The Protection And Therapeutic Effects Of Polydatin On Experimental High Altitude Pulmonary Edema Model Of Rats And Tts Mechanism

1. Back groundHigh altitude pulmonary edema （HAPE） is a life-threatening acute altitude disease which will be caused by anoxia if entering the plateau too quickly or doing heavy exercise in high-altitude area. The delayed rescue to the patient may cause death. Hypoxia in highland is generally considered one causative factor to HAPE. The rising arterial pressure, the damage of vascular endothelial cell and pulmonary capillary wall, and higher permeability of alveolar epithelial cell are the basic pathology characters of HAPE. The disease has an incident rate between 0.5%-9.9% and it is usually found in area of above 3000 meter-altitude. The higher the altitude goes, the higher the incident rate grows. In China, the area of highland of an altitude above 3000 meters occupies more than one forth in the total territory, thus makes the mechanism of HAPE and its prevention and therapy the hot issues in highland medical research. With the deepening of HAPE research in recent years, the clinical recovery rate has been obviously improved. There are different hypothesis however remaining insufficient to explain the pathogenic mechanism and the change of HAPE’S pathophysiology. The prevention and remedy of HAPE has many disadvantages of high cost, delayed rescue, inefficacious treatment, especially the available medicines which are difficult in selecting the pulmonary hypertension and reducing the pulmonary vascular resistance with some problems of few target points, heavy side-effect. Those factors mentioned above limit the application of these methods. Therefore, to research on the pathologic mechanism of HAPE caused by hypoxia in highland and to find the relative medicines are still an important research topic.With the constantly improvement of the research on naturally occurring drugs, more and more crude drugs and their active ingredients are discovered and recognized gradually. On the basis of the conventional therapy of western medicine, we can bring into the advantage of the naturally drugs and select the safe and effective active constituents. The clarification of the mechanism of these drugs will provide new thoughts to cure HAPE. Polydatin （PD）, isolated from Chinese traditional medicine giant knotweed rhizome which can improve the microcirculation, inhibit platelet aggregation, reduce plasma viscosity, and prevent the lipid peroxidative and pathogenic microorganism. The extracting solution of PD can significantly reduce the pulmonary hypertension caused by hypoxia and has certain selectivity on pulmonary circulation vascular. The prophase research of this experiment also proved that PD reduces the pulmonary hypertension and restrains the revascularization in lung by multiplying the content of NO in serum and lung of the rats and strengthening NOS activity, which illustrates that PD has a superior foundation toward HAPE prevention and cure.This essay is to set up HAPE model on rats by simulating high altitude condition and applying low pressure and hypoxia as well as composite motion, so that we can observe the influence of PD to hemodynamics, haemorheology, active factors in blood vessels and anaerobic cultivated pulmonary microvascular endothelial cells （PMVECs） in the model rats. It will also discuss the possible mechanism of the effect in entirety and cellular level to provide experimental basis for PD’s effect on HAPE and develop new methods for clinical therapy of HAPE.2 Objectives2.1 To observe the influence of different PD dose on Lung Hemodynamic Parameters of high altitude pulmonary Edema rats model （PASP、PADP、mPAP、HR）, blood gas （PaO₂、PaCO₂、SaO₂）, lung coefficient, and the right ventricular hypertrophy index.2.2 to observe the influence of different PD dose on rats’blood rheology, vasoactive substance and content of ET^-1、VEGF in rats’lung tissue, and exploring their mechanism;.2.3 to observe the influence of different PD dose on rats’pulmonary microvascular endothelial cells damage caused by anoxia, and exploring the mechanism of actions.3 Methods3.1 PD’s influence on hemodynamics and blood gas of HAPE rat model3.1.1 SD rats were randomly divided into control