Experimental Study On The Protective Effect Of Ginkgo Biloba Extract On Monocrotaline-induced Pulmonary Arterial Hypertension

BackgroundPulmonary arterial hypertension (PAH) is an extremely malignant disease which refers to the pulmonary artery systolic pressure is over30mmHg, or mean pulmonary artery pressure exceeds25mmHg at reat; mean pulmonary artery pressure higher than30mmHg in the activity, common and severe pathophysiological manifestations always be observed in such diseases, like left to right shunt congenital heart disease, hypoxic pulmonary disease, idiopathic pulmonary arterial hypertension etc. Although the mechanism of PAH has been studied over100years, because of its complex etiology, pathogenesis has not yet been fully elucidated.PAH does not have specific clinical manifestations, but it need to pay attention to differential diagnosis of PAH when the paitents have symptoms with dyspnea, angina, syncope and edema. PAH has been divided into idiopathic pulmonary arterial hypertension (or primary pulmonary arterial hypertension, IPAH) and secondary pulmonary arterial hypertension in clinic, incidence of IPAH is very low, the morbidity of general population is approximately (2-3) cases/1000000, about300-1000patients a year. Non-selective autopsy detection rate is0.08‰-1.3‰which shows more women than men (1.7:1), average age of36years old,6%of patients with familial heredity, and its poor prognosis reveals the average survival time is2-3years, the pulmonary arterial pressure in patients with IPAH can be higher than60mmHg. Secondary pulmonary arterial hypertension is more common, the pulmonary arterial pressure was significantly lower than that of IPAH, rarely higher than40mmHg. Secondary pulmonary arterial hypertension caused by many common diseases which includes persistent pulmonary arterial hypertension of the newborn, congenital heart disease, as left to right shunt congenital heart disease, Eisenmenger syndrome, left ventricular failure, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome (ARDS), chronic pulmonary thromboembolic disease, scleroderma, pulmonary veno-occlusive disease, cirrhosis of the liver, cardiopulmonary bypass surgery ect.Although PAH has lots of causes, pathogenesis is steal unknown, but after clinical and experimental studies have found patients with PAH have occurred pulmonary arterial remodeling in levels of pulmonary artery, the main pathological change is the membrane hypertrophy, dilatation of pulmonary elastic artery and intimal atherosclerosis in muscle, elastic and micro pulmonary artery, the inner, middle and outer membranes of pulmonary artery is thickening, meanwhile the arterial occlusion and concentric intimal thickening have also been occurred in pulmonary arteries. Larger vessels have plexiform lesions and eccentric intimal thickening. Severe pulmonary vascular remodeling have been found refer to prognosis of patients in different stages of the disease.According to the current findings, clinical treatment included general measures, drug therapy and interventional or surgical treatment. General treatment mainly refers to the treatment of the underlying disease, drug therapy includes different kinds of pulmonary vasodilators which have been widely used in clinical:calcium antagonists, prostacyclin derivatives, endothelin receptor antagonists, phosphodiesterase esterase inhibitors, these drugs play a role to block the different specific certain aspects of pathogenesis in PAH to achieve curative effects. Interventional and surgical treatment including percutaneous balloon atrial septostomy, lung transplantation and heart-lung transplantation which includes series of postoperative complications and problems: high risk of surgical, limited indication, particularly donor lung scarce, lifelong oral immunosuppressants treatment and other issues, the surgery always plays the last treatment of PAH.Endothelin-1(ET-1) is an important vasoconstrictor substances in body, and NO generated by eNOS has vasodilatory effects, both of them play an important role in the reconstruction of pulmonary hypertension and pulmonary vascular remodeling.ET-1acts on two main receptors:endothelin A receptor (ETAR) and endothelin B receptor (ETBR), ETAR presents in vascular smooth muscle cells, ETBR presents in vascular smooth muscle cells and pulmonary vascular endothelial cells. In pulmonary hypertension, the two receptor regulate endothelin-mediated vasoconstrictor effects.eNOS is a Ca+and calmodulin (CaM)-dependent enzyme, which has three isoforms:inducible nitric oxide synthase (iNOS), neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS), eNOS had first been found in endothelial cells in1991by the Forstermann and Pollock, which can specific apart L-arginine into generate L-citrulline and nitric oxide (NO), promoting the synthesis of NO. NO plays an important role in the cardiovascular system, including dilate blood vessels and inhibit vascular smooth muscle proliferation, take part in the development of hypertension, cause atherosclerosis etc. NO produce cyclic guanosine monophosphate (cGMP) by vascular smooth muscle cells to relaxing blood vessels: endothelial cells produce NO, NO act on cGS to make GTP become cGMP, cGMP activate cGMP activate kinase, which opens potassium channels in the cell membrane, increasing potassium efflux, reducing calcium influx and intracellular calcium, thus relaxing blood vessels.Ginkgo is biloba ginkgo plants, domestic and foreign scholars have extensive researched its chemical composition and pharmacological effects more than60years, especially ginkgo biloba leaves have attracted most concern. Ginkgo biloba has complex chemical composition, including flavonoids, terpene lactones, phenolic acids and polyisopentenol, they are the main active ingredient of ginkgo biloba to play a important pharmacological activity. Ginkgo biloba extract (EGb) use modern pharmaceutical production to extract active ingredients of ginkgo biloba from aditional Chinese medicine. Ginkgo biloba extract has widespread effect, the main roles include competitive inhibit PAF receptors on the platelet membrane to induce decrease platelet abnormal aggregation and thrombus formation; improve activity of superoxide dismutase (SOD) and anti-free radical effects;, inhibite neutrophil accumulation and decrease the expression of intercellular adhesion