Study On The Mechanism Of Atherosclerotic Lipid Plaque Formation And The Mechanism Of Lipid - Regulating Particles

Background and ObjectiveAtherosclerosis, acting as the common pathological basis of various ischemic cardiocerebro-vascular diseases, erodes human health and life silently and brings heavy economic burden to hundreds of millions of families and even the whole society. Atherosclerosis can be regarded as a complex of chronic disease states in majority and acute cardiocerebrovascular adverse events in minority, which is well summarized by the term "cardiometabolic disease". Since the application of intensive drug therapy or intervention is more passive when acute adverse events occur or are about to happen in the later stage, we should start treatment in time when chronic metabolic disturbance appears in the early stage for improving the pathological basis of atherogenesis at root. The present study aimed to determine the exact therapeutic effects of a Chinese herbal compound preparation called Tiaozhi Tongmai Granules, which has a potential clinical role in the treatment of atherosclerosis, on an atherosclerotic animal model and, subsequently, to elucidate the potential pharmacological mechanisms by which the granules protect against atherosclerosis from the perspective of intracellular cholesterol catabolism and efflux.MethodsExperiment1:Fifteen male New Zealand rabbits were randomly divided into balloon injury group (n=8) and sham-operated group (n=7). To evaluate the rabbit atherogenesis that the balloon injury combined with high-fat diet had formed, gross observation, Sudan IV and hematoxylin-eosin staining of vascular tissues were performed and the percentages of thickened intimal area, average intimal thickness as well as maximal intimal thickness were calculated. The determination if the lipid core exists was also performed.Experiment2:Fifty-six male New Zealand rabbits were randomly divided into four groups (n=14):normal group, normal diet; model group: high-fat diet and balloon injury; Chinese herb group: high-fat diet and balloon injury plus Tiaozhi Tongmai Granules (7.3g/kg/d); statin group: high-fat diet and balloon injury plus atorvastatin (1.1mg/kg/d). The assessments was performed at two time points (6th and12th week). The drug therapeutic effects were evaluated after hematoxylin-eosin staining and quantified by thickened intimal area percentage and maximal intimal thickness percentage. The ATP-binding cassette transporter A1(ABCA1) protein expression in macrophages of the common carotid arteries (CCA), thoracic aortae (TA) and in liver tissues were observed by immunohistochemical staining and evaluated using the mean optical density (OD) value in macrophages and ABCA1-positive hepatocyte number. The engulfment of plaque apoptotic cells labeled using TUNEL method by macrophages stained using immunohistochemistry i.e. phagocytosis was observed. Together, all above methods were to explore the potential pharmacological mechanisms by which the granules protect against atherosclerosis from the perspective of lipid trans-membranal transportation and inflammation.Experiment3: One hundred and twenty apoE knockout mice were randomly divided into three groups (n=40): control group, Chinese herb group and statin group. All mice were fed a high-fat diet to induce atherosclerosis. Tiaozhi Tongmai Granules were administered at a dose of16.5g/kg/d in Chinese herb group and atorvastatin5.1mg/kg/d in statin group. The assessments was performed at two time points (8th and16th week). To evaluate the impacts of two drugs on lipophagy of macrophages within plaques and to explore the potential pharmacological mechanisms by which the granules protect against atherosclerosis from the perspective of lipid catabolism, the colocalization of neutral lipid stain BODIPY and microtubule-associated protein1light chain3(LC3) and the colocalization of BODIPY and lysosomal-associated membrane protein1(LAMP1) within mice aortic plaques were viewed using fluorescence confocal microscopy and the Pearson’s coefficients were quantified. The protein expression of LC3, ULK1, phosphorylated ULK1(p-ULK1-Ser317), adenosine monophosphate activated protein kinase a (AMPKa) and phosphorylated AMPKa (p-AMPKa-Thr172) were measured by western blot and then the protein content ratio of LC3-Ⅱto LC3-Ⅰ, the ration