Effects Of Total Flavones Of Hippophae Rhamnoides On Cardiovascular Remodeling And Vasomotor Response In Hypertensive Rat

Objective:To observe the effects of total flavones of hippophae rhamnoides (TFH) on vasomotor response and cardiovascular remodeling and its possible mechanism in treating abdominal aortic coarctation-induced hypertension rats. Methods:(1) Experimental hypertensive rats were established by partially banding abdominal aortic artery and were randomly divided into Control group(n=15, potable water 10 ml·kg-1), TFH group (n=15,30 mg·kg-1), Captoprill(CP) group (n=15, 100 mg·kg-1),Com group (n=15, TFH 20 mg·kg-1+Captoprill 60 mg·kg-1) for 6 weeks with sham-operating as Sham group(n=15, potable water 10 ml·kg-1). (2) Using carotid artery cannulation, testing blood pressure before operation, 1week after operation and 7week after operation by Powerlab. (3) Measurement of cardiac left ventricular mass, left ventricular weight/body weight (g/100g):When the rats were anesthetized, Immediate removal of rat heart, cut the pulmonary vein, cut the pulmonary artery from 2mm above the pulmonary valve, cut on the aorta and inferior vena cava in the turning point of visceral pericardium. After removing the heart washed with 0.1mol·L-1,PH 7.4 PBS, dry with filter paper, weighing, isolated atrial and ventricular along the mitral valve plane, cut off the right ventricle, weighed. Calculation of left ventricular weight/body weight (g/100g) ratio. (4) Observe heart and thoracic aorta of rats pathological structure, calculate thickness of thoracic aorta:The rat left ventricle, thoracic aorta, in 4% formaldehyde fixed, paraffin embedded, sliced, HE staining, microscopy. Randomly selected sections for each rat five vessels to the vessel wall thickness of the membrane 3,6,9,12 point average as an indicator of vascular wall thickening, measured using computer image analysis software IPP6.0 statistics.(5) Testing plasma concentrations of nitric oxide:Acquisition of right ventricular blood 6ml, high-speed centrifugation separated serum,-20℃save ready. Serum tested with the NO test kit, nitrate reductase activity was detected in serum NO. (6) The effects of total flavones of hippophae rhamnoides (TFH) on vasomotor response:1) Preparation and perfusion of vascular ring:When the blood is acquisition, quickly cut above the right renal artery thoracic aorta, placed pass to 95% O2+5% CO2 gas mixture Krebs solution, after carefully removing the connective tissue surrounding blood vessels cut of about 4～5mm wide vascular rings. Isosceles triangle bracket (made of two silver 200μm in diameter, bottom and isometric vascular rings, the angle penetration silk ligation) into the vascular ring, then vascular ring into 10ml Krebs solution containing 37℃constant temperature bath, the bath continued to charge with 95% O2+5% CO2 gas mixture. Thread one end fixed at the bottom of the bath, one end with the tension sensor, sensor is connected with the amplification device, and then enter information into the computer. Avoid excessive traction during the operation to protect the integrity of endothelium. With norepinephrine bitartrate (NE) 10-6 mol·L-1 testing in vitro activity of thoracic aortic rings, select the thoracic aorta, it has response to contraction to the NE10-6 mol·L-1, resting load of 2g, balance of 90 min, every 30 min during the balancing replacement of a Krebs solution.2) Experimental Methods:Before each experiment, vascular rings were pretreated with 40 mmol·L-1KCl to platform of maximal contraction,then washed six times with the Krebs solution. Vascular rings were observed:When reach the maximum tension of pre-contraction platform, by sub-maximal contraction dose (10-5mol·L-1) of the NE, detection of endothelium-dependent vasodilation to acetylcholine (Ach) (3×10-7～10-4 mol·L-1) cumulative concentrations. The maximum contraction amplitude of NE at 10-5 mol·L-1 as the standard of 100%, the percentage of different concentrations of Ach induced vascular tone range to maximum contraction induced by NE reflects the changes in endothelium-dependent