The Damage Effects Of Low Temperature Combined With Hypoxia On Rat Pulmonary Microvascular Endothelial Cells

Objective:Research into the damage effect of the rat pulmonary microvascular endothelial cells (PMVECs) in single factor study of low temperature,hypoxia, compound of low temperature and hyoxia conditions, illustrate the differences between single factor and the compound stress injury, clarify the main influencing factors of compound stress, in order to further understand of the mechanism of high altitude pulmonary edema(HAPE).Methods:1.animal experiments:twenty-four healthy male Wistar rats were randomly divided into four groups with six rats in each group. Control group:exposed in25℃,21%O2for24hours; cold expose group: exposed in5℃,21%O2for24hours; hypoxia exposure group:exposed in25℃,10%O2for24hours; cold compound hypoxia exposure group: exposed in5℃,10%O2for24hours. The wet to dry ratio(W/D), pulmonary edema and hemorrhage score, NO synthase (NOS) and ET-1activity in serum, VEGF and caspase-12mRNA in lung tissue were conducted on each animal.2. cells experiments:primary PMVECs were obtained from isolated lung tissues of Wistar rats by complex phosphoesterasum digestion. The PMVECs were separately cultured in normal temperature (37℃), low temperature condition (28℃), hypoxia condition (3%O2), low temperature compound with hypoxia condition (28℃,3%O2) for24hours. Using MTT assay detect cell viability; detecting lactate dehydrogenase (LDH) and superoxide dismutase (SOD) activity; detecting the release of nitric oxide (NO); using RT-PCR to detect vascular endothelial growth factor (VEGF), endothelin-1(ET-1), cell adhesion molecule-1(ICAM-1), interleukin-1β (IL-1β), interleukin-6(IL-6), tumor necrosis factor-α (TNF-α), caspase-12gene expression levels; Using hoechst and TUNEL method to detect apoptosis. Using the one-way ANOVA method to clarify the differences between compound injury and single-factor injury, and the interaction effects between low temperature and hypoxia in PMVECs.Results:1.animal experiments:compared with the control group, the W/D ratio, the activity of ET-1, the VEGF and caspase-12gene expression levels were significant increased and the activity of NOS were significantly lower in the cold exposure group, hypoxia group and the complex group. Cold exposure and hypoxia had interaction effects in the above aspect.2. cells experiments:The monolayer of cultured PMVECs displayed the shape of pavingstone. Compared to the control group, the activity of LDH,the ET-1/VEGF/ICAM-1/IL-1β/IL-6/TNF-α/caspase-12gene expression levels were significant increased in low temperature group, hypoxia group and hypoxia combined with low temperature group. And the levels of NO concentration were reduced in these three groups. But the activity of SOD increased in low temperature group and the complex group, then reduced in hypoxia group. The results of One-wayANOVA showed that low temperature and hypoxia have positive effects on cell viability, the activity of LDH and SOD, the release of NO, the ET-1/VEGF/ICAM-1/caspase-12gene expression. But do not have interaction effects on inflammatory factors gene expression.Conclusions:The injury of low temperature compound with hypoxia is more serious than the single factor injury, the complex stress may be the primary cause of high-altitude pulmonary edema.