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Friday, March 05, 2010

METODE PENELITIAN



This research will be conducted in two steps :
1. In the first steps, we focus on the of interaction between quercetin and non-genomic topics through identification of the effect of quercetin on the levels of ET-1, ICAM-1, and VCAM-1 mRNA in human umbilical vein endothelial cells (HUVECs) by Reverse transcriptase PCR (RT-PCR) and Fluorescent In Situ Hybridization (FISH) methods.
2. Second steps, research will be performed in vivo using diet-induced obese rat (Rattus norvegicus strain Wistar) to elucidate the dual effect of quercetin as a results of 1st year research.


First step research

Samples. These research will be done in the in vitro study. HUVECs are obtained from umbillical cords as a waste products of patients who undergo parturation by informed consent in dr. Syaiful Anwar Hospital Malang. This research has been approved by institutional research and human ethical commitee from Medical Faculty Brawijaya University Malang.


The number of samples.
n ≥ (15 + p) : p
n ≥ (15 + 4) : 4
n ≥ 4,75

Cell Culture and Treatment
Human umbilical vein endothelial cells (HUVEC), isolated as described previously, are seeded in culture dishes containing M-199 medium (Life Technologies, Eggenstein-Leopoldshafen, Germany) and 10% fetal calf serum (Biochrom, Berlin, Germany) supplemented with penicillin (50 U/ml) and streptomycin (50 mg/ml). HUVEC are treated with human recombinant leptin (500 ng/mL) and incubated at 37°C in a 5% CO2 incubator for 6 hours (Quehenberger et al 2002). After induction of leptin, HUVECs are treated with quercetin (Sigma-Aldrich, St. Louis, MO) at concentrations ranging between 50 M, 125 M, dan 625 M for 6 h at 37°C in a 5% CO2 incubator. These concentrations selected for the in vitro studies are similar to levels found in plasma in human subjects that have ingested 150 mg or 300 mg of quercetin (Kobuchi et al (1999)). The cell are then incubated at 37°C in a 5% CO2 incubator.
As the continuation of the first year research, the second year research will be conducted to investigate the expression of transcription gene of several factors which involve in endothelial dysfunction through nutri-transcriptomic approach, including: mRNA of ET-1, ICAM-1 and VCAM-1. In this research, we will use human recombinant leptin 500 ng/mL, followed by optimum or dual quercetin dose as a result from preceeding first year research.

