gualou-Xiebai regulating autophagy of VSMCs.The phosphorylation levels of p-p I3K,p-AKT and p-m TOR protein in GLXB-L,M and H groups were significantly decreased in a concentration effect dependent manner after GLXB treated VSMCs in vitro.After CO administration of PI3K selective activator740Y-P and GLXB-H group,the phosphorylation levels of upstream signal molecules p-PI3K and p-AKT were significantly increased,p-m TOR was highly expressed,and autophagy was inhibited.CONCLUSION1.GLXB inhibits the development of aortic plaque in AS mice by inhibiting the abnormal proliferation of VSMCs.2.The proliferation rate of VSMCs in vitro is significantly increased due to the relatively insufficient autophagy.GLXB inhibits the abnormal proliferation of VSMCs stimulated by ox-LDL by inducing VSMCs autophagy.3.GLXB may inhibit the activation of m TOR in the downstream by inhibiting PI3K/AKT,and activate VSMCs autophagy indirectly.PI3K/AKT/m TOR may be the main target of GLXB induced autophagy.