Injuries Of High Altitude-cold Environment On Endothelial Cell And Effects Of Targeted Compound

Background:The plateau covers an extensive area in China, and most of them at our borders,their strategic significance are very important. The environmental characteristics ofHypothermia and hypoxia caused by the special geographical environment andgeomorphological features. In the plateau region, plateau environment will not onlydamage to human health, but also affect the mobility, combat capability, trainingcapacity of the troops, and therefore, solve the health issues of the plateau soldiers is anecessary part to enhance the army’s combat effectiveness. When people entered theplateau, due to the oxygen concentration plummeted, to make a series of pathologicalchanges in the body, susceptible to high altitude pulmonary edema (HAPE) and highaltitude cerebral edema (HACE), while the people long-term living in the plateau,because of long-term hypoxia compensatory response, susceptibility to pulmonaryhypertension, the plateau heart disease, and the initiating factor of all the diseases isthe disorder of the endothelial cells. In addition, in the cold environment of plateau,the time of field work may cause systemic hypothermia (35～28℃), it manifests asperipheral blood contraction, blood perfusion reduced, limb susceptible to frostbite, atthe time the first by the injury is stillendothelial cells. Therefore, it becomes the newstrategies for prevention and control plateau diseases that the protection of endothelialcells, the reversal of endothelial cell dysfunction. Natakalim(Nat) is a lead compoundswhich can specifically activate the SUR2B/Kir6.1subtypes of KATP. Previous studieshave proved iptakalim has a positive effect on high altitude pulmonary edema (HAPE)and high altitude cerebral edema (HACE). Now we have known natakalim canimprove the heart failure caused by the pressure overload through reversalsing the ofendothelial function disorders, but the impact on natakalim to hypoxia-inducedendothelial cell damage has rarely been studied. Therefore, researching the effects andmechanisms of hypothermia, hypoxia and complex conditions on the body, and researching the effects and mechanisms of New endothelial cell protective drugsagainst hypoxic injury has an important significance.Objective:Research into the damage rule of the rat thoracic aorta endothelial cells (RAEC)in moderate hypothermia, hypoxia, compound of hypothermia and hypoxia conditions,use the2×2factorial analysis method to clarify the differences between compositeinjury and single-factor injury, and to clarify the main influencing factors in complexconditions, to provide a theoretical basis for the further understanding of the specialenvironmental damage. In addition, homocysteine (HCY) and hypoxia as an injuryfactor, to research in the protective effect and mechanism of the drug on endothelialcells, aim at natakalim which open KATPchannel SUR2B/Kir6.1.Methods:Using tissue explants adherent culture, cultured in vitro SPF male wistar ratthoracic aorta endothelial cells, cell viability was measured by MTT assay, using aninverted microscope to observe the cell morphology, determined the active time ofhypothermia injury, create a cell model of hypothermia injury. In21%O2,28℃,30hconditions, study the injury effects of hypothermia to endothelial cell. In3%O2,37℃,30h condition, study the injury effects of hypoxia to endothelial cell. Research Thecombined effects of hypothermia and hypoxia, endothelial cells are divided intocontrol group (21%O2,37℃), hypothermia group (21%O2,28℃), hypoxia group(3%O2,37℃), complex group (3%O2,28℃). Cultureing all the groups at30h byMTT assay cell viability, by nitrate reductase assay the release of nitric oxide (NO),using RT-PCR to detect cell adhesion molecule-1(ICAM-1), vascular endothelialgrowth factor (VEGF), endothelin-1(ET-1) gene expression levels; hochst andTUNEL to detect apoptosis；western blot to detect HIF-1α protein expression levels;observed cell submicroscopic structure and changes of the organelles. Use thefactorial analysis method to clarify the differences between composite injury andsingle-factor injury, to determine the primary and secondary effects of hypoxiaintervention under the complex conditions of low temperature and hypoxia.In the protective effect research of natakalim on RAEC injury induced byhypoxia, the cells were divided into normal group，hypoxia model group, natakalimdose low, medium, high group, glibenclamide antagonistic groups. Determination of cell viability by MTT assay, nitrate reductase assay the release of NO, by xanthineoxidase (hydroxylamine method) Determination of SOD