UNIVERSA MEDICINA
May-August, 2012 Vol.
31 - No.
Catechins inhibit atherosclerosis in male rats on a high fat diet Erna Susanti*,**.
Achmad Rudijanto***, and Retty RatnawatiA
ABSTRACT
BACKGROUND
A catechin isolate from the green tea clone GMB 4, which shows antioxidant activity, may be a candidate drug for prevention of atherosclerosis.
The aim of this study was to analyze the effect of catechin on endothelial nitric oxide synthase .
NOS) and p110 phosphoinositide 3-kinase inhibitor (PI3K) expression and on p38 mitogen activated protein kinase (MAPK) activity in male rats fed a high fat diet.
METHODS
Twenty five male Wistar rats were divided into the following five groups: rats on standard diet.
rats on high fat diet.
rats on high fat diet catechin 3 mg/ rats on high fat diet catechin 6 mg/day.
and rats on high fat diet catechin 24 mg/day.
eNOS and p110 PI3K expression and p38 MAPK activity were measured by means of ELISA.
RESULTS
High fat diet significantly increased eNOS expression, decreased p110 PI3K expression, and increased p38 MAPK activity in male rats, in comparison with standard diet .
<0.
Administration of 3 mg/day catechin decreased eNOS expression compared to that in the high fat diet group without catechin .
<0.
The administration of catechin increased p100 PI3K expression to a similar extent as that in the high fat diet groups with catechin 6 mg/day and 24 mg/day.
Administration of catechin at all doses decreased p38 MAPK activity to the level of the standard diet group.
*Biomedical Study Program.
Faculty of Medicine.
Brawijaya University.
Malang **Academy of Pharmaceutics and Food Analysts AuPutra Indonesia MalangAy.
Malang ***Endocrine and Metabolic Division.
Faculty of Medicine.
Brawijaya University Physiology Laboratory.
Molecular Physiology Division.
Faculty of Medicine.
Brawijaya University.
Malang Correspondence Erna Susanti Academy of Pharmaceutics and Food Analysts AuPutra Indonesia MalangAy Jl.
Barito No.
5 Malang Phone: 62341Ae491132 Email: rna_far@yahoo.
Univ Med 2012.
31:81-7
CONCLUSIONS
High fat diet increases eNOS expression, decreases PI3K expression, and increases p38 MAPK activity.
Administration of catechin decreases eNOS expression, increases PI3K expression, and decreases p38 MAPK activity.
Keywords: Catechin, eNOS, p110 PI3K, p38 MAPK, high fat diet, male rats Susanti.
Rudijanto.
Ratnawati Catechins inhibit atherosclerosis Katekin menghambat proses aterosklerosis pada tikus jantan dengan diet lemak tinggi ABSTRAK
LATAR BELAKANG
Isolat golongan senyawa katekin dari teh hijau klon GMB 4, yang mempunyai aktivitas antioksidan, dapat dikembangkan sebagai kandidat preventif aterosklerosis.
Penelitian ini bertujuan menilai efek katekin terhadap penghambatan penurunan ekspresi p110 phosphoinositide 3-kinase inhibitor (PI3K) dan endothelial nitric oxide synthase .
NOS) serta penghambatan peningkatan aktivitas p38 mitogen activated protein kinase (MAPK) pada tikus jantan dengan diet tinggi lemak.
METODE
Dua puluh lima ekor tikus Wistar jantan yang terbagi dalam lima kelompok perlakuan, tikus dengan diet pakan tikus dengan diet tinggi lemak.
tikus dengan diet tinggi lemak katekin 3 mg/hari.
tikus dengan diet tinggi lemak katekin 6 mg/hari.
dan tikus dengan diet tinggi lemak katekin 24 mg/hari.
Ekspresi eNOS, p110 PI3K, dan aktivitas p38 MAPK dianalisis dengan teknik ELISA.
HASIL
Diet tinggi lemak meningkatkan ekspresi eNOS, menurunkan ekspresi p110 PI3K, meningkatkan aktivitas p38 MAPK secara bermakna dibandingkan kelompok diet standar .
<0,.
Pemberian katekin dosis 3 mg/hari meningkatkan ekspresi eNOS secara bermakna dibandingkan diet standar dan diet tinggi lemak .
<0,.
Pemberian katekin meningkatkan ekspresi p110 PI3K secara bermakna dibandingkan diet tinggi lemak .
<0,.
, akan tetapi baru dapat mencapai ekspresi pada diet standar di dosis 6 mg/hari dan 24 mg/hari.
