Total Flavones From Psidium Guajava Leaves Reduce Macrophage Foam Cell Formation

BackgroundAtherosclerosis (AS), an inflammatory disorder, is the phenomenon, which makes individuals susceptible to diseases like coronary heart disease (CHD), stroke, and so on. According to the statistics of WHO in 2007, the mortality of cardiovascular diseases of our country in 2002, which came to 299/100 thousand people, was much higher than that of tumour, which was 148/100 thousand people. Therefore, it is very significant to improve the treatment and prevention of AS.The pathological mechanisms of AS have not been fully understood, however, now it is widely accepted that AS is an inflammatory disorder. A variety of cells coordinate the evolution of inflammatory responses and establish acquired immunity to specific pathoens. Among the numerous complicated factors, monocyte-derived macrophages plays an essential role in the pathogenesis of atherosclerosis. Monocytes adhere to the endothellium and migrat to the sub-endothelial space. Once monocytes are in the endothelium, they differentiate into macrophages. Macrophages accumulate modified low-density lipoprotein (LDL) especially oxidized LDL (ox-LDL). Theses modified LDL are metabolized into cholesterol and cholesterol esters leading to foam cell formation. Foam cells are typical of fatty streaks in atherosclerosis. Besides, macrophages interact with other cells in the endothelium including T-cells and vascular smooth muscle cells via various receptors, cytokines released and matrix metalloproteinase (MMPs) by them. This leads to enhance inflammatory response and even causes acute conoary syndrome (ACS). Hence, reducing macrophage foam cell formation is one of the most important targets of AS prevention.Macrophages take up lipid mainly through scavenger receptors (SR), the membrane proteins expressed by macrophages. Out of all the scavenger receptors, the importance of type A (SR-A), CD-36 and LOX-1 in atherogenesis can be realized from the fact that macrophages uptake 90% lipid with these receptors. In 1997, Sawamura et al. discovered a new receptor for ox-LDL, the lectin-like ox-LDL recceptor-1 (LOX-1) in bovine aortic endothellial cells. He described LOX-1 as the major receptor for ox-LDL in endothelial cells of large arteries. The important role of LOX-1 in atherogenesis is supported by several lines of evidence.LOX-1 shows a strong activity in binding, internalizing, and proteolytically degrading ox-LDL. ox-LDL activaties LOX-1 and induces endothelial dysfunction/apoptosis, a major change in vascular biology seen at the beginning of atherogenesis. Besides ox-LDL, other mediators of atherosclerosis such as angiotensinⅡ(AngⅡ), sheer stress, advanced glycation end-products (AGE) all upregulate LOX-1. LOX-1 is dynamically upregulated by pro-atherogenic conditions, such as diabetes, hypertension and dyslipidemia. LOX-1 is present in atheroma-derived cells and is seen in large amounts in human and animal atherosclerotic lesions in vivo. We believe that LOX-1 is involved in multiple steps in atherogenesis and its complications.Chinese traditional medicine Psidium guajava L. belonging to Myrtaceae Psidium, is widely planted in Guangxi, Guangdong, Sichuan and so on. Its leaves is neutral in nature, sweet flavor, and have an effect on astringing to stop diarrhea and eliminating inflammation and hemostasis. Foreign studies also show that it functions in regulating blood lipid, treating AS, but the mechanisms remain unknown. The main components of Guava leaf are flavonoids, which are improtant active materials of many plants. A great many studies on flavonoids have shown that they can treat cardiovascular and cerebrovascula diseases. Hence, we consider that the anti-AS effect of Guava leaf might be closely related to its total flavonoids.ObjectiveTo investage the effect of total flavones from Psidium guajava leaves on macrophage foam cell formation induced by ox-LDL and its probable mechanism.Methods1. THP-1 cells cultureTHP-1 monocytes (American Type Culture Collection, USA) were maintained at 37℃in humidified 5% CO2 atmosphere, in suspension at cell density of 106 cells/mL in RPMI 1640 containing 10% fetal bovine serum (FBS) (v/v). Medium was changed every 2-3 days, and passages 3-5 were used for experiments.2. The differentiation of THP-1THP-1 cells were plated into 6 well plates at 5000-10000 cells/well and then cultured in phorbol 12-myristate 13-acetate (PMA) at a final concentration of 100ng/mL for 48 hours. The shape of the cells were observed under a microscope.3. Macrophage foam cell formation and identificationThe differentiated THP-1 cells were washed with serum-free RPMI and then incubated in serum-free RPMI 1640 containing 50μg/mL of ox-LDL for 24 hours. THP-1 macrophages were washed 3 times with D-Hanks, fixed with formaldehyde, and stained with oil red O. Lipid accumulation was observed under a microscope. 