Effect Of Tetramethylpyrazine On Pulmonary Edema Induced By Simulated Altitude Hypoxia In Rats

Background and Objective:High altitude pulmonary edema (HAPE) is a severe acute mountain sickness. Becase of its acute onset, rapid development, endangering a large, there will be potentially life-threatening if not treated in time. So far, the methods of prevention and treatment of HAPE still have some shortcomings, such as the high cost and the poor efficacy. With the needs of economic development and national defense in country at high altitudes, it is urgent to develop a new simple, effective control means of HAPE.An increase in pulmonary vascular permeability induced by acute hypoxia is an important pathophysiological step that occurs during HAPE. Endothelial barrier dysfunction induced by reactive oxygen species (ROS) is an important cause of pulmonary vascular hyperpermeability. Therefore, an anti-oxidative stress strategy may be a promising approach to prevent and treat HAPE. Tetramethylpyrazine (TMP) is an active constituent of the herb Ligustium wallichii Franch, a traditional Chinese remedy. It is commonly used in the treatment of a variety of ischemic cerebral and cardiovascular diseases due to its effectiveness and low toxicity. In recent years, a significant body of evidence has developed on the anti-oxidative pharmacological actions of TMP. TMP alleviates kidney and brain damage induced by I/R in rats via the scavenging of oxygen free-radicals, and significantly reduces lipid peroxidation in neuronal cells of the hippocampus in rats with hypoxia. In the meantime, some previous studies revealed that TMP has a protective effect in human umbilical vein endothelial cells (HUVECs) when they were exposed to hydrogen peroxide (H₂O₂) oxidative stress.The purpose of this study was to test the hypothesis that the anti-oxidative properties of TMP inhibited pulmonary vascular leakage in rats subjected to hypoxic conditions. Methods:1. RPMVECs were cultured by peripheral lung tissue-sticking method. Histological sections from peripHeral lung tissue pieces used for cell culture were examined, CD34, lectin from Bandeiraea simplicifolia and factor?related antigen in the cultured cells were quantified by immunocytochemical staining. In addition, the cell morpHology and ultrastructure were observed with inverted optical microscope and transmission electron microscope respectively.2. RPMVEC were randomly divided into normal control group and hypoxia for 2h, 4h, 6h, 12h, 24h and 48h group. The permeability of devices was used to detect cell monolayer permeability. In order to detect the cell permeability coefficient, RPMVEC were then randomly divided into normal control group, 24h hypoxia group, hypoxia + TMP50?g / ml group, hypoxic + TMP100?g / ml group and hypoxia + TMP200?g / ml group.3. RPMVEC were randomly divided into normal control group, control + TMP200?g / ml group, hypoxic group and hypoxia + TMP200?g / ml group. Intracellular ROS was detected by fluorescence microplate reader using ROS assay kit. Real time-PCR and Western blotting was used to detect the HIF-1?and VEGF-A mRNA and protein expression levels.4. We prepared HAPE rat model through hypoxia exposure and the complex movement pattern. After injection of TMP into rats, rats randomly divided into control group, control + TMP120mg/kg group, hypoxia group, hypoxia + DEXA 5mg/kg group, Hypoxia + TMP30mg/kg group, hypoxia + TMP60mg/kg group and hypoxia + TMP120mg/kg group. The rats arterial oxygen tension (PaO₂), arterial pressure, lung water content and pulmonary microvascular permeabilitywere measured,lung tissue for biopsy was performed by HE staining.5. Rats were randomly divided into control group, hypoxic group and hypoxia + TMP120mg/kg group. SOD activity and MDA content in lung tissue, TNF-?and IL-6 levels in serum and BALF, plasma catecholamine levels were measured, HIF-1?and VEGF-A mRNA and protein expression levels of lung tissue in each group were detected by Real time-PCR method, Western Blotting method and immunohistochemical staining. Results:1. Histological sections showed that tissue pieces were scissored from peripHery lung lobes accurately.?factor related antigen and CD34 expression detected by immunofluorescence was positive, and BSI binding test was positive, too. Transmission electron microscope showed lots of protuberance on cell membrane and pinosomes in cytoplasm.2. Compared with the normal control group, RPMVEC monolayer permeability of hypoxic group was significantly increased, and in a time-dependent manner; cell permeability of TMP50?g / ml group, TMP100?g / ml group, TMP200?g / ml is significantly lower than hypoxic group, and in a dose-dependent manner.3. Compared to the normal control group, intracellular ROS, HIF-1?and VEGF-A mRNA and protein expression of hypoxic group was significantly increased. After treated with TMP200?g / ml, cells Intracellular ROS, HIF-1?and VEGF-A mRNA and protein expression levels were significantly reduced than hypoxia treated alone.4. After exposured to simulated high altitude hypoxia in, rats PO₂ was significantly lower than normal control group and pulmonary artery pressure, lung water content and lung microvascular permeability was significantly higher than the normal control group; compared to model group, PO₂ of rats in pre-protection group was significantly higher, pulmonary artery pressure, lung water content and lung microvascular permeability was significantly decreased. Meanwhile the lung biopsy revealed that compared with model group, pulmonary edema of rats in pre-protection group was attenuated.5. Compared to the control group rats, SOD activity, MDA content, HIF-1?, VEGF-A mRNA and protein level in the lung tissue, TNF-?and IL-6 levels in serum and BALF and plasma catecholamine levels of rats in hypoxia group were significantly higher; however, SOD activity, MDA content, HIF-1?, VEGF-A mRNA and protein level in lung tissue, TNF-?and IL-6 levels in serum and BALF, plasma catecholamine levels of rats in pre-protection group were significantly lower than hypoxia group. Meanwhile immunohistochemistry showed that HIF-1?and VEGF-A expression levels of pre-protection group was lower than the hypoxia group. Conclusion:1. Exposure to hypoxia for 24h will lead to an increase in permeability of cell monolayer in RPMVEC, in time-dependent manner; TMP can significantly inhibit the permeability increase in cells under hypoxia in dose-dependent manner.2. TMP exerts its protective effect in RPMVEC through the inhibition of intracellular ROS generation, HIF-1?activation and VEGF expression.3. We successfully established pulmonary edema model under simulated high altitude hypoxia in rats. Pretreated with TMP can effectively attenuate the pulmonary edema under simulated high altitude hypoxia, in dose-dependent manner.4. TMP can be used to prevent high altitude pulmonary edema by the following means:?reducing the activity of free radical lipid oxidative in lung injury caused by altitude hypobaric hypoxia.?inhibiting injury and exudative inflammatory activation, catecholamine release and protecting the structural integrity of cells.?inhibiting the VEGF and HIF-1?expression and regulation process.