mRNA Isolation, purification dan In situ hybridization. Incubate the homogenates for 5 minutes at room temperature to permit complete dissociation of nucleoprotein complexes. Add 0,2 ml of chloroform per ml of monophasic lysis reagent. Mix the samples by vigorous shaking or vortexing. Separate the mixture into two phases by centrifuging at 12,000 rpm (10,00 g in a Sorvall SS-34 rotor) for 15 minutes at 40C.Transfer the upper aqueous phase to a frees tube. Precipitate the RNA from the aqueous phase: for each initial millilitres of monophonic lyses reagent, add 0.25 volume of isopropanol and 0.25 volume of RNA precipitation solution. After through mixing, store the final solution for 10 minutes at room temperature. Collect the precipitated RNA by centrifugation at maximum speed for 10 minutes at 40C in a microfuge. Wash pellet twice 75% ethanol, and centrifuge again. Remove any remaining ethanol with a disposable pipette tip. Store the open tube on the bench for a few minutes to allow the ethanol to evaporate. Do not allow pellet to dry completely. Add 50-100 l of DEPC- Treated H2O. Store the RNA solution at -700C. Suspend 0.5-1.0g of oligo(dT)-Cellulose in 0.1 N NAOH. Pour a column of oligo(dT) –Cellulose ( 0.5-1.0 ml packed volume) in a DEPC – treated discolumn (or a Pasteur pipette, plugged with sterile glass wool and sterilized by baking for 4 hours at 300 0C). Wash the column volumes of sterile DEPC- treated H2O. Wash the column with sterile 1 X column loading – buffer (dilute from 2X stock using sterile DEPC- Treated H2O) until the Ph of the effluent is< 8.0.Use Ph paper for this measurement. Dissolve the RNA in double – distilled, autoclaved H2O, and head the solution to 650C for 5 minutes. Cool the solution to room temperature quickly, and add 1 volume of 2x column-loading buffer.
Apply the solution of RNA to the column, and immediately begin to collect in a sterile tube the material flowing through the column. When all of RNA solution has entered the column, wash the column with 1 column of 1x column- loading buffer while continuing to collect the flow- through. When all the liquid has emerged from the column, head the collected flow- through to 650C for 5 minutes and reapply it to the top of the column. Again collect the material flowing through the column. Wash the column with 5-10 column volumes of 1x column loading – buffer, collecting 1-ml fractions into sterile plastic tubes. Use quartz or disposable methacrylate cuvettes to measure the absorbance at 260 nm of a 1: 20 dilution of each fraction collected from the column using 1x column loading – buffer as a blank. Precipitate the fractions containing a majority of the OD260 material by the addition of 2.5 volumes of ethanol. Elute the poly(A)+RNA from the oligo(dT)- cellulose with 2-3 column volumes of sterile RNase – free elution buffer. Collect fractions equivalent in size to 1/3 to ½ of the column volume. Use quartz or disposable methacrylate cuvete to measure the absorbance at 260 nm of each fraction collected from the column. Pool the Fractions containing the eluted RNA. The material obtained after a single round chromatography on oligo(dT)- cellulose usually contains approximately equal amounts polyadenylated and non polyadenylated species of RNA. Polyadenylated RNA may be further purified as described in the following step. To purify the poly(A)+ RNA further, heat the preparation of RNA to 650C for 3 minutes and then cool it quickly to room temperature. Adjust the concentration of NaCl in the eluted RNA to 0.5 M NaCl and carry out a second round of chromatography on the same column of oligo(dT)- cellulose. To the poly (A)+RNA eluted from the second round of oligo(dT)- cellulose chromatography, add 3M sodium acetate(pH 5.2) to a final concentration of 0.3 M . Mix well. Add2.5 volumes of ice-cold ethanol, mix, and store the solution for at least 30 minutes on ice. Recover the poly (A)+RNA by centrifugation at 10,000g (9000rpm in a sorvall SS-34 rotor) for 15 minutes at 40C. Carefully discard the supernatant, and wash the pellet with 70% ethanol. Recentrifuge briefly, remove the supernatant by aspiration, and store the open tube in an inverted position for a few minutes to allow most of the residual ethanol to evaporate. Do not allow the pallet to dry. Redissolve the damp pellet of RNA in a small volume of sterile, DEPC- treated H2O. Use quartz of disposable methacrylate cuvetes to measure the absorbance at 260 nm of each fraction collected from the column. Pool the fraction that contains RNA. (Sambroek and Russel, 2001).

Reverse transcription polymerase chain reaction (RT-PCR). Thaw template RNA, primer solution, dNTP Mix, 5x QIAGEN OneStep RT-PCR Buffer, and RNase-free water, and place them on ice. Prepare a master mix. Mix the master mix thoroughly snd dispense appropriate volumes into PCR tubes. Add template RNA (2 μg/reaction) to the individual PCR tubes. When using a thermal cycler with a heated lid, do not use mineral oil. Otherwise, overlay with appropriately 50 μL mineral oil. Program the thermal cycler according to the program outlined. Start the RT-PCR program while PCR tubes are still on ice. Wait until the thermal cycler has reached 50oC. Then place the PCR tubes in thermal cycler.

Fluorescence In situ hybridization (FISH) for gene detection ET-1, ICAM-1 and VCAM-1. Prepare cells in cell preparation. Prewar a humidified chamber or water bath to 37oC. prewar a water bath to 60oC. Prepare hybridization cocktail and make enough for both prehybridization and hybridization. Allow about 10 – 50 μl per specimen, per treatment. Prehybridization. Apply 10 – 50 uL hybridization cocktail to each sample. Save the remaining hybridization cocktail for addition of the biotin-labeled probe. Incubate for 30 minutes in a 37oC waterbath on top of a foam float or in a humidified chamber. Shake off hybridization cocktail from the slide and blot the slide with drying paper. Prewar an incubator or oven to 95oC. Hybridization. Add biotinylated probe to the remaining hybridization cocktail to a concentration of 1.0 ng/uL. Apply 10-50 uL hybridization cocktail containing probe to the specimen. Cover the slide with a gasketed HybCover and put the slide in a HybHolder. Alternatively, the slide may be covered with a coverslip. Place the slide to 95oC and incubate for 2 minutes to denature the target and probe DNA. Transfer the slide to a 37oC humidified chamber and incubated 1-2 hours. Immediately after starting hybridization, make enough 2X SSC for two incubations in Coplin jar. Prewarm 2X SSC to 60oC in one glass Coplin jar in a water bath. Place the remaining 2X SSC in a Coplin jar at room temperature. Following hybridization, remove the cover from the slide and place the slide in the Coplin jar containing prewarmed 2X SSC at 60oC for 5 minutes. Transfer the slide to the Coplin jar containing 2X SSC at room temperature and incubate for 5 minutes.

