The Effects of Telmisartan to Thickness of Aortic Tunica Media in 8 % Sodium Chloride-induced Wistar rats ( Rattus norvegicus )

Non-communicable disease have became pandemic disease in developing countries, such as cardiovascular diseases (CVD), diabetes, chronic obstructive pulmonary diseases (COPD) and cancers. Epidemiological studies have confirmed a positive correlation between salt intake and elevated blood pressure in up to half of patients with hypertension. The fibrotic process is associated with renin-angiotensin-aldosteron system, transforming growth factor-â (TGF-â), connective tissue growth factor (CTGF), matrix metalloproteinases and peroxisome proliferator-activated receptor gamma (PPARã). Telmisartan is antihypertensive drug that not only blocks angiotensin receptor but also leads to the decrease of blood pressure, activates peroxisome proliferator activated receptor gamma (PPAR-ã) and presumeably inhibits expression of transforming growth factor beta-1 (TGF-â1). Does telmisartan inhibit thickness of aortawall in excessive NaCl-induced Wistar rats are studied in this experiment. Twenty five male Wistar 2,5-3 months of age and 100 – 150 g BW rats were used in this experiment. They were grouped into 5, each contain of rats. Group I (G I) as placebo did not receive NaCl 8% nor telmisartan. G II received NaCl 8% only. G III, IV and V received NaCl 8% and telmisartan 3, 6 and 12 mg/ kg BW. The treatments were given every day for 8 weeks. At the day of 56 all rats were sacrificed by mean of neck dislocation and operated to take aorta. The thickness aortic wall were analyzed with imageJ 1.49c. Data were expressed as mean ± standard deviation. They were analyzed by parametric test (ANOVA) that followed by Post Hoc test for comparison of multiple groups or nonparametric test (Kruskal-Wallis). A value of p<0.05 was considered statistically significant. Ratio of aortawall thickness to lumen diameter in rat decreased significantly on telmisartangiven rat groups comparing NaCl 8% only (p<0,05). In conclusion, ratio of aortawall thickness ratio to lumen diameter were higher in aorta of NaCl 8% only male Wistar rats comparing to telmisartangiven male Wistar rats.


INTRODUCTION
Non-communicable disease have became pandemic disease in developing countries, such as cardiovascular diseases (CVD), diabetes, chronic obstructive pulmonary diseases (COPD) and cancers. 1 Epidemiological studies have confirmed a positive correlation between salt intake and elevated blood pressure in up to half of patients with hypertension. 2per tension is the most common cardiovascular disease and is a major public health problem in developed countries and developing countries.Although generally easily diagnosed, hypertension can cause death due to complications of untreated.Hypertension caused 6% of deaths in adults in the world. 3 The process of fibrosis associated with the renin-angiotensin-aldosterone system, transforming growth factor-â (TGF-â), connective tissue growth factor (CTGF), matrix metalloproteinases, and peroxisome proliferatoractivated receptor gamma (PPARã). 14Peroxisome proliferator-activated receptor gamma is a nuclear receptor that has three subunits, ie subunits á, â and ã.Some derivatives of lipids, especially unsaturated fatty acids are ligands of this receptor. 15Activation of PPARã will prevent bonding between p-300 proteins with Smad proteins leading to decreased production of TGF-â1 and decrease the incidence of fibrosis. 16n addition, PPARã would prevent bonding between Smad3 protein (R-Smad) with response elements and Co-Smad protein that will inhibit the production of TGF-â1 and decrease the incidence of fibrosis. 17B e c a u s e m o r b i d i t y a n d i n c i d e n c e of hypertension is still high, various types of drugs have been recommended for the control of hypertension. 18 Telmisartan is an angiotensin receptor blocker (ARB) in addition to the unique properties inhibit the angiotensin II type 1 receptor (AT1), also serves as a partial PPARã agonist. 21e aim of this research is to know the effect of telmisartan to prevention of fibrosis aorta of Wistar rats (Rattus norvegicus) that are induced by 8% NaCl.

Animal model maintenance and treatment
Twenty five male Wistar 2,5-3 months of age and 100-150 gram body weight rats were used in this experiment.They were maintained in individual pen and given feed pellet and drinking water adequately.Placed in room temperature 20-24 °C, dark-bright cycle for 12 hours.Before its treatment, animal model was acclimatized for maximal seven days.They were grouped into 5, each contain of rats.Group I (G I) as negative control or vehicle which did not receive NaCl 8% nor telmisartan.G II received NaCl 8% only.Group III, IV and V received NaCl 8% and telmisartan 3, 6 and 12 mg/ kg BW.The treatments were given every day for 8 weeks.

