| Cardiovascular diseases included dyslipidemia, hypertension, coronary heart disease. Atherosclerosis (AS) which is mainly involved large, medium-sized arteries is the pathogenesis basis of cardiovascular disease. The early lesion of AS is characterized by lipid points and lipid stripes, which is closely related to the macrophage-derived foam cells. Ross believed that the various pathological changes of the AS were the different stages of chronic inflammation of the arteries. The inflammation injuries increase the permeability of endothelial cells and the adhesiveness of leukocytes. These would release the chemokines and promote the monocytes into the intima changing into macrophages. The macrophages absorb OxLDL by the scavenger receptor, then change into foam cells. This macrophage-derived foam cells can increase lipid deposition, reduce cholesterol shipped out and promote the formation of fatty streaks. It is the potential sites of the development of AS. Therefore, to reduce macrophage-derived foam cells is very important for the prevention and treatment of AS.Apigenin is mainly from the celery leaves, which belongs to the umbrella plant. It also exists in many of herbal medicine. It is a flavonoid compound, with the anti-inflammatory, antioxidant, antitumor, neuroprotective and immunoregulatory abilities. The early animal experiments, we found that apigenin can reduce the atherosclerotic plaque area of ApoE-/- mice. These suggested that apigenin had a certain effect on the prevention of atherosclerosis and this may be by reducing the quantity of macrophage-derived foam cells.Macrophages have emerged as protagonists of atherosclerosis and its complications.Macrophage apoptosis has been identified as aprominent feature of atherosclerotic plaques. However, its role in AS has been controversial for a long period. There is evidence that the survival of mcrophages promotes atherogenesis. However, there is also observation that macrophage apoptosis is atherosclerogenic as the released cytokines from apoptotic macrophage stimulate the surrounding cells, such as vascular smooth muscle cells. Recently, it has been reported that macrophage apoptosis in the early phase of the atherosclerotic process negatively regulates the progression of atherosclerotic lesions by preventing the differentiation of macrophages to foam cells and reducing the cytokine production. So, macrophage apoptosis might be dichotomous in AS process:it is antiatherogenic during theearly stages of AS, but accelerates plaqueprogression in more advanced lesions.Autophagy is a process in which de novo formed membrane enclosed vesicles engulf and consume cellular components. The macrophage autophagy plays an essential role in atheroprotection during early AS and becomesdysfunctional in the more advanced stages of AS. Autophagy has been shown to engage in complex interplay with apoptosis.Generally, autophagy blocks the induction of apoptosis, and apoptosis-associated caspase activation shuts off the autophagic process.The dialogue between macrophage autophagy and apoptosis has been reported to influence vascular inflammation, oxidative stress, and plaque necrosis.Oxidized low-density lipoprotein (OxLDL) activates the expression of tumor necrosis factor a (TNF-a) and interleukin 1(3 (IL-1β). The secretion of proinflammatorycytokines, such as IL-1β and IL-18 were enhanced in atg16L1 or atg7 deleted macrophages in response to LPS. Accumulating evidences suggested that apoptosis is an important control point in the development and resolution of inflammation.Our previous work showed that apigenin induced cancer cell apoptosis.Apigenin significantly inhibited LPS-induced secretion of IL-6, IL-1β, and TNF-α in macrophages. So, both in vivo and in vitro test were carried out to address the function of apigenin in regulating cytokines through the interactions between macrophage apoptosis and autophagy. It is seemingly controvertial that oxLDL induces secretion of proinflammatory cytokines and protects macrophages from apigenin induced both apoptosis and autophagy. Excessive activation of autophagy contributes to cell death ,OxLDL protects lipid-laden macrophages from cell death through restraining excessive autophagy. So, oxLDL is pro-survival molecules to maintain immune homeostasis between autophagy and apoptosis and surport a mild macrophage activation and pro-inflammatory cytokine secretion in the process os AS. Apigenin induces both autophagy and apoptosis [39]. The autophagy might be a self-protecting response as macrophage fighting to keep cells alive under apigenin induced "life-threatening" conditions.Autophagy can also attenuate cell death by selectively reducing the abundance of pro-apoptotic proteins in the cytosol. So, inhibition of autophagy upregulates the apoptosis induction efficiency of apigenin.OxLDL maintains the survival of macrophages and activates the secretion of pro-inflammaory cytokines to initiate AS process. Apigenin induces apoptosis of foam cells and resolves pro-inflammaory cytokines to ameliorate AS. Combination of apigenin with autophagy inhibition may be a