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Prevention of High Altitude Pulmonary Edema in Mice by Ethanol Extract from Portulaca Oleracea and Its Mechanism

Author: TanYue
Tutor: LiMin
School: Second Military Medical University
Course: Nutrition and Food Hygiene
Keywords: Ethanol Extract from Portulaca Oleracea high-altitude pulmonary edema nuclear factor-kB inflammation
CLC: R285.5
Type: Master's thesis
Year: 2013
Downloads: 14
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Abstract


Acute mountain disease (AMD) is known as a series of syndrome which is the most common symptom after rapid ascent to high altitude. It is reported when persons reached altitude over4000m, the incidence of AMI was65%. AMI is the term for acute mountain sickness (AMS), high altitude cerebral edema (HACE) and high altitude pulmonary edema (HAPE). HACE and HAPE is based on AMS do not get effective treatment, so as to develop characteristic obvious symptoms, severe life-threatening disease. HAPE is a lethal form of high-altitude disease. Due to edema, may lead to abnormal alveolar cavity fluid accumulation, hinder the pulmonary ventilation function and diffusion of oxygen, and together with abnormal sympathetic nervous activity so the tissues can not get enough oxygen to complete physiological necessary energy metabolism. In this case, Body will die of progressing dyspnea in a few hours. According to data display the incidence of HAPE is higher than HACE, with as many as10%of people who ascend rapidly to high altitude developing the condition. At the same time, although several decades years of effort, besides relieving symptoms people are not cure of HAPE, and the research of HAPE prevention received widespread attention.Because the precise mechanism of HAPE has not been elucidated, pharmacologic prophylaxis is recommended method to prevent HAPE. At present, drugs are used to prevent HAPE include two sorts, western medicine and traditional Chinese medicine. Western medicine such as Benzedrine, Acetazolamide and Dexamethasone can alleviate the symptoms to some extent, but the side effects are limited in their use; Lactones ginkgo, ginseng saponins and rhodiola as traditional Chinese medicine can improve hypoxia tolerance ability. Their lack of resources and the expensive price are difficult to be popularisation. So a drug which is good prevention, number of sources and low side effects is necessary.The Portulaca oleracea L.(PO) belonging to Portulaceae and Portuaca is one of the wild plants not only as a kind of food but also a drug approved by Ministry of Public health. In our previous research, we found that the ethanol extract of Portulaca oleracea L.(EEPO) significantly extended the survival time of mice in closed environment and protected against hypoxia-induced neuro damage. Moreover, recent pharmacological studies have reported that a wide range of pharmacological effects of PO, such as anti-inflammatory, anti-oxidant, anti-aging, protective effect on vascular endothelial cells and so on. Therefore, the aim of the present study is to investigate prevention of HAPE in mice by EEPO. Furthermore, we studied the possible mechanism by which EEPO ameliorates pulmonary permeability.ObjectiveTo study the prevention of hypobaric hypoxia induced high-altitude pulmonary edema in mice by EEPO and the possible underlying mechanism by which EEPO ameliorates pulmonary permeability.Method1. The replication of HAPE model in mice1.1Experimental animals and treatmentThe70male Bal b/c mice weighting18to20g were randomly assigned to one control group and six model groups(n=10) according to body weight after the first5days’ acclimation. All mice of the model groups exposed to different altitude of5000m,6000m and7000m in hypobaric chamber for different periods of time, namely3h and6h, while the mice of control group were in normal atmosphere. Mice were sacrificed in fast state after exposure.5mice in each control and model group were anesthetized and lungs were washed with PBS through pulmonary artery. The lungs were removed and tracheal cannula was placed.0.9%NaCl lml was gently instilled into the lungs through tracheal cannula, with drawn and reinstilled4times. Other5animals were anesthetized and lungs were washed with PBS through pulmonary artery and removed. The right sides of lungs preserved in10%neutral-buffered formalin and for histopathological analysis and the lift sides were weighted as wet weight. After72h in calorstat at80℃, the lungs were weighted again as dry weight.If laboratory animals have been dead in experimental process, the group will be repeat again, and the dead mice were in mortality.1.2MortalityMortal ity=dead mice/exposure mice*100% 1.3Determination of wet-to-dry ratioWet-to-dry ratio=wet weight/dry weight1.4Determination of total protein in BALThe concention of total protein was determined using a commercially available kit according to the manufacturer’s protocol.1.5Lung histopathological AnalysisLungs were fixed with10%neutral formalin, embedded in paraffin, and