Primer
ET-1:
F 5'-CAGACAAAGAACTCCGAGCC-3;
R 5'-GGTCTTGATGCTGTTGCTGA-3' (GenBank:NM_001955).

ICAM-1:
F5'-GGTGTATGAACTGAGCAATGTGCAAGAAG-3',
R5'-AGCACCGTGGTCGTGACCTCAG-3'(GenBank:NM_000201),

VCAM-1:
F5'-AGATTGGTGACTCCGTCTCATTGACTTG-3'
R 5'-CTGAACACTTGACTGTGATCGGCTTC-3' (GenBank:NM_001078)


Second step research

This study will be carried out in accordance with the Brawijaya University on Malang Indonesia guidelines for animal care and protection.


Experimental type and design
The type of this research is true experimental laboratory research. The experimental animal will be divided randomly into treated and control group. Research will be carry out using control group post test only design and a grouping of experimental object will be done using complete randomized design.

Animal and Diet
Male Wistar rats, will be supplied by Pharmacology Laboratory on Medical Faculty of Brawijaya University Malang, weighing initially about 150 gram, will be housed at 25 ± 1oC with a 12 h light cycle (8:00 a.m. – 8:00 p.m.). To develop a diet-induced obesity model, animal will feed on a cafeteria diet (high-fat) whose component chow, wheat, cholesterol, cholate acid and pig fat with proportions 3:2:1:1:1, which was given to each rat daily. The macronutrient composition of the cafeteria diet was 103 gram of protein, 357 gram of carbohydrate and 330 gram of lipids, which provided 4670 kcal/kg of diet. Animals will be offered this diet for 90 days. All animals have ad libitum access to water and food during the experimental period.

Reagents
Quercetin dihydrate and other chemicals will be obtained from Sigma Chemicals (USA).

Animal and Treatment.

Animals will be assigned into two experimental groups, control obese groups (n=6) and normal diet (control rats) groups (n=6). Obese and control rats will be randomly assigned to four groups, two of which received a daily dose of quercetin, either 0, 2, 10 and 50 mg/kg of body weight, mixed in the vehicle (1 ml of 1% methylcellulose), whereas the other group receive just the vehicle. The rats will be treated orally by gavage for 10 weeks (Rivera et al, 2008).

Henceforth, the obese and control groups given the variation dose of quercetin are referred to as:

1. OQ1 (obese rats treated with 2 mg/kg/day of quercetin) and CQ1 (control rats treated with 2 mg/kg/day of quercetin),

2. OQ2 (obese rats treated with 10 mg/kg/day of quercetin) and CQ2 (control rats treated with 10 mg/kg/day of quercetin),

3. OQ3 (obese rats treated with 50 mg/kg/day of quercetin) and CQ3 (control rats treated with 50 mg/kg/day of quercetin),

4. The groups given the vehicle (control groups) will be designated as OC (obese control rats group) and LC (lean control rats group).

Administration of quercetin will be stopped two days before the end of the experiments, in order to study its long-term consequences without the involvement of its acute administration effects. Over the experimental period, the rats will free access to tap water and diet, and food intake will be measured daily.


Dependent variables:
• Leptin receptor, ERK 1/2, NFB density and levels in both adipocyte visceral adipose tissue (VAT) and aorta of rat,
• Plasma glucose, insulin, TNF, lipid profile, F2-isoprostan, H2O2, leptin and adiponectin levels
• Vascular wall VCAM-1, ICAM-1 density


Methods

Samples collection and storage
At the end of the experimental period, rats will be fasted overnight, blood will be obtained from the tail vein to analyze biochemical parameters, and animals will be sacrificed. Aorta and visceral adipose tissue (VAT) samples will be excised, cleaned, and frozen until analysis. Plasma will be obtained by blood centrifugation at 2000 g for 15 min, aliquoted and frozen.

Plasma analytical procedures
Plasma glucose, triglycerides, HDL, LDL and total cholesterol concentrations will be measured by colorimetric methods using Spinreact kits (Spinreact, Spain).
Plasma insulin concentration will be quantified using a rat insulin enzyme immunoassay kit (Amersham Biosciences, Buckinghamshire, UK).
Homeostatic model assessment of insulin resistance (HOMA-IR) will be calculated from fasting glucose and insulin levels as a measure of insulin resistance (Matthews et al, 1985).
The plasma concentration of the F2-Isoprostan will be quantified using a rat F2-Isoprostan enzyme immunoassay kit (Assaydesign, USA).
Plasma leptin and adiponectin concentration will be determined using a mouse/rat leptin and adiponectin ELISA kit (B-Bridge International, Mountain View, CA).
Plasma H2O2, as marker reactive oxygen species (ROS) will be measured using a rat H2O2 enzyme immunoassay kit (Amersham Biosciences, Buckinghamshire, UK).