activity, RT-PCR method todetect ICAM-1and VEGF in ET-1gene expression levels; by ELISA Determinationof ICAM-1, VEGF protein expression levels. Realtime-PCR assay of vascular celladhesion molecule (VCAM-1), monocyte chemotactic factor (MCP-1) and ICAM-1gene expression levels, in the research of the protective effect of natakalim onhomocystein induced RAEC injury.Results:1. Culture and identification of vascular endothelial cells.The rat aortic endothelial cells which were cultured by the method of aorticexplants adherent were identified by CD31immunofluorescence, its positive rate wasabove98%.2. Effects of cold on endothelial cells.The response curve, which was drawed according to the results of MTT, betweenthe hypothermia reaction time and cell survival rate showed: starting from the24h, thesurvival rates of endothelial cells in the hypothermia group compared with the controlgroup began to decrease, and they had a significant difference, after24h survival ratesof endothelial cells continued to decline. In addition, according to the determinationof cell viability and morphological observation. Determined30h as the active time forhypothermia injury, therefore, does incubation of cells30h at28℃as the study ofhypothermia injury model in vitro. The study found that hypothermia has a significantrole to reduce the cell proliferation rate and inhibit the cell NO release, in addition,hypothermia has a certain effect on apoptosis and permeability, but it has certainstimulative effect on the release of inflammatory factors and the expression ofendothelin, in hypothermia hypothermia we can see dilatation of endoplasmicreticulum, even become some bubbles.3. The influence of altitude hypoxia on the endothelial cells.Hypoxia can promote apoptosis, inhibit the release of NO, enhanced theexpression of inflammatory factors and endothelial. It also can lead to vascularpermeability increased, the expression of HIF-1α increased, when the cells are in thehypoxia environment, changes in cell ultrastructure manifested as mitochondrialswelling and rupture, plasma membrane vesicles increased.4. Effects of hypothermia combined with hypoxia on endothelial cells. The composite factors of hypothermia and hypoxia have interactions in respectsof cell survival rate, releasing of endothelial active factors(NO, ET-1), expression ofinflammatory factors, HIF-1α expression, cells apoptosis, arganelles change. There isno interaction in respects of permeability. As follows: build a modeling, whenendothelial cells up to60%, MTT results showed that the cell survival rate of thecombined group are lower compared with control group, hypoxia group andhypothermia group, indicating that hypothermia and hypoxia have positive interactiveeffects on cell viability,.under the complex conditions，hypothermia is the main factor,has more significant impact on cell survival. Build another modeling, whenendothelial cells up to100%, the cell survival rate of combined group are lowercompared with control group, and there is no significant difference compared withhypothermia group and hyoxia group. Hypothermia and hypoxia have no interactiveeffects on cell viability rate, under the complex conditions, hypoxia has a significantimpact on cell viability rate only. In the aspect of NO released, the NO emission of thecomposite group dropped significantly compared with the control group, hypothermiagroup and hypoxia group, indicating that hypothermia and hypoxia have positiveinteractive effects on the release of NO, under the complex conditions, hypothermia isthe main factor, has more significant impact on the release of NO. In the aspect ofapoptosis, there were both the phenomenon of apoptosis, under hypoxia conditionsand complex conditions, and the apoptotic cells of the composite group were less thanin the hypoxic group, indicating that hypothermia and hypoxia have negativeinteractive effects on apoptosis. Under the complex conditions, hypoxia is the mainfactor, but hypothermia has a protective effect to apoptosis. In the aspect ofinflammatory factor expression, the ICAM-1gene expression of the combined groupwas increased compared with control group and hypothermia group, but lower thanhypoxia group. Factorial analysis shows that hypothermia and hypoxia have negativeinteractive effects on the ICAM-1gene expression. Under the complex conditions,hypoxia is the main factor, has more significant impact on the expression ofinflammatory factors. In the aspect of cell permeability, the expression levels of thecell VEGF gene of the combined group was significantly increased compared withcontrol group and hypothermia group, but it reduced compared with the