promising strategy to induce foam cell apoptosis.Method1. Animal experimentAll procedures involving laboratory animal use were in accordance with the guidelines of the Instituted Animal Care and Use Committee of Southern Medical University. All protocols were submitted and validated by Animal Care Ethics Committee of Southern Medical University. The apolipoprotein E gene knockout(ApoE-/-) mice (Laboratorial Animal Center of Beijing University, Beijing, China) and C57BL/6 mice (Laboratory Animal Center of Southern Medical University, Guangzhou, China) were maintained under controlled conditions(22℃, 12-h/12-h dark/light cycle) in a conventional animal colony. Apigenin (lOmg) was suspended in lml vehicle material (0.5% methyl cellulose and 0.025% Tween 20) by sonication for 30 s at 4℃. Apigenin gavage (100mg/kg/d) and western diets (containing 20% fat and 0.15% cholesterol) feeding were started simultaneously in 6-week-old mice and sustained for 8 weeks. ApoE-/- mice intragastrically administered with vehicle material and feeding with western diet were served as the model, while C57BL/6 mice feeding with general diet were served as the blank control. Mice were sacrificed, blood was collected, aortas were separated at the end of the experiment.2. Macrophage preparation of purified rate testTo demonstrate macrophage preparation of purified rate, the macrophages were obtaied from normal C57BL/6J mice.The cells were seperated into two groups. Cells stained by F4/80 were assayed with FACS Calibur flow cytometer (BD Biosciences, USA).3.The expression of LC3BII in arteries of ApoE-/- miceThe expression of LC3BIIin arteries of ApoE-/ -mice tested by immunofluorescence, which was double stained by DAPI, were observed under a fluorescence microscope.4. Oil red O staining assaysTo demonstrate fat within atherosclerotic lesions, the lengthwise aortas were stained with Oil-Red-O. Macrophage-derived foam cells were seeded on 24-well plates. Rinsing with PBS before staining, paraformaldehyde was added for 10 min then staining with oil red O for 15 min at room temperature.1 ml per well hematoxylin was added for 10 min. Cells were captured by a fluorescence microscope (Eclipse-Ti, Nikon, Japan).5. Measurement of serum cytokinesThe levels of TNF-α, IL-1βand IL-6 in the serum were were measured using mice cytokine multiplex kit (Millipore, USA). Quantification of cytokines was performed with the Luminex system (Merck Millipore, Bioscience Division, USA).6. Cell viability assaysThe C57BL/6J mice were euthanized and the abdominal cavity was lavaged with 8 ml of ice-cold RPMI 1640 to collect resident peritoneal macrophages. Cell viability were detected by MTT assay in macrophage-derived foam cells as described previously.7. TUNEL/DAPI doubling staining assayCells were labeled with terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) stain and were counter stained with DAPI. The apoptotic index was calculated as the percent of TUNEL-positive apoptotic nuclei dividedby the DAPI-stained nuclei.8. Annexin V/PI assayCells double stainingwith annexin V/propidium iodide (PI) staining (FITC Annexin V Apoptosis Detection Kit I, BD Pharmingen, USA) were assayed with FACS Calibur flow cytometer (BD Biosciences, USA).9. Monodansylcadaverine staining assayMonodansylcadaverine (MDC), alysosomotropic fluorescent compound,which selectively labels autophagic vacuoles, was used to assess autophagy induction by apigenin though confocal microscope (C2 Plus, Nikon, Japan).10. Western blottingEqual amounts of protein lysate were separated on 12% SDS gel and then electro-transferred to the PVDF membrane. The membrane was incubated with the anti-LC3II (cell signaling technology, CST,1:1000 dilution), anti-Atg5 (CST,1:1000 dilution), anti-Atg3 (CST,1:1000 dilution), anti-Atg7 (CST, clone dl2b11,1:1000 dilution), Anti-Caspase-3 (CST,1:1000 dilution), anti-cleaved-Caspase-3(CST,clone aspl755ile,1:1000 dilutionn),anti-Bax (CST,1:1000 dilution),anti-Bcl-2(CST,clone 50e3,1:1000 dilution)over night at 4℃. This was followed by incubation with horseradish peroxidase (HRP) conjugated secondary antibodies. The results were visualized with chemiluminescence (ECL, GE Healthcare Bio-science, Sweden) and captured with a CCD system (Imagestation 2000MM, Kodak, USA). The semi-quantitative analysis of these images wasperformed using Molecular Imaging Software Version 4.0, which was provided by Kodak 2000 MM System. The opticaldensity was normalized against β-actin.11. Statistical AnalysisData are expressed as the mean ± standard error, for each experimental group. Allthe data were analyzed using the SPSS statistical package (version 13.0, SPSS Inc, USA). Mean values were compared by one-way ANOVA analysis of variance and multi-comparison was done. Data were tested via homogeneity test for variance. If the variances were homogenous, mean values were compared by ANOVA analysis and differences between two groups were analyzed by the least significant difference test; if the variances were not homogenous, mean values were compared by Welch test and differences between two groups were analyzed by Dunnett’s T3 