4. CCK-8 analysisAfter differentiated by PMA, the macrophages were washed with serum-free RPMI 1640 and then cultured for 24 hours in 100μL serum-free medium containing 7 types of concentrations of total flavones from Psidium guajava leaves:0μg/mL, 15μg/mL,30μg/mL,60μg/mL,120μg/mL,250μg/mL,500μg/mL.10μL CCK-8 were added to each well. After 4 hours, the absorbance value of each group was analyzised by ultraviolet-visible spectrophotometer at the wave length of 540 nm.5. Experimental protocolsThe macrophages were randomly divided into five groups:(i) Control group: cells were treated without any drug intervention. (ii) ox-LDL group:cells were incubated in serum-free medium containing 50μg/mL ox-LDL. (iii) ox-LDL+20μg/mL total flavones from Psidium guajava leaves. (iv) ox-LDL+40μg/mL total flavones from Psidium guajava leaves. (v)ox-LDL+80μg/mL total flavones from Psidium guajava leaves.6. Abstraction of the total ptotein of the macrophagesThe macrophages were treated with different drugs for 24 hours, then washed with D-Hanks 3 times. NP-40 buffer was used to dissolve the cells membrane. The protein of each group was harvested by centrifuging in 10000-15000g for 3-5min and stored in -80℃. The concentration of protein was determined with BCA assay using bovine serum albumin (BSA) as a standard.7. Cellular lipid analysisTo ditermine intracellular lipid contents, each group of macrophages were washed with D-Hanks 3 times and then dissolved with NP-40 buffer. The cellular total cholesterol (TC) was measured using an enzymatic method.8. Western blottingForty micrograms of protein extracts was mixed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. They were boiled for 5min, and underwent electrophoresis on a 10% SDS polyacrylamide gel. They were then transferred onto PVDF membranes. The membranes were blocked with 5% non-fat dry milk in TBST buffer and then incubated overnight with primary rabbit antibody against LOX-1 at 4℃. They were then washed 3 times in TBST (10 min for each wash) befor incubating the secondary antibody for 1 hours. The results of analyzed by KODAK Image Station 4000MM exposure imaging system and quantified using Image tool V3.0 software.9. Statistical analysisResults were expressed as mean±SD of at least three independent experiments. One-way ANOVA was applied to test the level of significance for the expression among different doses using post-hoc analysis with Bonferroni correction. When the variance of arrhythmia, the use of robust estimation Welch, then the use of T3 compared methods.A value of P<0.05 was considered as significant. All the analyses were performed using SPSS 13.0.Result1. The effect of different doses of total flavones from Psidium guajava leaves on the viability of macrophagesAccording to the result of CCK-8, total flavones from Psidium guajava leaves with high dose affected the viability of macrophages. Compared with the control group, the viability of the 500μg/mL group decreased by 31.97%, the difference of which was significant (P<0.05). It indicated that this dose of total flavones from Psidium guajava leaves was toxiferous. Though the difference between the control group and the 200μg/mL group was not significant, the viability of the latter also decreased. Hence, we selected 20,40 and 80μg/mL three doses within the safe scope.2. The effect of total flavones from Psidium guajava leaves on total cholesterol content of macrophagesAfter induced with ox-LDL for 24 hours, the total cholesterol (TC) of ox-LDL group increased significantly (P<0.05), which demonstrated that macrophages formed foam cells with the induction of ox-LDL. However, the TC content of all the total flvones groups were lower than ox-LDL group. And the higher the dose was, the lower the TC was. The difference was significant (P<0.05). It indicated that total flavones had an dose-effect relationship in the function of decreasing TC.3. The effect of total flavones from Psidium guajava leaves on the expression of LOX-1 protein macrophages foam cellsAs shown by western blot, ox-LDL markly increased ox-LDL-triggered LOX-1 expression in THP-1 macrophage foam cells (P<0.05). In contrast, the expression of LOX-1 of the total flavones groups all reduced (P<0.05), and the dose-effect relationship of this effect also existed. That manifested that total flavones from Psidium guajava leaves inhibited the up-regulation of LOX-1.ConclusionsTotal flavones from Psidium guajava leaves have an function of reducing macrophages uptaking ox-LDL and thus inhibiting foam cells formation through the depression of LOX-1.