Pemberian katekin berbagai dosis terbukti menurunkan aktivitas p38 MAPK secara bermakna dibandingkan diet tinggi lemak .
<0,.
, bahkan mencapai aktivitas p38 MAPK diet standar.
KESIMPULAN
Diet tinggi lemak meningkatkan ekspresi eNOS, menurunkan ekspresi PI3K, dan meningkatan ekspresi p38 MAPK.
Pemberian katekin dapat menurunkan ekspresi eNOS, meningkatkan ekspresi PI3K, dan menurunkan aktivitas p38 MAPK.
Kata kunci: Isolat katekin, eNOS, p110 PI3K, p38 MAPK, diet tinggi lemak, tikus jantan
INTRODUCTION
According to WHO estimates, annually 8 million males and 3.
4 million females worldwide die from coronary heart disease.
Atherosclerosis is the principal contributor to the pathomechanism of coronary heart disease.
One of the risk factors causing the progressivity of atherosclerosis is dyslipidemia resulting from a high fat diet and obesity.
A high fat diet may trigger the development of chronic inflammation leading to endothelial The contribution of endothelial dysfunction to the pathogenesis of atherosclerosis is via uncoupling .
of endothelial nitric oxide synthase .
NOS).
Uncoupling of eNOS triggers the formation of superoxide anions (O 2-) and decreases NO Superoxide anions may react with NO to form peroxynitrites for inactivation of tetrahydro biopterin (BH 4 ), which in turn increases accumulation of asymmetric dimethylarginine (ADMA), the endogenous inhibitor of eNOS.
In addtion to causing Univ Med endothelial dysfunction, accumulation of toxic lipid metabolites, i.
fatty acyl coenzyme A (CoA), diacylglycerol, and ceramides in tissues and arteries causes the development of insulin .
The latter condition triggers abnormal insulin signal transduction in endothelial cells, involving two main pathways, the phosphoinositide 3-kinase inhibitor (PI3K) and mitogen activated protein kinase (MAPK) pathways.
Insulin resistance decreases PI3K signaling and increases MAPK signaling.
Continuous stimulation of MAPK activity causes vascular smooth muscle cell (VSMC) proliferation, increased collagen synthesis, overproduction of growth factors and pro-inflammatory cytokines, thereby accelerating the development of .
Among the naturally occurring substances that have antioxidant potential are the catechins isolated from the tea plant (Camellia sinensi.
The Gambung Research Center for Tea and Quinine have developed green tea GMB4 clones with higher levels of catechins .
% - 16%) extracted from the third level leaf buds.
The number of hydroxyphenolic and galloyl groups and the epimerization structure of the isolates affect their activity despite similar levels of total There are several studies in support of the activity of tea catechins on vascular function, such as the study conducted by Anter et al.
which shows that the polyphenol fraction of black tea stimulates eNOS catalytic activity.
The mechanism involves stimulation-mediated activation of p38 MAPK and PI3K/Akt pathways leading to eNOS phosphorylation at serine 177 and dephosphorylation at Thr-495.
This activity results in the activation of calmodulin-dependent eNOS and increased NO .
Other studies have found strong evidence of NO involvement in the induction of vasorelaxation by tea polyphenols.
Catechins with their galloyl groups are natural inhibitors of tyrosine kinases that can modify the activity of various signaling kinases such as extra cellular signal-regulated kinase 1 and 2 (ERK1/.
Vol.
31 No.
protein kinase B (Ak.
PI3K and p38 MAPK.
The hydroxyphenolic groups of catechins contribute free radical scavengers, inhibition of lipid peroxidation and hydrolysis of fat, while their galloyl groups contribute to the production of prostacyclins, reduction in vascular cell adhesion molecule-1 (VCAM-.
expression and inhibition of VSMC proliferation.
On the basis of the results of abovementiomed studies, the aim of the present study was to analyze the effect of administration of a catechin isolate from the green tea clone GMB4 on the expression of eNOS and p110 PI3K, and on p38 MAPK activity in male Wistar rats on a high fat diet.
METHODS
Design of the study A laboratory experimental study with controlled post test design, conducted from June 2010 to March 2011 in the Physiology Laboratory of the Faculty of Medicine.
Brawijaya University.
Malang.