test. A value of P<0.05 was considered statistically significant.Result1. Apigenin effectively improved artery atheromatous plaque area in miceThe aorta plaque area of apigenin groupcompared with model group significantly decreased, which compared with model group difference was statistically significant (P=0.000)..2. Apigenin effectively reduced the cell inflammation factor expressionIn the animal level, group O+A compared with model group, the expression of TNF-α and IL-1β significantly decreased, and the difference was statistically significant (P= 0.000), on the other hand,the expression of IL-6 difference, was not statistical significant (P= 0.523). In cellular level, the expression of TNF-α level of group O+A was significantly reduced compared by groupO in apigenin in 6 hours, and between two comparative difference was statistically significant (P=0.000). The expression of IL-6 and IL-10 level in 12 hours began to decrease, the comparative difference was statistically significant (P= 0.000).3. Apigenin inhibited macrophage cells into foam by detection of oil red O stainingMacrophages in animal experiments were tested by oil red O staining detection. The oil red O positive area of apigenin-treated group compared was lower than model group significantly, and the comparative difference was statistically significant(P= 0.000).4. Determined by MTT method detectedapigenin in the concentration of 25μM, 48h of cell death rate of IC50.MTT results showed that the dose in 25μM,48 h was half IC50 fatality rate,which was analysed from significant difference between group O+A and group O (P= 0.000). And with the increase of time and concentration, the cell vitality of group agradually reduced compared with O+A group (P= 0.000).5. ELISA detectedapigenint-effect to the expression of cell inflammatory factors in different timeThe expression of TNF-α level of group O+A reduced significantlycompared with O groupin 6 hours,and the differences was statistically significant (P= 0.000). The expression of IL-1β and IL-6 levels in 12 hours began to decrease, and the differences was statistically significant (P= 0.000).The expression of IL-1β and IL-6 levels began toreduce in 12 hours.6. z-VAD inhibited the apigenin influence on the cell viability, and blocked the apigenin effect on expression of cell factor inflammationDetermined by MTT results showed thatgroup O+A+Z cell apoptosis ratewas significantly higher than that of group O+A of illustrating the Z-VAD inhibited the celery element influence on the cell vitality, at the same time O+A+Z group of cells in clear cell inflammatory factor, the expression of TNF-a levels in the O+A group(P=0.000).7. Flow cytometry technology analysis of apigenin on macrophage derived foam cell apoptosis and the effect of inducing apoptosisThe cell apoptosis rate of group 0 compared withgroup O+A had significantly difference (P= 0.000). The cell apoptosis rate of group A was significantly higher than DMSO group and group O. After joining autophagy inhibitor 3-MA, apoptosis rate of group O+A+M increased significantly compared with group O+A (P= 0.000).8. Apigenin induced apoptosis protein expression tested by Western blotCompared with group O, caspase 3 in group O+A fragmented, and the difference was statistically significant (P= 0.00).Compared with groupO+A,group O+A+ Zof caspase 3 fragmentation decreased, and the difference was statistically significant(P=0.000).9. Apigenin induced autophagosome by MDC staining detectionMDC staining results showed that group the green with granular autophagosome of group O+A increased significantlycompared with the group O, and the difference was statistically significant (P=0.000). Compared with group O+A, autophagosome expression in O+A+M group reduced, and the difference was statistically significant (P=0.00), which showed that autophagybe suppressed.10. Apigenin induced autophagy protein expression by Western blotCompared with group O, LC3B Ⅱ, Beclin 1, Atg-7, Atg-5 expression of groupO+A increased. Compared withgroup O+A, LC3B Ⅱ, Beclin 1, Atg-7, Atg-5 expression in groupO+A+M decreased(P=0.000).11. Autophagy inhibition elevates apoptosis induced peritoneal macrophages apoptosisDetermined by MTT results showed that after joining autophagy inhibitor 3-MA cell vitality was reduced, but was not obvious as join z-VAD. Streaming results showed that apoptosis rate of group O+A+M rose significantly compared with group O+A (P=0.00).TUNEL method test results showed that the apoptosis rate of group O+A+M rose significantly (P=0.00)compared with O+A. Results showed that the Western blot method O+A+M group compared with O+A group, and enhancing the expression of Caspase-3, Bax/Bcl-2 percentage (P=0.00).Conclusions1.Apigenin improved the ApoE-/-mice artery atherosclerotic lesions.2.Apigenin reduced inflammation disease factors in the process of atherosclerosis though inducing macrophage derived foam cell apoptosis.3.Apigenin induced macrophage derived foam cell autophagy, and autophagy inhibition elevated apoptosis induced by apigenin to macrophages. |
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