| Background:Vascular endothelial cells (VECs) not only play a natural barrier role, but also have metabolic and endocritic functions. The dysfunction of VECs is related to many diseases of different systems, such us cardiovascular, respiratory, reproductive, neuroendocrine system,etc. The evaluation methods of VECs dysfunction mainly include detection of the circulating endothelial cells, examination of the active substances produced by VECs, examination of the Ca2+ concentration and mitochondria membrane potential in VECs and so on. The previous studies indicate that VECs are not only"target"cells but also"stimulating"cells. Therefore, to study the mechanisms of VECs injury and prevention is important to the prevention and treatment of above diseases.Hypoxia is an extremely common pathogenic factor for injury of VECs. Angiotensinâ…¡(Angâ…¡) is an important vascular bioactive peptide of rennin -angiotensin system,which has direct constriction effect to vessels of vascular bed. Angâ…¡may also cause the hypofunction of VECs, that is related to hypertension, arteriosclerosis, cardiac insufficiency, pulmonary hypertension and other diseases. In modern aviation activities, accidents or near-accidents caused by hypoxia is not seldom, the research of hypoxia injury and its protection is still important to aviation medicine. Therefore, under the guideline of enhancing the endogenous ability of anti-injury, it is very important to study the protective effects of the vegetative extractives to the hypoxia injury.Objective:To investigate the protective mechanism of ginkgo biloba extract (GBE) to VECs by observing the changes of intracellular free calcium([Ca2+]i), the mitochondria membrane potential(MMP) and the variation of ET content in the VECs culture supernatant in VECs hypoxia injury model. Methods:1. The VEC strain of human aorta was used to establish hypoxia-induced injury model. The cultured VECs were divided into seven groups: control group, simple hypoxia group, hypoxia+GBE group, simple Angâ…¡group, Angâ…¡+GBE group, hypoxia+Angâ…¡group, and hypoxia+Angâ…¡+GBE group.2. The VECs were loaded with Fluo-3/AM, the fluorescence intensity of Fluo-3 was measured to reveal the [Ca2+]i by laser scanning confocal microscopy.3. The VECs were loaded with Rhodamine 123, the fluorescence intensity changes of Rhodamine 123 were measured to reveal the MMP by laser scanning confocal microscopy.4. The change of endothelin content in the cell culture supernatant was measured by radioimmunoassay.Results:1. Compared with control group, intracellular free calcium concentration of the hypoxia group or the Angâ…¡group increased, MMP of the hypoxia group or the Angâ…¡group decreased, and ET content increased in the cell culture supernatant of the hypoxia group or the Angâ…¡group significantly. After combined effects of hypoxia and Angâ…¡, the [Ca2+] and the ET content increased more, the MMP decreased further.2. Compared with corresponding injury groups, the [Ca2+]i and ET content of GBE protective group decreased, the MMP of GBE protective group increased significantly.3. Compared with control group, the [Ca2+]i, MMP and ET content of GBE protective groups did not return to non-injury level, and the differences were significant.Discussion:Ca2+ is an important intracellular messenger of regulating the cell functions, Ca2+ overload is the"final common pathway"of the cell damage caused by many factors. Intracellular Ca2+ concentration is regulated by the ATP-dependent calcium pump, Na+/ Ca2+ exchange and normal Ca2+ channel. Hypoxia causes the inhibition of oxidative phosphorylation function and the sodium-potassium pump dysfunction, and the rise of the intracellular Na+ concentration causes the cell membrane depolarization, which causes the transitory opening of voltage-dependent Ca2+ channel and the internal flow of Ca2+. Meanwhile, Ca2+ is released due to the mitochondrial dysfunction, which causes the overload of Ca2+ in the cell and cause the opening of mitochondrial permeability transition pore(MPT), and induced the decline of MMP. The hypoxia promotes the production of active oxygen, and the excessive production of active oxygen may injure the membrane of mitochondria directly or indirectly, which may lead to MMP decrease. The increase of oxygen free radicals in VECs and the reinforcement of the lipid peroxidation may lead to the enhancement of the ET excretion by VECs. Our results suggest again that hypoxia may lead to increase of intracellular free calcium concentration and ET scretion, decrease of MMP of VECs.Angâ…¡is a kind of strong vessel-constriction factor and it may stimulate the transcription of VEC endothelin precursor and promote the release of ET. ET and Angâ…¡have positive feedback regulatory effect each other. In normal condition, the low concentration of Angâ…¡can maintain the structure and tonicity of vessels; and under pathological condition, the increase of the Angâ…¡concentration may lead to the injury of endothelial cells. Our results showed that Angâ…¡can lead to the increase of intracellular free calcium concentration and ET secretion of VECs and decrease of MMP. The mechanism is not clear completely. Angâ…¡acts on the AT1 receptor of VEC, and increases the IP3, which may induce Ca2+ release from the endoplasmic reticulum; Angâ…¡could inhibit the large-conductance calcium-activated potassium channel, reducing the Na+-K+ ATP-ase activity in the VEC membrane. The Na+-K+ pump dysfunction would cause the transitory opening of voltage dependent calcium channel and the Ca2+ inward. The Ca2+ overload and oxidative stress caused by Angâ…¡could decrease the MMP. From the mechanisms of hypoxia and Angâ…¡-induced injury to the VECs, we can see that oxidative stress is the common characteristics. The different mechanisms of hypoxia and Angâ…¡make biological effects simultaneously, and show synergetic action to increase the Ca2+ concentration and decrease the MMP within the VEC further, the content of the ET in the culturing supernatant liquid increases further.The intracellular Ca2+ aggregation is the important stage of the cell irreversible injury, and the functional change after the VECs injury is the important factor that may lead to related diseases occurring and development. Therefore, avoiding or relieving the VECs injury by oxidative stress and preventing as well as relieving the intracellular Ca2+ aggregation is the key-role for the prevention and treatment of cell irreversible injury. The GBE can inhibit the platelet activating factor activity, remove the free radicals, and act against Angâ…¡to inhibit VEC large-conductance calcium-activated potassium channels, it may activate the Na+-K+ATP-ase activity of VEC membrane, reduce the Ca2+ channel activity, block the Ca2+ inward and inhibit the release of sarcoplasmic reticulum Ca2+, and so effectively inhibit the Ca2+ overload. The decrease of Ca2+ concentration will avoid or decrease the opening of MPT, so the GBE may maintain the MMP and block the over-expression of ET mRNA eventually, which may reach the result of decreasing ET level, and shows protective effects. However, the protective effects are not complete, because the biological activity of GBE is mainly antioxidation, it is not a complete Ca2+ channel block agent, which may partially block the Ca2+ inward; limit effect of Ca2+ pump activation; cannot inhibit the stored calcium release completely; the pharmacological effect may show time-effect and quantity-effect relationship, therefore, the protective effects of GBE to VECs are limited, which indicates that jointly utilize the VECs protection drugs and choose proper drug concentration is the emphasis for the future studies.Conclusion:Upon the results, we can conclude:1. Hypoxia and Angâ…¡may lead to increase of intracellular free calcium concentration and ET secretion and decrease of MMP of VECs.2. Hypoxia and Angâ…¡have synergetic effects on VECs injury, that is, AngII could enhance injury of hypoxia to VECs.3. GBE can prevent VECs from the injury of hypoxia and Angâ…¡partially. |
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