of p-ULK1-Ser317to total ULK1and the ration of p-AMPKα-Thr172to total AMPKa were quantified to elucidate the molecular mechanisms of autophagy regulation by the drug.Results1Establishment of advanced atherosclerotic rabbit model and assessments of atherogenesisBalloon injury group form obvious atherosclerotic plaques in CCA and had significantly higher thickened intimal area percentage, average intimal thickness percentage, maximal intimal thickness percentage (P all<0.001) as well as more animals with lipid core than those of sham-operated group.2Impacts of Tiaozhi Tongmai Granules on atherogenesis, ABCA1protein expression and efferocytosis in rabbitsCompared with model group at the6th week, Chinese herb group and statin group at the6th week all displayed significant reductions in total cholesterol (TC)(P=0.027,0.012) and low-density lipoprotein cholesterol (LDL-C)(P=0.039,0.028) levels, marked increases in ABCA1-positive hepatocyte numbers (P all<0.001) as well as significantly enhanced macrophage efferocytosis in the CCA (P<0.001,0.01), and only statin group displayed a markedly reduced maximal intimal thickness percentage in the CCA (P=0.018). Model group at the12th week displayed a significant reduction in efferocytosis of macrophages in the CCA compared with model group at the6th week.Compared with model group at the12th week, Chinese herb group and statin group at the12th week all presented significant reductions in TC (P=0.011,0.003), LDI-C (P=0.017, 0.010), thickened intimal area percentage in the CCA (P=0.001,0.022), as well as prominent increases in the ABCA1protein OD value of both the CCA (P=0.001,0.039) and TA (P=0.001,0.025), positive hepatocyte number (P all<0.001) and macrophage efferocytosis of both CCA (P<0.001,0.021) and TA (P<0.001,0.004). Only Chinese herb group at the12th week had a markedly reduced maximal intimal thickness percentage compared with model group at the12th week (P=0.006) and only statin group at the12th week presented marked increases in ALT and ALP level (P=0.001,0.004).Compared with statin group at the12th week, Chinese herb group at the12th week had significant increases in positive hepatocytes number (P=0.001)and macrophage efferocytosis in the CCA (P=0.032).3Impacts of Tiaozhi Tongmai Granules and atorvastatin on plaque lipophagy in ApoE knockout miceCompared with control group at the16th week, Chinese herb group and statin group at the16th week all displayed significant increases in colocalization of BODIPY and LC3(P all <0.001) as well as colocalization of BODIPY and LAMP1(P all<0.001) in. Statin group at the16th week also displayed marked increases in the protein content ratio of LC3-Ⅱ to LC3-I, the ratio of p-ULK1-Ser317to total ULK1and the ration of p-AMPKa-Thr172to total AMPKa (P=0.001,0.038,0.009).Compared with control group at the8th week, statin group at the8th week presented significant increases in the protein content ratio of LC3-Ⅱ to LC3-Ⅰ, the ratio of p-ULKl-Ser317to total ULK1and the ration of p-AMPKa-Thr172to total AMPKa (P=0.029,0.046,0.003). Control group at the16th week presented marked reductions in all three protein content ratios compared with control group at the8th week (P=0.001,0.043,0.009).Conclusions1Balloon injury combined with high-fat diet feeding can rapidly and efficiently establish obvious advanced rabbit atherosclerotic lesion, which well simulates the initiation and progression of human atherosclerosis.2Tiaozhi Tongmai Granules can promote overall lipid metabolism represented by cholesterol catabolism, efflux and clearance in plaque macrophages and alleviate excessive accumulation of cholesterols in the vascular walls, thereby producing an anti-atherogenic effect that is most likely mediated by simultaneously enhancing the functions of membranal transporter ABCA1in both vascular macrophages and hepatocytes and by activating macrophage lipophagy. Particularly, the granules have an advantage over atorvastatin in reducing plaque eccentricity in the later stage. In addition, the granules have an obvious lipid-lowering effect and lower side effects of long-term administration of the medication.3Atorvastatin can activate macrophage lipophagy within plaque via AMPK-ULP1-LC3 pathway and enhance cholesterol catabolism of macrophages to reduce foam cell formation, thus producing an anti-atherogenic effect in a novel way.