stained with hematoxylin/eosin and analyzed via microscopy.2. Protective effect of EEPO on hypobaric hypoxia induced HAPE in mice2.1Preparation of EEPO and SilymarinEEPO was prepared and provided by Department of Traditional Chinese Medicine, Changhai Hospitial, Shanghai. EEPO was dissolved in0.9%normal saline and prepared three different doses (2.0g/ml,4.0g/ml,8.0g/ml).Ginsenoside was provided by Department of Traditional Chinese Medicine, Changhai Hospitial, Shanghai. Ginsenoside was dissolved in0.9%normal saline (8.0g/ml).2.2Experimental animals and treatmentAfter the first5days’ acclimation, the90Male Bal b/c mice weighting18to20g were randomly assigned to six groups(n=15):normal control group (normoxia and normal saline), HAPE model group(hypoxia and normal saline), ginsenoside group (hypoxia and8.0g/ml ginsenoside), low-dose EEPO group(hypoxia and2.0g/100g EEPO), medium-dose EEPO group(hypoxia and4.0g/100g EEPO) and high-dose EEPO group(hypoxia and8.0g/100g EEPO).All mice in EEPO groups were given EEPO solution2.5ml/100g (body weight) in the morning while the mice of control group were given normal saline and the mice of ginsenoside group were given ginsenoside solution2.5ml/100g (body weight) for7days. Mice were exposed to altitude of6000m for6h. Mice were sacrificed after exposure.5mice in each group were anesthetized and lungs were washed with PBS through pulmonary artery. The lungs were removed and tracheal cannula was placed.0.9%NaCl1ml was gently instilled into the lungs through tracheal cannula, with drawn and reinstilled4times. Another5animals were anesthetized and lungs were washed with PBS through pulmonary artery and removed. The right sides of lungs preserved in10%neutral-buffered formalin and for histopathological analysis and the lift sides were weighted as wet weight. After72h in calorstat at80℃, the lungs were weighted again as dry weight. Blood was collected and serum was separated for biochemical analysis.2%Evans Blue solution was injected into mice30minutes before tissue collection. Lungs were perfused free of blood with PBS, blotted dry, and weighted. Lung tissues were homogenized in PBS and incubated formamide for48h. The optical density of the supernatant was determined at620nm.2.3Determination of wet-to-dry ratioSame as in part1.2.4Determination of total protein in BALSame as in part1.2.5Determination of vascular permeabilityThe Evans Blue in lung homogenate was exposed as μg of Evans Blue dye per g lung tissue.2.6Lung histopathological AnalysisSame as in part1.3. Study on mechanism of the protective of EEPO on HAPE in mice.3.1Experimental animals and treatmentSame as in part2.3.2Determination of biochemical parametersThe Malondialdehyde (MDA), Reduced glutathione (GSH) and super oxide dismutase (SOD) were determined using commercially available kits according to the manufacturer’s protocol. 3.3Determination of pulmonary IKK and p65protein levels in cytoplasm and nucleusIKK and p65protein levels in mice were determined by Western blot.3.4Determination of pulmonary IL-1β, TNF-α, P-selectin, E-selectin, ICAM-1and VCAM-1mRNA levels in miceReal-Time PCR was performed using IQ5Real-Time PCR Detection System. Two step RT-PCR method was performed using Real Time PCR Master Mix. Primers used to analyze all the transcripts have been reported elsewhere.3.5Determination of pulmonary IL-1β and TNF-α protein concentration in miceThe protein concentration was determined using commercially available ELISA kits according to the manufacturer’s protocol.3.6Determination of pulmonary P-selectin, E-selectin, ICAM-1and VCAM-1protein levels in miceP-selectin, E-selectin, ICAM-1and VCAM-1protein levels in mice were determined by Western blot.4. Statistical analysesThe results are expressed as means±SD. Statistical analysis was carried out by using SPSS16.0statistical software. T-test, One-way analysis of variance (ANOVA) and nonparametric test were performed for the analysis of the biochemical indices. Differences were considered significantly at P<0.05level.Results1. The replication of HAPE model in mice1.1Mortality All of mice were survival in the groups of5000m3h and5000m6h. The mortality of7000m3h group is10%. And the mortality of5000m6h,6000m6h and7000m6h groups is15%,35.5%and46.7%separately.1.2Comparation of the ratio of wet-to-dryThe wet-to-dry ratio were higher in5000m3h and6000m3h group compared with control group (P<0.05). A wet-to-dry ratio of7000m3h,5000m6h,6000m6h and7000m6h groups is significantly higher than control group (P<0.01).1.3Comparation of total protein in BALThe total protein in BAL of mice in5000m3h group was no statistical differences compared with control group (P=0.8).6000m3h group’s is higher than control group (P<0.05). Other model groups were significantly higher than control group (P<0.01).1.4Histopathological AnalysisOptical microscope found the lung tissues in model group pathological changed and pathological grading. The animals which exposed to altitude of5000m,6000m and7000m for6h were determined obvious lung