hypoxiagroup. Factorial analysis shows that the impact of hypothermia and hypoxia to cellVEGF gene expression was no significant interaction effect, under the complexconditions, hypoxia is the main factor, has very significant impact on VEGF gene expression. In the ecpression of ET-1gene, level of the cell ET-1gene of thecombined group was significantly increased compared with control group andhypothermia group, but it reduced compared with the hypoxia group. Factorialanalysis shows that hypothermia and hypoxia have negative interactive effects on theET-1gene expression. Under the complex conditions, hypoxia is the main factor, hasvery significant impact on ET-1gene expression. In the expression of hypoxia-inducible factor (HIF-1α), combined group of cells of HIF-1α expression levelscompared with control group and hypothermia group is very significant transientincrease, but it reduced compared with the hypoxia group. There is a negativeinteractive effect when hypothermia combined with hypoxia on expression level ofHIF-1α. Under the complex conditions, hypoxia is the main factor, has verysignificant impact on ET-1gene expression. In the aspect of cell ultra structure, suchas mitochondrial swelling and rupture, endoplasmic reticulum has appeared whenhypothermia combined with hypoxia. The complex injuries are more serious thansingle injuries.In short in a composite damage, low temperature in cell proliferation force andNO release quantity as the major influencing factors, and low oxygen composite withbonus effect. Low oxygen in cell apoptosis, inflammatory factor release, permeabilitychanges, HIF-1α for expressed in the main influence factors, and low temperaturecomposite with antagonist function, or low temperature has some of the low oxygeninjury protection.5. Natakalim protective effect of hypoxia induced endothelial cell injuryCultured endothelial cells24h under the conditions of3%oxygen concentration,cell viability was reduced by22.3%compared with the control group, NO releasedecreased by39.9%,61.8%reduction in the activity of SOD, ICAM-1, ET-1, VEGFgene expression were significantly up-regulated (P <0.05, P <0.01); exposure tohypoxia for12hours, the ICAM-1, VEGF protein expression up-regulated(P <0.05, P<0.01). And then0.1,1,10μmol/L, natakalim and endothelial cells were incubated8h, cultured endothelial cells in3%oxygen concentration under the conditions of24h,compared with model group, the three dose groups of natakalim can be reversedhypoxia-induced vascular endothelial cell function changes, including raisingendothelial cell viability and the release level of NO, enhanced the activity of SOD,significantly inhibits hypoxia induced expression of ICAM-1, ET-1, VEGF geneoverexpression (P<0.05, P <0.01) and ICAM-1, VEGF protein expression up-regulated(P <0.05, P <0.01). Use glibenclamide as antagonist incubated withendothelial cells6h, and then10μmol/L natakalim incubation after8h, placed in theincubator oxygen concentration of3%for24h, on the aspects of cell viability, NOrelease, SOD activity, glibenclamide could reverse the protective effect of thenatakalim’s significantly.6. Natakalim protective effect of homocystein induced endothelial cell injury:0.1mmol/L of HCY incubated rat aortic endothelial cells6h, compared with normalcontrol group, and VCAM-1and ICAM-1, MCP-1gene expression levelssignificantly raised.0.001,0.01,0.1,1,10μmol/L natakalim and endothelial cellswere incubated8h, giving0.1mmol/L of HCY incubated6h, compared with modelgroup,0.001to10μmol/L, natakalim arecould significantly inhibit HCY inducedexpression of VCAM-1, ICAM-1, MCP-1gene overexpression, but did not show asignificant dose-dependent effect.0.001μmol/L natakalim can play an inhibitory in theover-expression of endothelial cell inflammatory factor caused by homocystein,indicating that natakalim has a potent protective effect to endothelial cells.Conclusion:The injury of hypothermia and hypoxia is different from the single factor injury，there are interaction effects in survival rate, releases of NO, expression ofinflammatory cytokines and ET-1gene, protein expression of HIF-1α, apoptosis; butthere are not interaction effects in expression of VEGF gene. Combined injuries ofhypothermia and hypoxia have additive effect in inhibition of cell proliferation;inhibit the release of NO, played a certain protective effect of hypothermia on hypoxicinjury in the expression of inflammatory factors, ET-1gene, HIF-1α protein, and cellapoptosis. Natakalim，it is a lead compound which use KATPas a target，can reversevascular endothelial cell dysfunction which is caused by homocystein and hypoxic,have potent endothelial protective effects.