Medicinus - Dexa Medica

More documents

Recommendations

Info

18 Bioactive Protein Fraction DLBS1033 Exerts its Positive Pleiotropic Effects in the Vascular Cells via Down Regulation of Gene Expression Frans Kurnia* , ** and Raymond R. Tjandrawinata** * Department of Protein Biochemistry, Division of Protein Biochemistry and Molecular Pharmacology ** <strong>Dexa</strong> Laboratories of Biomolecular Sciences (DLBS), Industri Selatan V Block PP No. 7, Jababeka Industrial II, Cikarang, Bekasi 17520, Indonesia ABSTRACT There is accumulating evidence suggesting that cardio- and cerebrovascular diseases have a positive correlation with the events of inflammation that in addition to plaque instability occurs in the vasculature. Providing a therapy that exerts its anti-inflammatory action and inhibition of proliferation and migration of smooth muscle cells may be promising and effective in fighting the cardiovascular disease (CVD). The bioactive protein fraction DLBS1033 is shown to posses such activities. DLBS1033 down-regulates inflammatory markers including JAK1, STAT1, TNFα, NF-kB as well as decreasing vascular smooth muscle cells proliferation and migration. As Matrix Metaloproteinase-9 (MMP-9) expression was downregulated by DLBS1033, it was suggested that DLBS1033 also permitted plaque stabilization to occur in the vasculature. Taken together, these data suggest DLBS1033 exerts its positive pleiotropic effects on the vascular cells primarily via its anti inflammatory activities, plaque stabilization, vascular smooth muscle cells inhibition of proliferation and migration. INTRODUCTION Cardiovascular disease (CVD) is one of the leading causes of death worldwide. CVD covers a wide array of disorders, including diseases of the cardiac muscle and other vascular system supplying the heart, brain, and other vital organs. CVD related with occlusion of a blood vessel (thrombus) or the rupture of a blood vessel. 1 Several studies have demonstrated that inflammation is also involved in development of CVD. Increased plasma levels of inflammation markers have been identified in patients with a risk of atherosclerotic diseases. Activated leukocytes emigrate, adhere to the endothelial wall and migrate through the arterial wall, resulting in the transfer of macrophages rich in oxidized lipoproteins that trigger the onset of atherosclerotic plaque formation. Atherosclerosis is also characterized by degeneration of extracellular matrix (ECM). The resulting is dissolution and fragmentation of collagen and elastin, leading to invasion of vessel wall, that could lead to development of atherosclerotic plaque. 2 Thickening of the intimal area in blood vessels MEDICINUS 24(1), January 2011 original article research occur spontaneously with age or in response to increased intraluminal pressure. 3 Intimal thickening can also be triggered in response to injury, for example, after balloon dilatation, stent implantation or during atherosclerosis formation. 3,4 All these forms of intimal thickening are associated with increases in numbers of vascular smooth muscle cells (VSMC) and the amount of VSMC-associated extracellular matrix (ECM). Most intimal VSMC probably originate by migration from the underlying media. Intimal thickening occurs in blood vessels in response to injury or atherosclerosis. The balance of migration and proliferation of vascular smooth muscle cells (VSMC) over death by apoptosis has an important impact on the final size of intimal thickening and may also affect atherosclerotic plaque stability. 5 Treatment of the disease involves medical therapies that restore blood flow by inhibiting platelet aggregation or dissolving the thrombus occluding the arterial lumen. 1 Thrombolytic agents, such as streptokinase and urokinase have been used to treat stroke and cardiovascular disease. Those enzymes could dissolve blood fibrin clots and, to a certain
extend, could also inhibit the platelet activation and aggregation. 6 We have shown previously the activity of DLBS1033 as antithrombotic and thrombolysis agent. 7 DLBS1033, a bioactive fraction containing 8 different proteins have four different modes of actions in treating the cardiovascular disease. However, the effects of DLBS1033 on the structure of the vasculature have not been explored, particularly those pertaining to its effects on inflammation, smooth muscle cells as well as plaque structure. This present research was aimed at studying the effects of DLBS1033 at the molecular levels. MATERIALS AND METHODS Cell Culture RAW 264.7 cells obtained from American Type Culture Collection (ATCC) were grown in a 75 ml flask containing DMEM medium, 10% Fetal Calf Serum (FCS), and 1% Penicillin/Streptomycin at 37 o C, 5% CO 2 . Human aortic smooth muscle cells (HVSMC; Clonetics, San Diego, CA) were cultured in SMC basal medium according to the supplier's instructions. Preparation