molecule-1(ICAM-1) by antioxidant to reduce ischemia-reperfusion injury of the lung; reduce endothelial cell damage caused by intracellular Ca2+concentration, decreased MMP secretion and increased ET extent,; reduce the formation of pulmonary fibrosis through the decrease levels of tumor necrosis factor (TNF-a) and lipid peroxidation (LPO), enhances reactivity of pulmonary ring to5-HT and lower expression of lung tissue matrix metalloproteinase-9(MMP-9) and tissue inhibitor-1(TIMP-1).PurposeThis experimental study explore protective effect of ginkgo biloba extract on monocrotaline induced PAH by the established model of SD rats; observe expression of eNOS, ET-1in lung tissue from SD rats with Ginkgo biloba extract act on PAH model.Method1. establishment of animal model and grouping:30male SD rats with weigh180-200g, being randomly divided into three groups with random number table: control group, pulmonary arterial hypertension model group (PAH group) and treatment group,10SD rats in each groups. PAH model group and treatment group rats were injected back subcutaneously using1%monocrotaline (Anhydrous ethanol as solvent mixture mixed with normal saline to2:8)60mg/kg to replicate model of PAH, on the second days after injection began to gavage of2ml0.9%sodium chloride injection (PAH model group) or60mg/kg of Ginkgo biloba extract(treatment group) each day; the control group was injected back subcutaneously with the same amount of solvent, on the second days after the injection began daily to gavage of2ml0.9%sodium chloride injection.2. specimen collection and measurement of data:on the22days, intraperitoneal narcotic injection with3%sodium pentobarbital30mg/kg, through vein external carotid to measure right ventricular systolic pressure (RVSP) and mean pulmonary arterial pressure (mPAP) by the right heart catheterization. Rats are Blooding to death and determine weight ratio of right ventricle/left ventricle plus septum (RVHI), taking the right middle lobe and left lower lobe lung tissue immediately, observing lung tissue and pulmonary arterioles basic organizational structure and calculate the diameter of50-150μm thickness of wall thickness representing the percentage (WA%) and vascular wall area and vascular cross-sectional area ratio (WV%) by HE staining of lung tissue; Observing the expression of endothelial nitric oxide synthase (eNOS) and endothelin-1(ET-1) in the lung tissue of rats and take the positive rates by immunohistochemical methods.Result1. The change of hemodynamic results:Both vaules of RVSP and mPAP values in PAH model group (39.2±6.10mmHg,23.0±2.54mmHg) and treatment group (31.6±2.68mmHg,20.6±2.55mmHg) were higher than control group (24.2±2.39mmHg,12.5±1.43mmHg), but the treatment group lower than the PAH model group, the differences were statistically significant (P<0.05). The values of RVSP and mPAP from high to low are:model group> treatment group> control group. RVHI in both of PAH model group (40.5±0.08%) and treatment group (36.9±0.02%) were significantly higher than control group(25.8±0.04%), the difference was statistically significant (P<0.05), but the difference between the PAH model group and the treatment was not significant (P=0.117>0.05).2. Observation of HE tissue section:Observation from light microscopy, in the control group pulmonary artery endothelial cells keeped flat, cell distribution was uniform, vessel lumen and intact alveolar structure were complete; In PAH model group, pulmonary endothelial cells were swelling and shedding, vascular smooth muscle cells were prominent proliferation and hypertrophy significantly, vessel lumen were irregularly thickening, stenosis and occlusion, the alveolar cavities wre infiltrated by a large number of inflammatory cells; In the treatment group, thickness of vessel lumen was between the control group and the PAH model groups, partial endothelial cells were loss, proliferation of vascular smooth muscle cells were lighter than the PAH model group, alveolar infiltration with inflammatory cells were also seen in the view, but degree of inflammatory cell infiltration was lesser.3. Observation of pulmonary vascular remodeling indicators:using Image-Pro Plus6.0pathological image analysis system observed small pulmonary arteries in diameter from50to150μm, measuring pulmonary arterial intravascular and extravascular circumference, calculating the percentage of wall thickness and vessel diameter (WA%)=(extravascular circumference/2π-intravascular perimeter/27π)/(extravascular circumference/2π)×100%and ratio of vascular wall area and vascular cross-sectional area ratio (WV%)=(extravascular circumference2/4π-intravascular perimeter2/4π)/(extravascular circumference2/4π)×100%, the values of WV%and WA%were calculated. Compared with the control group, the values of WA%and WV in PAH model group and treatment group was significantly higher, the difference was statistically significant (P<0.01). while the treatment group compared with the PAH model group, the values of WA%and WV in treatment group was significantly lower than PAH model group, the difference was statistically significant (P<0.01).4immunohistochemical staining with eNOS and ET-1expression and scores of positive rates:lung tissue use immunohistochemical staining of eNOS and ET-1and immunohistochemical positive rate scores by Rahman, positive rate scores in the three groups were analyzed by immunohistochemical with statistically pairwise comparison, the result showed that pairwise comparisons of positive rate scores of eNOS and ET-1were statistically significant in three groups(P<0.01). In three groups, the positive rate of eNOS strength as follows:control group> treatment group> PAH model group, the positive rate of ET-1from high to low:PAH model group>treatment group>control group.Conclusion1Monocrotaline can successful induced pulmonary arterial hypertension model with back subcutaneously injection in SD rats.2By observation of hemodynamic change and pulmonary small artery remodeling index, Ginkgo biloba extract can reduce right ventricular systolic pressure, mean pulmonary arterial pressure and pulmonary vascular remodeling to delay the formation of pulmonary arterial hypertension.3Ginkgo biloba extract can reduce endothelial cell injury, maintain expression of eNOS and restrain the synthesis of ET-1to slow the progression of pulmonary arterial hypertension.