groups, HAPE model group (ig.0.5% Sodium carboxymethyl cellulose, 5mL·Kg^-1), PD groups (ig. 20、40、80mg·Kg^-1 PD), positive PF control group (ig.50mg·Kg^-1 PF), respectively. Control group was fed in normal environment. Others were fed in hypopiesis hatch in which the HAPE rat model was set up with an simulated 5000 meter-altitude environment.3.1.2 Measure rats’lung hemodynamics （PASP、PADP、mPAP） by floating right heart catheter. Lead-electrodes will be placed to detect and record heart rate （HR）.3.1.3 Measure left common carotid artery blood’s blood gas （PaO₂、PaCO₂ and SaO₂） with ABL-300 blood gas analyzer.3.1.4 Calculate the lung tissue coefficient after lung tissue moisture content was blotted.3.1.5 Take out the heart of the rat, cut off the atriums, auricle and tissues around them, and separate the right ventricle （RV）. What was left were left ventricle （LV） +septum （S）. Weigh RV and LV+S, calculate the ratio of RV/ （LV+S）, and get the RVHI.3.2 PD’s influence on haemorheology and vasoactive substance of HAPE model rats3.2.1 The methods of rat-grouping, medication and HAPE modeling are the same as that in 3.1.1.3.2.2 Obtain 4 ml blood through arterial cannulation after anesthetizing the rats with 1% Nembutal. Then measure the blood viscosity, hematocrit, erythrocyte aggregation, fibrinogen content, platelet pggregation function and platelet adhesiveness function.3.2.3 Obtain some blood from rat’s artery and measure ET^-1, radio-immunity measure TXA2, TXB2 and 6-keto-PGFla using Enzyme linked immunosorbent assay3.2.4 Pan Mill right lung tissue to homogenate, take supernate after centrifugation and measure ET^-1 by ELISA. Pan Mill left lung tissue to homogenate, take supernate after centrifugation and measure VEGF by ELISA.3.3 The influence of PD on rat lung PMVECs damage caused by anoxia3.3.1 Add 200μl low carbohydrates DMEM （5% fetal bovine serum, 100U/ml penicillin and 100μg/ml streptomycin） in every well of 96 plates while culturing SD rat lung PMVECs in vitro. After incubating 24 h, the cells were divided into normoxia control group （21%O₂,5%CO₂,74%N2） , normoxia + PDA low, medium and high-dose groups(30μmol·L^-1、60μmol·L^-1、120μmol·L^-1) , hypoxia control （21%O₂,5%CO₂,74%N2） , hypoxia + PDA low, medium and high-dose groups (30μmol·L^-1、60μmol·L^-1、120μmol·L^-1) . 3.3.2 Detect PD’s Toxicity on PMVECs under normoxia by MTT.3.3.3 Add proper medium in PMVECs, set normoxia, hypoxia conditions in triple gas incubator, and then MTT measure absorbance of every group.3.3.4 Measure the LDH activity in cell culture supernatant with LDH kit.3.3.5 Addproper medium according to experimental groups and measure VEGF expression by immunohistochemisty by using SABC.3.3.6 Add proper medium according to experimental groups and measure VEGF content by ELISA.4. Results4.1 The influence of PD on hemodynamics and blood gas of HAPE model ratsModel group PASP、PADP、mPAP are averagely higher than normal control （P<0.01）; PD low-dose group PASP、PADP、mPAP are higher than normar control （P<0.01）; PD medium and high-dose groups and PF positive control group can largely reduce pulmonary hypertension （P<0.05 or P<0.01） compared to model group, and its effect equals that of the masccline medicine. The analysis of blood gas shows the results as following: compared with normal control, model group PaO₂ and SaO₂ are reduced （P<0.05） while PaCO₂ hasn’t changed; Compared with model group, PD medium and high-dose groups and PF positive control group can raise PaO₂ and SaO₂ （P <0.05）.The pulmonary weight and coeffient in model rats group have increased respectively 33.91% and 65.83% compared to normal control, which means that the pulmonary edema degree is obvious （P<0.05）. Comparing with that in model group, both weight and coeffient in PD medium and high-dose groups and PF positive control group are reduced （P<0.0 or P<0.01）, while PD medication groups has no obvious difference with PF positive control group （P>0.05）. Comparing with normal control, the right ventricular hypertrophy index of the model group becomes higher （P<0.05 or P<0.01）; Comparing with model group, PD medium and high-dose groups can effectively inhibit the raising of cardiac index of the rat caused by low pressure and hypoxia so that it can prevent the form of