| Background:Acute coronary syndrome (Acute coronary syndrome, ACS) is one kind of diseaseswhich seriously jeopardized the health of human beings. Many studies showed that theinstability of plaque within coronary artery is the key factor during the occurrence of ACS.Conseqently unstable plaque (UP) represents the essential pathology of ACS. The erosionand rupture of unstable plaque and secondary thrombosis is the leading cause of adversecardiovascular events induced by ACS. Further investigation on unstable plaque formationand rupture is of great importance for the prevention and treatment of ACS as well asreducing mortality. But the mechanism involved in "instability"of plaque is still unclear.Recent studies suggested that macrophage apoptosis plays an important role ininducing atherosclerotic plaque instability. In late atherosclerosis, ERS is closely related tomacrophage apoptosis. ERS is known as the disorders of endoplasmic reticulumhomeostasis and normal physiological events due to some reasons. Severe and long termof ERS induced apoptotic signaling activation, and ultimately resulted in apoptosis. Ourprevious study also showed that ERS could lead to macrophage apoptosis, which wasinvolved in unstable atherosclerotic plaque formation.However, ERS promoted degradation of unfolded and misfolded proteins, which in turn attenuate stress itself.It wasreported that moderate ERS protect cardiac injury against ischemia/reperfusion. While theeffect of moderate ERS on atherosclerotics remains unclear.Autophagy refers to the metabolic pathway in which long half-life proteins and someorganelles within the cell are degraded by the lysosomal system. The autophagy may beinitiated by starvation, cell differentiation and growth, mainly in order to maintain cellhomeostasis and survival. It was reported autophagy activation in macrophagy canstabilize atherosclerosis plaque. However, during the development of atherosclerosis, therelationship between endoplasmic reticulum stress and autophagy as well as the ultimateimpact on the stability of atherosclerotic plaque remain unclear. The latest research foundthat ERS could induce moderate autophagy, thereby reducing myocardialischemia-reperfusion injury.Therefore, we established unstable atherosclerotic plaquemodel with ApoE knockout mouse and examined the effect of different dosestunicamycin-induced endoplasmic reticulum stress on atherosclerotic plaque stability, andto observe the degree of ERS and autophagy. In vitro application of different dosestunicamycin on macrophages was also performed and the effect of endoplasmic reticulumstress of varying degree on formation of the foam cells and apoptosis was alsodetected.Moreover,the possible molecular mechanism,especially the role of autophagyplayed was also explored.To this end, we designed and completed the followingexperiments.Objectives:1. To establish ApoE-/-unstable plaque model in vivo and to observe the effect ofdifferent doses tunicamycin on atherosclerotic plaque stability, and to observe thedegree of ERS and autophagy.2. To establish the foam cells model in vitro to simulate the atherosclerotic plaqueformation in vivo and observe the effect of different doses tunicamycin on foam cellformation.3. Further，To investigate the mechanism of low-dose tunicamycin on the formation of foam cells, especially the possible role autophagy played.Methods:1. Forty6-8weeks old ApoE-/-mice were fed with high-fat diet for1week and wererandomly divided into the control group and the surgery group. Mice in surgery groupwere subjected to perivascular carotid collar placement (PCCP), after which fatfeeding was continued. After9weeks, the animals were sacrificed and H&E stainingof carotid artery was performed to observe the formation of atherosclerotic plaque.2. To observe the effect of different doses tunicamycin on atherosclerotic plaque stability,mice were randomly divided into four groups.Saline, saline,low-dose and high-dosetunicamycin were treated (intraperitoneal injection) respectively in mice in control,PCCP, low-dose and high-dose tunicamycin groups. Two weeks later, mice