injury.2. Protective effect of EEPO on HAPE in mice2.1Wet-to-dry ratioAt the end of experiment, the pulmonary wet-to-dry ratio of mice were significantly higher in HAPE model group compared with normal control group (P<0.05). After oral administration of EEPO for7days, The pulmonary wet-to-dry ratio decreased in low-dose EEPO, middle-dose-EEPO and high-dose EEPO compared with HAPE model group (P<0.05). Ginsenoside group can reduced it compared with HAPE model group (P<0.05).2.2Total protein in BALThe concentration of total protein in BAL was significantly higher in HAPE model group compared with normal control group (P<0.01). Protein concentration levels decreased in low-dose EEPO, middle-dose-EEPO and high-dose EEPO compared with HAPE model group (P<0.05). Data showed that the EEPO had a dose-dependent effect on decreasing total protein in BAL. It was also decreased in silymarin compared with HAPE model group (P<0.05).2.3Vascular permeabilityThe results of the experiment shown the vascular permeability of mice were significantly higher in HAPE model group than normal control group (P<0.01). Preventive administration of EEPO lower vascular permeability compared with HAPE model group (P<0.05). It was also decreased in Ginsenoside group compared with HAPE model group (P<0.05). However, there were no difference in vascular permeability between Ginsenoside group and middle-dose-EEPO group.2.4HistopathologicalThe Pathological grading showed the lungs in HAPE model group has obvious pathological damage. EEPO and Ginsenoside made lung tissues to avoid hypoxia damage in different degree.3. Study on mechanism of the protective of EEPO on HAPE in mice.3.1Effects of EEPO on biochemical parameters in HAPE miceThe results indicated that hypoxia exposure up-regulated MDA level and down-regulated GSH, SOD levels (P<0.05). EEPO and Ginsenoside administration significantly reduced MDA level and rose GSH, SOD levels (P<0.05). Data showed that the EEPO had a dose-dependent effect on biochemical parameters.3.2Effects of EEPO on IKK and p65protein levels in HAPE miceAt the end of experiment, in HAPE model group, the protein of IKK in cytoplasm and p65in nucleus were rose by hypobaric hypoxia exposure (P<0.01). Different doses of EEPO can effective relieve IKK and p65protein levels (P<0.05).3.3Effects of EEPO on pulmonary IL-1β and TNF-a mRNA levels in HAPE miceThe expressions of IL-1β and TNF-a in HAPE model group are higher than in control group (p<0.01). Differental administration of EEPO could reduce IL-1β and TNF-α mRNA levels. There is no difference between medium dose EEPO and positive control group. 3.4Effect of EEPO on pulmonary P-selectin and E-selectin mRNA levels in HAPE miceThe real-time PCR results showed that EEPO had remarkable effect on P-selectin and E-selectin mRNA levels compared with HAPE model group (P<0.01). EEPO were administrated before hypobaric hypoxia exposure led to down regulation of mRNA levels.3.5Effect of EEPO on pulmonary ICAM-1and VCAM-1mRNA levels in HAPE miceComparing with control group, ICAM-1and VCAM-1were higher in model group (p<0.01). EEPO could weakened the high levels of ICAM-1and VCAM-1mRNA. At the same time, ginsenoside had effect on reducing their mRNA expressions.3.6Effects of EEPO on IL-1β and TNF-α protein in HAPE miceThe concentration of IL-1βand TNF-a protein increased in HAPE model group (P<0.05). EEPO can reduce higher concentration in HAPE groups (P<0.05), and higher EEPO dose matched lower concentration.3.7Effects of EEPO on P-selectin, E-selectin, ICAM-1and VCAM-1protein levels in HAPE miceAll of pulmonary P-selectin、E-selectin、ICAM-1and VCAM-1protein levels were significantly higher in HAPE model group compared with normal control group (P<0.05). After oral administration of EEPO for7days, pulmonary P-selectin, E-selectin, ICAM-1and VCAM-1protein levels decreased in EEPO groups compared with HAPE model group (P<0.05). EEPO showed a dose-dependent effect on these protein levels.Conclusions1. Obvious lung damage was found in6000m6h and7000m6h groups. The pulmonary edema and vascular permeability also had significantly changed in these conditions. Considering the stability of model and mortality, we decided hypobaric hypoxia exposure at6000m for6h as the following study condition.2. EEPO decreased pulmonary edema and vascular permeability. At the same time, the lungs pathology damage was also improved. Different dose EEPO indicated different levels of HAPE prevention.3. EEPO may affect pulmonary edema by decreasing inflammatory reaction in pulmonary via improving ability of resistance to oxidative stress injury, suppressing activity of NF-kB pathway, and decreasing inflammatory gene transcription and protein expression in hypobaric hypoxia induced pulmonary edema.4. The pathogenesis of HAPE is complicated, and is not accurate clear. The effect of EEPO on of HAPE is still studied. The preventive effect of EEPO on AFL might through not one target. Further study will be necessary.

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