right ventricular hypertrophy （P<0.05）.4.2 The influence of PD on HAPE rat model’s haemorheology and vasoactive substanceCompared with control group, the model groups’plasma viscosity, and whole blood high and low viscosity were raised significantly （P<0.01）. Compared with the model group, all PD groups and PF positive control groups have significant difference on plasma viscosity reduction, and high and low viscosity decrease （P <0.05）, while PD low–dose group has a less influence on blood viscosity.Compared with model rats, hematocrit, Platelet Aggregation and Fibrinogen content in all PD groups and PF positive control group were decreased significantly （P<0.01）. Platelet Aggregation and platelet adhesion reaction in all PD groups were decreased significantly （P<0.01）. Blood plasma ET-1 levels and TXB₂ levels in Medium, PD high-dose groups and PF positive control group were decreased obviously. The PGF1αand PGF1α/TXB₂ were increased significantly （P<0.01）.ET-1 contents in Medium, PD high dose-groups and PF positive control group were decreased significantly （P<0.05）. However, they were not reach normal levels when compared with control group （P >0.05）. VEGF contents in PD high-dose groups and PF positive control group were decreased obviously （P<0.05）.4.3 The influence of PD on rat lung PMVECs damage caused by anoxiaIn normoxia condition, cell survival being measured by MTT, has no difference with control group.Compared with normaxia group, the PMVECs in hypoxia control group decreased significantly （P<0.05）. Compared with hypoxia control group, PMVECs activities in all PD groups increased significantly （P<0.01）. While PD dose increases, the anti- hypoxia-inhibition of PMVECs was strengthened with a relation of dose effect.Compared with normaxia group, LDH activity in hypoxia group was increased significantly （P<0.05）. All PD dose group can reduce LDH’s release caused by hypoxia （P<0.05）, with a relation of dose-dependent.Through the inspecting of VEGF immunohistochemisty after dyeing, we found that the normoxia group was not clear; while hypoxia control has a deep dyeing. The dyeing in PD medium and high-dose groups became weakened, which shows that PD has certain restraining effect on PMVECs’VEGF expression caused by hypoxia.The ELISA method was used under low normoxia and hypoxia conditions to detect the change of VEGF content after the interference by PD to PMVECs. The VEGF working curve was set up with capillary as abscissa and density as ordinate. C=-102.1045A+779.4202, r=0.9997. The linear relation stayed well when the content of VEGF was between 0～1000pg/ml. Under hypoxia, VEGF was increased （P<0.01）; Under hypoxia, medication groups’VEGF content went down （P<0.05）; and medication groups has no many difference with control group under normoxia （P>0.05）.5. Conclusion5.1 PD can lower down the pulmonary hypertension of HAPE model rats and improve the pulmonary vasoconstriction condition. PD can raise PaO2 and SaO2 in a shor time, improve the oxygenation, adapt the acute hypoxia, and reduce the pulmonary vascular risistance, thus to release the degree of the pulmonary hypertension and pneumonedema. This is one of PD’s mechanisms of action.5.2 PD can significantly reduce the viscosity of model rats’blood and the adhesiveness of erythrocyte aggregation and platelets, improve the blood circulation, and show an obvious dose-effect relationship. Meanwhile, it largely reduced the TXA2 level in blood plasma, increased plasma PGI₂ and the ratio of PGI₂/ TXA2, and lowered the active factors level of plasma ET-1, lung ET-1, and VEGF in dose-dependent way. The blood circulation improving effect of PD may speed up the blood flow and weaken the vascular resistance when the blood with high viscosity and red blood with reduced deforming force flew through vascular bed in the model rats, and therefore improve the microcirculation of pneumoangiogram, reduce the pulmonary artery pressure of the rats. This is the second one of its mechanism of action.5.3. PD has effect against hypoxia inducing PMVECs oxidative damage and it can directly restrain the adding of VEGF expression caused by hypoxia, which means that PD has a good protection effect to PMVECs in hypoxia condition. This is the third mechanism of action.