Catechins Green Tea GMB4 Clone Increases mRNA of ABCA1 through LXR Signaling in Cultured Macrophage Exposed OX-LDL

Inhibition of atherogenesis through inhibition of lipid metabolism has not been explored while other inhibitions through inflammation, endothelial dysfunction and free radicals have done. Inhibition of atherogenesis via inhibition of lipid metabolism can be done through the mechanism of Reverse Cholesterol Transport (RCT). Signaling pathways that play a role in this mechanism is LXR signaling. LXR activation by an LXR agonist led increasing cholesterol efflux. Catechins based on bioinformatics study showed as a potent candidate LXR agonists that can be used as an inhibitor of atherogenesis. This study aims to prove that the administration Catechins green tea Clone GMB4 can prevent atherosclerosis through increasing mechanism cholesterol efflux from macrophage by taking effect of ABCA1, ABCG1, SRB1 gene expression in cultured macrophages were exposed ox-LDL. Long-term goals of the outcome of the research are the use of Catechins Green Tea Clones GMB4 as an inhibitor of atherogenesis so that it can be used as a complementary therapy for the treatment of atherosclerosis and cardiovascular diseases. The research is divided into 5 groups, namely the culture of macrophages without exposed ox-LDL, culture exposed ox-LDL and groups of Catechins dose I, II, III. In vitro study showed that administration of Catechins increases mRNA of ABCA1, whereas mRNA ABCG1 and SRB1 decreased at all three doses given. The result of protein profilling was identified a protein with a molecular weight of 70 kDa by SDS-PGE with silver staining.


Introduction
Cardio vascular disease (CVD) is first cause of death in the world. Atherosclerosis is the main contributor cause of death because the trigger of myocardial infarction and ischemic stroke. Based on WHO data, CVD mortality rate is estimated at 36.3% (1 of 2.3 of the total number of deaths) in the United States in 2004. While the costs for CVD in 2007 was reported at $431.8 billion [1]. The process of atherogenesis can be initiated by high LDL that cause endothelial injury that trigger endothelial dysfunction. The next process, NADPH oxidase oxidized LDL endothelial cells. LDL modification that caused enzymatic and oxidative reaction triggers the release of inflammatory lipid which induces endothelial cells express leukocyte adhesion molecules. Modified LDLs were scavenged by scavenger receptors of macrophages that develop into foam cells [2].
One pathway inhibits atherosclerosis through a mechanism of reverse cholesterol transport (RCT). Reverse cholesterol transport is the process efflux cholesterol macrophages to HDL or lipid-free apolipoprotein like Apo-A1 or Apo E [3].
Further, HDL or Apo A1 is uptaked by the liver where free cholesterol in the liver to be excreted into the bile ducts in the form of cholesterol that is not changed or after converted into bile acids. The next process is the transport of intestinal cholesterol to be excreted through the feces. One of the signaling pathways that contribute to the inhibition of atherosclerosis through mechanisms RCT is LXR. Liver X receptors act as sensors of cholesterol that works to lower cholesterol levels through increasing the expression of a target gene associated with RCT that are the protein transporter ATP-binding cassette subfamily A member 1 (ABCA1), ATP-binding cassette subfamily G member 1 (ABCG1), Apo A1 and hepatic scavenger receptor class B type I (SRB1) [4]. LXR signaling is activated by an agonist effect on effluks cholesterol in macrophages which can be seen by inspection parameter ABCA1, ABCG1, Apo A1 and SRB-1. LXR clicking the upregulation of expression of ABCA1 transporter and ABCG1 whose role is to transport cholesterol from the plasma membrane to the extracellular acceptor. ABCA1 is a major protein for cellular cholesterol to Apo effluks acceptor such as ApoA1 and the first step in RCT. ABCA1 transporter is also a fully working as a single molecule to transfer cholesterol and phospholipids in the plasma membrane into pre high density lipoprotein (HDL) and lipid-free ApoA1. While working as a homodimer ABCG1, the transfer of cholesterol to HDL is not a lipid-free ApoA1 (Julve et al., 2011). Intervention therapy for improving the expression of ABCA1 and ABCG1 is an effective strategy to increase RCT macrophages and potentially reducing atherosclerosis. While Scavenger receptor B1 is a key receptor responsible for the selective uptake of cholesterol ester (CE) from HDL to the liver, the hepatic SRB1 positive regulator known as RCT. Without the SRB1, macrophages RCT will not run. The role of SRB-1 against the uptake of cholesterol in the liver and affect RCT research Macrophages are the first inflammatory cells that cause atherosclerotic lesions that are major components of atherosclerotic plaque. In the pathogenesis of atherosclerosis, monocyte infiltration of blood to the intima and subintima, a process which are activated by the accumulation of lipoproteins containing apolipoprotein B (apoB-LPs) subendothelial. The presence of chemokines would cause the monocytes bind to the endothelium by effecting of P-selectin, E-selectin, Lymphocyte Function-Associate Antigen-1 (LFA-1), verry late antigen-4 (VLA-4), Vascular Cell Adhesion Molecule-1 (VCAM-1) and Intracellular Cell Adhesion Molecule-1 (ICAM-1), which in turn will monocytes enter the subendothelial so that is known diapedesis. The next phase occurs macrophage differentiation influenced by Macrophage Colony Stimulating Factor (MCSF). M1 differentiated from high Ly6C monocytes that cause inflammation, which is activated by Lipopolisacharide (LPS) in the presence of interferon-γ (IFNγ), which triggers the production of interleukin-2 (IL-2), Interleukin-23 (IL-23), interleukin-6 (IL-6), Interleukin-1 (IL-1) and Tumor Necrosis Factor-α (TNFα) at high levels. While M2 differentiate from low Ly6C monocytes that play a role of inflammation. This differentiation is effected by IL-4, IL-13, IL-1 and vitamin D3 so that lead to produce IL-10, expression of scavenger receptors, mannose receptors and arginase in large quantities. In the development of atherosclerosis there is an imbalance between M1 and M2 [8]. Atherogenesis process can be inhibited by a mechanism RCT. The RCT mechanism activated by LXR signaling. LXR/RXR heterodimer binds to LXR response element (LXRE) containing sequences hexamerik (AGGTCA). LXR/RXR can be activated by LXR agonists and 9-C is Retinoic Acid (9cRA), RXR specific ligands. These receptors can also be activated synergistically with the ligand for the two receptors. Activators of endogenous LXR is oxysterol, oxidized cholesterol derivatives, namely 22-(R)-, 20-(S)-, 24-(S)-hydroxycholesterol dan 24-(S), 25-Epoxycholesterol which induces transcriptional activity of LXR at physiological concentrations, Coactivator for LXR transactivation are Grip 1, Ap 160 Coactivator, TRRAP, PGC-1α. PGC-1α not only as a key regulator of hepatic gluconeogenesis but as Coactivator LXRα. In the absence of ligand, nuclear receptor represses gene transcription by recruiting corepresor proteins such as nuclear receptor corepressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT). Transrepression by LXR depends on the interaction NCoR and SMRT [9]. Activation of LXR signaling increases choleterol efflux through increasing protein transporter ABCA1, ABCG1 and SRB1. ABCA1 gene is expressed in high amounts in liver, testis, small intestine, adrenal glands, heart, brain and macrophages. ABCA1 gene expression is regulated by intracellular cholesterol levels. mRNA of ABCG1 is expressed at moderate to high levels on macrophages, spleen, lung, thymus, placenta, brain and at low levels in other tissues, liver. One of the natural substances that can potentially increase cholesterol efflux from macrophage is Green Tea GMB4 clone. Green tea is developed by the Centre for Development of Tea and Quinine Gambung with the high content of Catechins. Based on the results of analysis total Catechins from Green Tea clones GMB4 are 14% -16% [10].

