PI3K/Akt Path Way Of Rat Cerebral Endothelial Cells Activated By Hypoxia And Regulation Neurovascular Unit

Part one Expression of PI3K/Akt pathway in rat brain microvascular endothelial cells after hypoxiaObjectiveCurrently, compared with neurodegenerative diseases in the aging brain, pathogenesis of cognitive disorders in the development brain are not exactly the same. From the current information available, most research place particular emphasis on the social and biological influences that contribute to cognitive development. However, the study on molecular mechanism of cognitive dysfunction especially the signal transduction pathway based on the feature of development brain is less.PI3K/Akt signal channel is a classic anti-apotosis and promoting survial signal transduction pathways. Recent studies found that PI3K/Akt channel is connected with learning and cognition. In the chronic cerebral ischemia model, detected that Akt and PTEN expressing in the blood endothelial cells were closely related to the degree of learning and memory injury, and this demonstrated that Akt and PTEN on CMECs can influence the ability of learning and cognition by means of regulate the microenvioment of cerebral.This study objected to establish a hypoxic model of CEMCs in vitro. Firstly find the change of PI3K/Akt signal pathway after hypoxic, secondly observe the activities of CMECs intervened by LY294002, a inhibitor of PI3K/Akt signal pathway, MethodsCulturing of CMECs, and establish its in vitro hypoxia model. Set up a normal control, a hypoxia group, a LY intervented group, and a hypoxia with LY294002 intervented group. LY294002 is the classical inhibitor of PI3K/Akt pathway which can inhibit the activation of PI3K. Measure cells'activity by MTT and detect cells'apoptosis by Annexin V-FITC. Using immunofluorescence and western blot technique, to observe and test the protein expression of sevsral interrelated factors in PI3K/Akt pathway,contain Akt,P-Akt,P-PTEN,P-FOXO1.ResultsSuccessfully cultured CMECs, and successfully established the invitro hypoxia model of the two cells. The inhibitoty rate of CMECs caused by LY294002, was positively correlated to the concentration and intervene time. The result of MTT and Annexin V-FITC demonstrated that put the cells in the anoxic box for 6 hours can activate the cells degranluation, and the cells viability was well. Hypoxia CMECs treated with LY294002 whose cell viability and apotosis had obvious difference compared with the control and hypoxia groups. The result of immunofluorescence and western blot technique demonstrated that the PI3K/Akt channel was activated and the express of some interrelated proteins channged. There was an inhancement in the expression of P-Akt, P-FOXO1 and a lower expression of P-PTEN in the hypoxia group while compared with the control. In the LY group,the expression of P-Akt, P-FOXO1 were decreased while P-PTEN increased.ConclusionOxygen-deficience can activate the PI3K/Akt path way of CMECs, and the protein expression of the intertrelated factors on the path way changed. LY294002 can inhibit the cell viability of CMECs, and the inhibitoty rate was positively correlated to the concentration of LY294002 and intervene time. Part twoThe effetc of difference kinds of conditioned mediums of CMECs on normal or hypoxic damaged neuronsObjectiveSynaptic transmission and.its plastically in brain plays a key role in the cognition. Information transferring and disposing among synaptic of neurons is the primarily progress of cognition. However, the internal environment, what neurons lived by, is the basis of neurons to function. NVU is a dynamic micro-environment structure which composed of several cells. Cerebral micro vessel endothelial cells (CMECs) is one of the most important key-component of NVU, effective regulation of the functional status of the CMECs contribute to changes in morphology and function of other cells, especially the neurons, and then adjust the balance of NVU's micro-envioment. Therefore, the balance of NVU's micro-envioment in revelant parts of brain is taken for the key link of cognition and its development. Cerebral hypoxia and ischemia in newborn is one of the most common cause contribute to children's cognitive impairment. Up to now, the mechanism of CMECs regulate the microenviroment of barin cognitive-related parts after brain hypoxia and ischemia is unknown, and the relationship between CMECs and cognition or cognitive dysfunction is not clear.We objected to study the effects of difference kinds of conditioned mediums of CMECs on normal or hypoxic damaged neurons, and to probe into the regulation of viability and apoptosis of neurons.MethodsCulturing of neuron cells, establish its in vitro hypoxia model. Collect the conditioned medium from four different cultured endothelial cell groups, which were normal CMECs, the normal ones treated with LY, the injured ones damaged by cerebral hypoxia, and the injured ones treated with LY. Then the conditioned medium were added into the cultures of normal and hypoxia injured neurons. The effect of each type of conditioned mediuln on the activities of neurons was determined through the measurement of MTT, and the apoptosis of neurons was tested through the measurement of Hoechst33258 dyeing. ResultsSuccessfully cultured neurons. The different conditioned medium of CMECs, can put different effect on the neurons. The conditioned medium of normal CMECs can help promote survival and anti apotosis of neurons. However, the conditioned medium of hypoxia CMECs would aggravate the damage.ConclusionDifferent conditioned medium of CMECs can put different effect on the neurons. Changes of NVU's microenvioment can effect the neuron' cell viability, and the CMECs can secrete some bioactive factors and via PI3K/Akt path way to regulate the neuron.