relaxation. After L-nitro arginine methyl ester (L-NAME) (10-4 mol·L-1) were incubated for 30 minutes, with the NE10-5 mol·L-1 pre-steady state contraction of vascular, detection of endothelium-dependent relaxation effects by Ach (3×10-7～10-4 mol·L-1). Determination of the contraction of vascular changes in different concentrations of NE (10-7～10-4 mol·L-1), the reaction of induced contraction of aortic rings expressed as a percentage of maximum contraction by 40 mmol·L-1KCl. (7) Detection of aortic endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), nuclear factor-KB (NF-KB) and left ventricular myocardial NF-KB:Thoracic aorta and left ventricular myocardium fixed in 4% formaldehyde, paraffin-embedded sections as 4mm, xylene dewaxing, hydration graded ethanol, PBS wash,3% hydrogen peroxide incubated at room temperature, antigen retrieval, dropping antibodyⅠin specimens, wet box at 4℃overnight, incubation with antibodyⅡ, color with DAB, stained with hematoxylin, by gradient alcohol dehydration, xylene transparency, then, sealed with neutral gum, observation and recording with a microscope. With PBS instead of antibodyⅠas a negative control. Experiment according to kit instructions step. The average optical density value of integrated optical density of positive substance (IOD)/measured tissue area express the changes of organization-related protein expression by IPP6.0. (8) Thoracic aorta and cardiac angiotensin-converting enzyme (ACE) was determined:After open the chest, remove the rat heart and thoracic aorta in normal saline at 4℃, washed twice, remove residual blood and connective tissue,-20℃save ready. Tissue were weighed and cut into pieces,add 4-8 times the buffer plus(Ingredients:0.05mol/L Tris, pH7.4,0.2% bovine serum albumin,75mmol/L NaCl,50μmol/L ZaSO4), homogenized for 30 seconds×3, homogenate at 3900r/min (1800×g) centrifugation for 15 minutes×2, discard supernatant, precipitation add additional buffer,resuspended, filter with gauze, determination of ACE activity in filtrate. Experiment according to kit instructions step. Determination of OD values at 450nm wavelength, according to standard and sample OD values, calculate the content of the samples ACE in rat. Results:(1) TFH and CP significantly reduced systolic blood pressure, left ventricular weight, left ventricular weight/ body weight ratio, aortic medial thickness and tissue ACE activity (all P<0.05)in hypertensive rats,elevated serum NO levels (P<0.05), and there is a synergistic effect of TFH and CP. (2) The results of immunohistochemistry show that:TFH and CP significantly increased the expression aortic eNOS and iNOS (P<0.01), decreased the expression of tissue NF-κB (P<0.01) in hypertensive rats, and there is a synergistic effect of TFH and CP. (3)Endothelium-dependent relaxation in treatment group was significantly increased compared with control group (all P <0.05), no difference between the treatment group. After NO synthase inhibitor—L-NAME pretreatment, on Ach-induced relaxation response, Com group was significantly higher than the Control group (P<0.05). On NE-induced contraction, Com group was significantly lower than the Control group (P<0.05). Contraction of TFH group and CP group less than Control group (P<0.05) when NE concentration in the lower level. Conclusion:(1) TFH has the exact effect of antihypertensive.(2) TFH long-term use can improve vascular endothelial function, reduced vascular sensitivity to vasoconstrictor substances in hypertensive rats. (3) Long-term use of TFH have the exact reverse the effects on the cardiovascular remodeling caused by hypertension. (4)TFH cardiovascular protection mechanisms may be related to improved endothelial function, increased nitric oxide synthase activity and stimulate increased endothelium-derived NO, inhibition of angiotensin-converting enzyme, angiotensinⅡand NF-KB inflammatory pathways.(5) TFH is equal to the cardiovascular protective effect of captopril, two drugs are synergistic. If the combination will be better able to control blood pressure, reversed cardiovascular remodeling.