Western Blot Analysis
Aortic and hepatic protein levels of markers involved in RCT (ATP binding cas- HCl, 1 mmol/l ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 0.1 mg/ml PMSF, 1 g/ml aprotinin, 10 g/ml leupetin, pH 7.4, briefly sonicated. Consentration of protein were measured by nanodrop instrument. Samples (50 g/lane) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Blots were placed in Tris-buffered saline, 0.05% Tween 20 (TBST) supplemented with PBS containing 3% BSA for 2 hrs at room temperature and then incubated with ABCA-1, ABCG1 and SRB1 overnight at 4˚C. The blots were washed three times with TBS-Tween, and the membranes were incubated with horseradish peroxidase-conjugated antibodies for 1 hr at room temperature and washed again as described previously [12] [13].

Statistical Analysis
Data are presented as mean ± SD of five replication. Statistical differences between multiple groups were determined by analysis of variance (ANOVA) Statistical comparations were made using of Tukey test. Difference were considered significant at p < 0.05.

Result
Results Isolation of macrophages from the Mouse Peritoneum Macrophage (MPM). To determine the number of the isolated cells was measured by hematositometer as shown in Table 1.
Statistical analysis showed that there is significant increase mRNA ABCA1 significantly in dose 50 μM and 100 μM Catechins with p value 0.003 and 0.000 where the value is less than 0.05, whereas the mRNA ABCG1 significant decrease in the third dose Catechins given. mRNA SRB1 significant decrease in dose of 50 μM compared with the group that was exposed ox-LDL with p value 0.039 ( Table 2).      mg/L is an optimal concentration for the formation of foam cells [19].  [21]. Widely accepted that HDL protects atherosclerosis by way of removing excess cholesterol from the cells by an active process mediated by membrane transporter ABCA1 [22].

Conclusions
Based on the results of research and discussion, it can be summed up as follows: 1) Catechins increase mRNA ABCA1 and decrese mRNA ABCG1 and SRB1 in cultured macrophages were exposed ox-LDL.
2) Catechins increase the expression of the protein with a molecular weight of 70 kDa based on the results of SDS-PAGE with silver staining in cultured macrophages were exposed ox-LDL.