Telmisartan suspension
Fourty or eighty miligram telmisartan was crushed by mortal, and then add water until 40 mL for 40 mg telmisartan and until 80 mL for 80 mg  telmisartan.Telmisartan suspension was taken by orogastric gavage suitable to rats dosage that have been determined to be entered directly to rat stomach.

Histological analysis
Histological picture of aorta stained by picrosirius red.Aortawall collagen fraction volume and ratio of thickness aortawall to lumen diameter was determined with ImageJ software. 24

Histopathology preparation
Tissue of aorta was fixed by 10% buffer formalin for 24-48 hours at room temperature, and then dehydration was committed respectively on 75%, 80% and 95% alcohol each 45 minute followed by 100% each for 1 hour with 3× repletion.Furthermore clearing process was executed by xylene each for 1 hour with 2× repetition and then tissue was infiltrated by paraffin each 1 hour with 3× repetition.The next process, tissue was blocked in paraffin block.Tissue that had been blocked then sectioned with 4-8 µm thickness.Tissue that had been sectioned then transferred to usual object glass for picrosirius red staining.Object glass was allowed to dry for a night and keep object glass until ready to be used.

Picrosirius red staining procedure
Aorta sections were deparafinized then continued to hydration process with water.Aorta sections were entered into A reagen (picric red sirius) for 1 hour.Furthermore, aorta sections were entered to absolute ethyl alcohol 3×, and then entered to xylene for 15 minutes.Aorta sections were washed and then mounted by Canada balsam.

Statictical analysis
Data are expressed as mean ± standard deviation.They are analyzed by parametric test (one way ANOVA) that followed by post hoc test for comparison of multiple groups or nonparametric test (Kruskal-Wallis).A value of p<0.05 was considered statistically significant.

Effect 8% sodium chloride and telmisartan to ratio of aortawall thickness to lumen diameter in rat aorta
All group aorta sections have been stained by picrosirius red.Aortawall thickness to lumen diameter ratio in aortic rats were quantified with ImageJ software.Aortawall thickness to lumen diameter ratio in aortic rat measurement results in Figure 2 and Table 1.
Kruskal-Wallis test showed significant difference (p <0.05).Analysis Mann-Whitney test showed the group II had ratio of aortawall thickness to diameter lumen more thicker than the group III, IV, and V with significance p<0.05.

DISCUSSION
Xu and Liu (2013) showed that telmisartan can lower systolic arterial pressure by blocking the angiotensin II receptor I, activate P13K/Akt/eNOS and AMPK pathways, increase the release of NO and decrease oxidative stress in SHR rats. 27ng and Sanders (1999) showed that endothelial cells and the production of TGF-â1 glomeruli in vivo is modulated by dietary salt which aortic endothelial in rats feeding with saline containing 8% causes increase in the expression of TGF-â1. 28 Nataatmadja et al. (2013) showed that the administration of losartan, an angiotensin receptor blocker drugs, can decrease the expression of TGF-â on intravesikular and ekstravesikular in cultured vascular smooth muscle of aortic aneurysm. 29 Wu  et al. (1997) showed that the mice that carried the subtotal nephrectomy increased TGF-â1 gene transcription in the glomerulus and tubulointersisial, where the provision of valsartan and ramipril can reduce the expression of TGF-â1. 30In this research showed that the collagen volume fraction were not different in all groups.Zhu et al. (2004) showed that the administration of sodium reduces renin and angiotensin II in contrary receptor of angiotensin I increased. 31In normotensive Wistar and WKY rats administration of excess salt can cause an increase in the mass of the left heart ventricle, remodeling and fibrosis in the heart and kidney. 32Telmisartan blocks the angiotensin receptor, but also can serve as a PPARã partial agonist. 33Angiotensin II is known to stimulate the production of TGF-â and plasminogen activator inhibitor-1, which causes an increase in the accumulation of matrix. 34Zhang et al. (2014) showed that telmisartan can inhibit collagen synthesis and metabolic imbalance of collagen. 35n this study it was found that the ratio of aortawall thickness to lumen diameter more thicker in group II than group III, IV, and V (p<0.05),thus telmisartan can inhibit aortic wall thickness.Shang et al. (2012)  showed that telmisartan can inhibit the increase in the ratio of the thickness of the tunica media with the lumen (p <0.05). 36Increase of thickness of the aortic wall due to cell hypertrophy in vascular smooth muscle cells that showed by Gu et al. (1998) that administration of high concentrations of sodium can cause hypertrophy in both cells in myocardial cells and vascular smooth muscle cells. 37Cell hypertrophy suspected of protein synthesis through increased influx of sodium leads to exchange of Na+/H+, so that the cell becomes alkaline, where the pH is high increase in cell growth in several cell types. 38