of DLBS1033 DLBS1033 powder was diluted in phosphate buffer pH 8 and the suspension was centrifuged at 5000 x g for 10 minutes. The supernatant was then collected and filtered before use as described previously by Jessica et al. 7 Treatment of DLBS1033 Before treatment, the cells were poured into six-well plate containing DMEM medium, 10% FCS, and 1% Penicillin/Streptomycin and incubated at 37oC for 18 hours. On the next day, the medium was replaced with DMEM medium containing only 1% Penicillin/ Streptomycin and 0.5 μg/ml DLBS1033. The cells were then incubated again until the next day for RNA isolation. Cell Viability Assay The number of viable cells was determined using Cell Titer 96 Aqueous Assay (Promega, Madison, WI). This assay was performed according to the manufacturer’s protocol. The absorbance at 490 nm was measured with a microplate reader type 680 (Biorad, Hercules, CA). Each experiment was performed in quadruplicate. The statistical analysis methods were carried out in ProStat. RNA Isolation and RT-PCR RNA isolation was done using TRIzol reagent (Invitrogen) which was then processed in RT-PCR. RT-PCR was used to determine mRNA expression of genes that involve in inflammation and platelet stabilization. 0.1 μg total RNAs were added to 25 μl reaction mixture containing 5 μl AMV Reverse Transcriptase 5x reaction buffer (250 mM Tris-HCl, 250 mM KCl, 50 mM MgCl 2 , 2.5 mM spremidine and 50 mM DTT), 30 units AMV Reverse Transcriptase, 40 units Rnasin Ribonuclease Inhibitor, 0.5 mM dNTPs, and 0.5 μg oligo(dT) 15 primer. The cDNA was then processed for PCR screening. PCR for Screening and Analysis PCR for screening the target gene was done using GoTaq MasterMix (Promega). PCR was performed with 2.5 μl cDNA in 25 μl reaction mixture containing 12.5 μl MasterMix, 1 μl 10 μM sense and antisense primer each. PCR conditions were initial denaturation 95 o C for 2 min followed by 30 cycles of 95 o C 1 min, annealing for 1 min, 72 o C for 1 min and extension 72 o C for 10 min. PCR products were resolved in 2% agarose gel containing ethidium bromide. Images were taken by a Gel Doc apparatus (BioRad) and analyze with 2D software (BioRad). Binding Assay with Monocytic Cells RAW 264.7 cells were preincubated in DMEM containing 2% FBS and 5 µg/ml fluorescent BCECF-AM at 37°C for 30 min in foil-covered tubes. Fluorescently labeled cells were washed twice to remove unincorporated dye and were then resuspended in DMEM containing 0.2% BSA. Loaded monocytic RAW cells (5x104) were added to each well of VSMC and incubated at 37°C. After 30 min, unbound monocytes were withdrawn and VSMC layers with attached monocytes were gently washed twice with DMEM and lysed with 0.2 ml of 0.1% Triton X-100 in 0.1 M Tris per well. Fluorescence (excitation 485 nm, emission 535 nm) was measured using a microplate reader (BioTek, Winooski, VT, United States). Cell Migration Assay original article research Chemotaxis cell migration assay was performed using modified Boyden chambers with polyvinylpyrrolidone-free polycarbonate filter membranes, 8-μm pore size. 25,000–30,000 cells in serum-free SMC basal medium were added to the upper well of the Boyden chamber. Angiotensin II was added to the bottom of Boyden chamber as a chemoattractant, MEDICINUS 24(1), January 2011 19
Page 3 and 4: Chief Editor Dr. Raymond R. Tjandra
Page 5 and 6: MEDICINUS Editors receive original
Page 7 and 8: para ilmuwan lainnya. 7,8,11-13 Rad
Page 9 and 10: AO juga dapat dibedakan berdasarkan
Page 11 and 12: ngan perawatan kulit terkini adalah
Page 13 and 14: Sekilas tentang Seloxy AA Aging mer
Page 15 and 16: Effect of DLBS3233, an Insulin Sens
Page 17 and 18: Figure 1. Effect of DLBS3233 (9 mg/
Page 19: commonly used method to assess insu
Page 23 and 24: NF-kB, TNFα, VCAM-1 and P-selectin
Page 25 and 26: ily inflammatory function, especial
Page 27 and 28: Rizaldy Pinzon SMF Saraf RS Bethesd
Page 29 and 30: Tabel 2. Profil kepatuhan terhadap
Page 31 and 32: Malaria pada HIV : Suatu Tantangan
Page 33 and 34: saja, sehingga pengobatan dilanjutk
Page 35 and 36: malaria, dan proteksi ini meningkat
Page 37 and 38: dosage and the effectiveness of a d
Page 39 and 40: ples of these are: caryophilli flos
Page 41 and 42: Pengaruh Bahan Tambahan atau Eksipi
Page 43 and 44: intoleransi terhadap terhadap pheny
Page 45 and 46: yaitu 109 watt dalam nano second. D
Page 47 and 48: dilaporkan, tetapi sementara waktu
Page 49 and 50: Vasospasmus Puting Payudara pada Wa
Page 51 and 52: dan merokok. Mereka juga disarankan
Page 53 and 54: dr. Amaranila Lalita Drijono, SpKK
Page 55 and 56: MEDICINUS 24(1), January 2011 53
Page 57 and 58: Peran Menguntungkan Clopidogrel dal
Page 59 and 60: medical news Cokelat Dapat Menguran
Page 61 and 62: Tips Sukses Pencegahan Ulkus Diabet

Medicinus - Dexa Medica

Create successful ePaper yourself

Delete template?

Save as template?