Animals In acordance with the Federer formula, a total of 25 male Wistar rats (Rattus norvegicu.
aged 7-8 weeks and weighing 130 - 155 grams, were divided into five intervention groups, which were fed the following diets: standard diet, high fat diet, high fat diet catechin 3 mg/day, high fat diet catechin 6 mg/day, and high fat diet catechin 24 mg/day.
Before undergoing the interventions, the rats were adapted to laboratory conditions for 7 days.
The interventions were conducted for 60 days, after which the rats were sacrificed to obtain their aortas.
Preparation of animal feed The standard feed consisted of PARS chicken feed .
ontaining water, protein, lipid, fiber, ash, calcium, phosphorus, antibiotics, and coccidiostatics, to a total content of 66.
6%, and 4% wheat flou.
The high fat diet was a combination of 57.
3% standard feed (PARS) and Susanti.
Rudijanto.
Ratnawati 8% wheat flou.
, with the addition of 9%, cholic acid 0.
1% and pork fat Measurement of p110 PI3K and eNOS expression and p38 MAPK activity The ELISA assay was performed as follows: after determination of the required number of microtiter wells, a standard curve was prepared, for which 100 y l of assay buffer was pipetted into the blank well, and 100 y l phosphorylated p38 MAPK or p110 PI3K standards 1-7 were pipetted into the designated treatment wells.
Protein from aortic tissue samples isolated by means of radio immuno precipitation assay (RIPA) buffer was used for measurement of p38 MAPK and p110 PI3K For the treatment samples, 100 y l of treatment sample was pipetted into each The microtiter plates were incubated at 370C for 2 hours.
Then the wells were washed with wash buffer 3 x 400 y l.
Next, each of the wells, with the exception of the blank, was filled with 100 y l conjugate.
After further incubation of the microtiter plates at 370C for 30 minutes, the wells were each washed again with wash buffer 3 x 400 y l.
Subsequently the substrate, i.
100 yl 3,3Ao,5,5Ao -tetramethylbenzidine (TMB), was pipetted into each well, and the microtiter plates were again incubated for 30 minutes at room temperature.
To stop the conjugation reaction, 100 AAl HCl was pipetted into each well and left to react for 5 minutes.
Absorbance readings were performed at an optical density (OD) of 492 nm.
For NOS and p110 PI3K expression, the measurements were in yg/mL and pg/mL, respectively, while p38 MAPK activity was expressed in pg/mL.
Catechins inhibit atherosclerosis Statistical analysis Normally distributed data on p38 MAPK activity and p110 PI3K expression from each intervention are presented as mean and standard Non-normally distributed data are presented as the median, or as the mean and standard deviation after appropriate Data analysis was by means of one-way ANOVA followed by the Tukey test, using SPSS for Windows version 17.
Ethical clearance This study was accorded ethical clearance by the Commission of Medical Research Ethics.
Faculty of Medicine.
Brawijaya University.
Malang.
RESULTS
Based on the ANOVA results, there was a significant difference in aortic eNOS expression between intervention groups .
<0.
From the results of the Tukey test it was concluded that high fat diet significantly increased eNOS expression in comparison with the standard diet group .
<0.
Catechin administration at a dose of 3 mg/day significantly increased eNOS expression in comparison with the standard and high fat diets .
<0.
Administration of catechin at a dose of 6 mg/day significantly reduced eNOS expression .
<0.
in comparison with the high fat diet, although the reduction did not reach the level of the standard Administration of catechin at a dose of 24 mg/day did not significantly reduce eNOS levels as compared with the high fat diet .
>0.
(Table .
Table 1.
Mean eNOS and p110 PI3K expression in the intervention groups Univ Med Vol.
31 No.
Table 2.
Mean p38 MAPK activity in the intervention groups According to ANOVA, mean aortic p110 PI3K expression showed significant differences between intervention groups .
<0.
From TukeyAos test it was concluded that the high fat diet significantly decreased p110 PI3K expression, in comparison with the standard diet .
<0.
Administration of catechins significantly increased p110 PI3K expression in comparison with the high fat diet .
<0.
but reached the expression level of the standard diet only at the doses of 6 mg/day and 24 mg/ day (Table .
As shown in Table 2, aortic p38 MAPK activities differed significantly between intervention groups .
<0.
TukeyAos test indicated that the high fat diet significantly increased p38 MAPK activity in comparison with the high fat diet .
<0.
Administration of catechins at various doses was shown to effect an exceedingly significant reduction in p38 MAPK activity in comparison with the high fat diet .
<0.
, and even reached the p38 MAPK activity level of the standard diet.
DISCUSSION