weresacrificed.H&E staining was conducted for observation of carotid plaque morphology,oil red O staining for observation of lipid accumulation within plaque, immunohis-tochemistry for detection of macrophage accumulation within plaque,Western-blot forthe detection of GRP78, Atg7and p62expression levels.3. Macrophage foam cell model was created with oxidized low-density lipoprotein(ox-LDL).In order to investigate the effect of different doses tunicamycin(TM) onmacrophages, RAW264.7cells were randomly divided into four groups:(1) controlgroup,(2) ox-LDL group,(3) ox-LDL+0.1TM group(with0.1μg/mL TM),(4) ox-LDL+1.0TM group(with1.0μg/mL TM);To further investigate the mechanisminvolving the effect low-dose tunicamycin on foam cells, RAW264.7cells wererandomly divided into five groups:(1) control group,(2) ox-LDL group,(3) ox-LDL+0.1TM group,(4) ox-LDL+0.1TM+3-MA group,(5) ox-LDL+Rapa group; oilRed O staining for the test of foam cells content; TUNEL staining for the detection ofmacrophage apoptosis; Western-blot for the detection of GRP78, Atg7and p62expression levels.Results:1. ApoE-/-mice was fed with high-fat diet for nine weeks following surgery, H&Estaining showed atherosclerotic plaques with large lipid pool within carotid in surgerygroup, while the smooth arterial wall in control group neck, without plaque formation, confirming the success of animal model.2. H&E staining showed that compared with PCCP only and high-dose tunicamycingroup, low-dose tunicamycin group had significantly smaller lipid pool within carotidplaque and the plaque was more stable; Oil Red O staining showed that comparedwith PCCP only and high-dose tunicamycin group, low-dose tunicamycin group hadsignificantly lower lipid content within carotid plaque (P <0.05); macrophagesimmunohistochemical staining showed compared with PCCP only and high-dosetunicamycin group,there were significantly fewer macrophages within carotid plaquein low-dose tunicamycin group (P <0.05).3. Oil Red O and TUNEL staining showed that ox-LDL of50mg/L successfully inducedthe foaming of macrophages with a large number of red lipid droplets and promotedmacrophage apoptosis (P <0.01vs control group). Low-dose tunicamycin treatmentsignificantly inhibited phagocytosis of ox-LDL by macrophage and significantlyreduced intracellular lipid red droplets and macrophage apoptosis (P <0.01vsox-LDL). However, the autophagy inhibitor3-MA significantly attenuated theprotective effect of autophagy induced by low-dose tunicamycin (P <0.01vs0.1TM+ox-LDL), while autophagy inducer rapamycin could produce the similar protectiveeffect as low-dose tunicamycin.4. After cells were exposed to ox-LDL, GRP78and p62protein level increased, whileAtg7protein level decreased (P <0.05vs control group). Low-dose tunicamycin couldsignificantly reduce GRP78and p62protein expression level, but significantlyincrease Atg7protein level (P <0.05vs ox-LDL and1.0TM+ox-LDL). When theautophagy inhibitor3-MA was co-administered with low-dose tunicamycin, Atg7protein levels decreased, while P62protein levels increased (P <0.05vs0.1TM+ox-LDL) with no significant change in GRP78protein expression. However,autophagy inducer Rapa significantly reduced P62protein level, but increased Atg7protein level with no significant change in GRP78protein expression.Conclusion:1. We successfully established the atherosclerotic plaque of high instability model inApoE-/-mice;2. Low-dose tunicamycin treatment significantly reduced lipid pool, decreased lipidcontent and macrophage number within atherosclerotic plaque, and induced moderate endoplasmic reticulum stress, regulated autophagy; which might stablize theatherosclerotic plaques;3. Low-dose tunicamycin was able to inhibit the foaming of macrophage and apoptosiscaused by ox-LDL, and induced moderate endoplasmic reticulum stress; meanwhile,autophagy increased;4. The effect and mechanism of Low-dose tunicamycin might induce moderateendoplasmic reticulum stress, and ERS activate appropriate autophagy, which maycontribute to stabilization of atherosclerotic plaques. |
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