TNF-α level in adipose tissue and plasma
VAT obtained from each rat will be homogenized in phosphate-buffered saline. Homogenates will be incubated at 37 °C for 20 min, and centrifuged at 15,000 g for 15 min. The levels of TNF-α in the tissue supernatants will be determined using an ELISA kit (Assaydesign, USA) specific for rat TNF-α.

Leptin receptor, ERK 1/2, NFB expression in biological tissue
VAT lysates will be prepared by homogenization of visceral VAT obtained from each rat in modified RIPA buffer (50 mmol/l Tris-HCl, pH 7.4, 1% Triton X-100, 0.2% sodium deoxycholate, 0.2% sodium dodecylsulfate, 1 mmol/l sodium ethylenediaminetetraacetate, 1 mmol/l phenylmethylsulfonyl fluoride, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin). Aortic homogenates will be prepared in a buffer containing 10 mmol/l N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES) (pH 7.4), sucrose (0.32 mol/l), EDTA (100 μmol/l), dithiothreitol (1 mmol/l), phenylmethylsulphonyl fluoride (1 mg/ml), and leupeptin (10 μg/ml). Homogenates will be centrifuged at 15,000 g for 30 min and supernatants used for assessing protein expression by western blot. Protein concentration in supernatants will be determined by the Biuret protein assay. Fifty microgram of protein of VAT and 40 μg of protein of aortic homogenates from each sample were separated by sodium dodecylsulfate–polyacrylamide (8%) gel electrophoresis in a mini-gel system (Bio-Rad Laboratories, Madrid, Spain), and then transferred electrophoretically onto nitrocellulose membranes overnight. After blocking the filters in 5% nonfat dry milk-Tris-buffered saline-0.1% Tween 20 (TBST), they will respectively incubated with a mouse anti-eNOS monoclonal antibody or with a rabbit anti-iNOS antibody (BD Transduction Laboratories, San Jose, CA), diluted at 1:2500 in 5% albumin-TBST. The filters will then washed three times for 10 min in TBST and incubated respectively with secondary peroxidase–conjugated goat antimouse or Antirabbit antibody (Sigma, USA), diluted at 1:2000 in 5% nonfat dry milk-TBST. Incubations were performed at room temperature for 2 h. After washing membranes, films will be scanned and densitometric analysis will be performed on the scanned images using Corel Image-Release CS.13 software.

Result of leptin receptor, ERK 1/2, NFB by Western blot will be compared with ELISA. Leptin receptor, ERK 1/2, NFB concentrations from VAT and aorta lysates will be measured by enzyme immunoassay kit (Assaydesign, USA).

VCAM-1 and ICAM-1 expression will be identified by immunofluorescence.
Antigenic determinants masked by formalin-fixation and paraffin-embedding often may be exposed by epitope umasking, enzymatic digestion or saponin, etc. Tissue will be rinsed sections in PBS-Tween 20 for 2x2 min. Serum Blocking: incubate sections in normal serum blocking solution – species same as secondary antibody (for example: primary antibodies are mouse and rabbit, and secondary antibodies are horse anti-mouse, and goat anti-rabbit, so horse and goat serum block should be used). Primary Antibodies: incubate sections in the mixture of two primary antibodies (mouse and rabbit) at appropriate dilution in antibody dilution buffer for 1 hour at room temperature. Rinse in PBS-Tween 20 for 3x2 min. Secondary Antibodies: incubate sections in the mixture of two fluorescent conjugated secondary antibodies (FITC conjugated Horse anti-Mouse and Texas Red conjugated Goat anti-Rabbit) in PBS for 30 minutes at room temperature). Rinse in PBS-Tween 20 for 3x2 min. Counterstain with DAPI if desired for 20 minutes at room temperature. Rinse in PBS-Tween 20 for 3x2 min. Coverslip with anti-fade fluorescent mounting medium and seal with nail polish. Store slides in dark at 4 °C.

Statistical analysis
Results will be expressed as mean ± S.E.M. of measurements. For statistical analysis, one-way ANOVA.


--
Shigenoi Haruki

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