MiR-181b Affects The Formation Of Atherosclerotic Plaque In The Carotid Artery Of Apolipoprotein E Knockout(ApoE^-/-) Mice By Autophagy Regulation

Objective.MicroRNA 181b?miR-181b?has been found to be to be associated with thedevelopment of atherosclerosis in which autophagy plays vital roles.This study was to investigate the correlation between miR-181b and autophagy in the pathophysiology of atherosclerosis.Methods.?1?40 6-week-old male SPF ApoE^-/-mice were given common diet for 2 weeks.Two weeks later,carotid artery atherosclerosis plaque was induced in ApoE^-/-mice through the nest of the silicone tube ring on the right common carotid artery,and high-fat diets?HFD;0.25%cholesterol+15%fat?were given for another 8 weeks.?2?Over expression?miR-181bOE?and knock-down?miR-181bKD?models were constructed by the injection of miR-181b lentiviral vector into the caudal vein.During 8weeks of high-fat diet feeding,mice were injected with normal saline?control?or mi R-181b lentivirus vector 3 times each week.Mice at the age of 8 weeks were divided into 4 groups with 10 mice in each group,including control group(ApoE^-/-+high-fat diet+sham operation group+10?l normal saline),model group(ApoE^-/-+high-fat diet+surgery+10?l normal saline),miR-181b over expression group?OE group?(ApoE^-/-+high-fat diet+surgery+10?l miR181b-LV,titrated to 2 x 10⁷TU each mice)and mi R-181bknock-downgroup?OEgroup?(ApoE^-/-+high-fatdiet+surgery+10?l mi R181b-RNAi-LV,titrated to 2 x 10⁷ TU each mice).?3?Separated and cut the femoral artery of ApoE^-/-mice for blood sampling and tested the content of triglyceride?TG?,total cholesterol?TC?and low-density lipoprotein cholesterol?LDL_c?in the serum of each ApoE^-/-mice.?4?Sacrificed ApoE^-/-mice after blood sampling and separated and drew the common carotid artery on the cannula side,fixed and made Paraffin sections,then performed HE staining,observed the changes of vessel wall structure in each group with an optical microscope.?5?After the isolated common carotid artery on the cannula side was fixed,made ultrathin sections and stained,then observed the ultrastructure of macrophages in the common carotid artery tissue of the mice in each group with transmission electron microscopy.?6?Extracted the total protein in the common carotid artery on the cannula side of of ApoE^-/-mice in each group,and detected the expression of autophagy-related proteins in the common carotid arteries of the mice in each group by Western blot.Results.?1?Levels of total cholesterol,triglyceride,and LDL cholesterol were not significantly different in the plasma of the ApoE^-/-mice in each group.?2?Histopathological observation showed that compared with the model group,the largest area of the stenosis site of the plaque in the miR-181b KD group(0.43±0.08?m² vs.0.71±0.13?m^2,Fig.1;P<0.05)and plaque volume?0.33±0.14?m³ vs.0.85±0.11?m³;Figure 1;P<0.05?significantly reduced.In contrast,compared with the model group,the plaque size?0.98±0.11?m²vs.0.71±0.13?m²,Fig.1;P<0.05?and volume?1.11±0.15?m³vs.0.85±0.11?m³;Fig.1;P<0.05?in the miR-181bOE group significant increased.?3?Observation from transmission electron microscopy showed that there was no autophagic vacuolization in the atherosclerotic plaques of the mice in the control group,and a small amount of lipid droplets were seen.While the autophagic vacuoles and lipid droplets in the atherosclerotic plaques of the mice in model group increased compared with the control group?Figure 2?.We had further observed the effect of miR-181bOE on autophagy in atherosclerotic plaques and found that the atherosclerotic plaque in mi R-181bOE mice significantly reduced compared with the model group,while the autophagosome in miR-181bKD atherosclerotic plaque was significantly higher than that in the model group,and a large number of autolysosomes were seen.?4?Results from Western blot showed that MiR-181bOE could lower the expression of LC3-II,activate the expression of p70S6K,and inhibit the formation of autophagosomes.In contrast,miR-181bKD could activate the expression of LC3-II,lower the expression of p70S6K,and promotes the formation of autophagosome.Conclusion.miR-181b could positively regulate the expression of the downstream molecule p70s6k of the mTOR signaling pathway and negatively regulate autophagy,thus having activated the formation of atherosclerotic plaque.