| Ischemic stroke is the disease of nerve dysfunction due to disruption of the cerebralblood flow. In old people, the disease incidence is rising, which will do harm to humanhealth, resulting in disability and high fatality rate. According to the survey of the relatedresearch, the number of ischemic cerebral elderly patients has reached around1.2-1.5million, the death reached to0.8-1million in China. In surviving patients, more than half ofthe people have varying degrees of neurological disability. With the increasing aging of thepopulation, the ischemic stroke number will also increase. Therefore, ischemic cerebralstroke has become the current major disease to our health. The study of ischemic cerebralstroke pathogenesis and the related treatment strategies have an important significance inclinic.The current pathogenesis and treatment of ischemic cerebral stroke have proposed thenew concept-neurovascular network. The theory suggest that vascular endothelial cells,nerve cells, glia cells and extracellular matrix surrounding composition such as organiccomplex formed of neurovascular unit. Influence and regulation between complex formed anetwork structure. The concept that ischemic stroke is not a vascular events or single neuralevents, but between blood vessels and nerves, liver cells and the interaction between matrixand cells. Therefore, after ischemic stroke, all the nerve cells and cellular elements shouldbe protected. Previously, much research focus on nerve cells and vascular endothelial cells,but the glia cells are lack in-depth discussion. However, microglia mediated inflammatoryreaction play an important role in ischemic stroke.Microglia can feel hypoxic stimulus signal and activate the corresponding signalpathway under anoxic environment. In this process, it can express the corresponding geneto maintain the cellular stability, and against the low oxygen to the adverse effects. Whencells are under hypoxic signal stimulation, a number of genes can express, includinghypoxia-inducible factor (HIF-1). Studies confirmed that HIF-1could activate the purpose gene transcription, regulate intracellular oxygen balance and play an important role inhypoxia adaptation. In harmful environment, such as lack of nutrition and hypoxia stress,cell can promote HIF-1expression to regulate corresponding gene activation.Autophagy is a kind of lysosomal degradation pathway in survival, development,differentiation and metabolism process. Its main role is to protect the stable environment,and avoid being influenced by outside factors. Much study have shown that autophagy isinvolved in a variety of diseases, such as liver disease, heart disease, muscle disease, cancer,aging, immune disease and neurodegenerative disease. Research reported that autophagychanged significantly and affected the cell pathology and physiology in the process ofhypoxia.However, the biology of microglia in cerebral ischemia hypoxia environment is stillunknown. Can HIF-1be expressed in microglia? Can microglia autophagy be occured?How about the specific condition and regulating mechanism? Whether the interference withthe above link has a protective effect of ischemic neurological function? In order to confirmthe above questions, we design a series of experiments to identify the effect of HIF-1expression of microglia autophagy in cerebral ischemia stroke and the effect of biologicalsignificance.The studies include:(1) In oxygen glucose deprivation model, microglia survival wasdetermined by PI and MTT assays at0-48hs;(2) In OGD model, microglia inflammatorysuch as cytokine IL-8and the TNF-α were detected by RT-PCR and ELISA assays at0-48hs;(3) In OGD model, HIF-1α expression was detected by western blot at0-48hs;(4) InOGD model, microglia survival was determined by PI and MTT assays with inhibitor2ME,YC-1and HIF-1α RNAi to block the expression of HIF-1α;(5) In OGD model, microgliainflammatory such as cytokine IL-8and the TNF-α were detected by RT-PCR and ELISAassays with inhibitor2ME, YC-1and HIF-1α RNAi to block the expression of HIF-1α;(6)In OGD model, microglia autophagy was detected by western blot, laser confocal and flowcytometry;(7) In OGD model, microglia autophagy was detected with inhibitor2ME, YC-1and HIF-1α RNAi to block the expression of HIF-1α;(8) In OGD model, microglia survivalrate was detected respectively with autophagy inhibitor3-MA and BafA1, activator Rapa,and beclin1RNAi;(9) In MCAO model, microglia autophagy in the ischemic area and mice neural function were observed.(10) In MCAO model, microglia autophagy in the ischemicarea and mice neural function were observed after inhibitor3-MA injection.Results:(1) In OGD model, microglia survival decreased at0-48hs;(2) In OGDmodel, microglia inflammatory increased at0-48hs;(3) In OGD model, HIF-1α expressionincreased at0-48hs;(4) In OGD model, microglia survival increased with inhibitor2ME,YC-1and HIF-1α RNAi to block the expression of HIF-1α;(5) In OGD model, microgliainflammatory increased with inhibitor2ME, YC-1and HIF-1α RNAi to block theexpression of HIF-1α;(6) In OGD model, microglia autophagy increased by western blot,laser confocal and flow cytometry;(7) In OGD model, microglia autophagy decreased withinhibitor2ME, YC-1and HIF-1α RNAi to block the expression of HIF-1α;(8) In OGDmodel, microglia survival changed with autophagy inhibitor3-MA and BafA1, activatorRapa, and beclin1RNAi;(9) In MCAO model, microglia autophagy increased in theischemic area and neural function decreased in mice. However, after attenuating autophagywith inhibitor3-MA, ischemic area and mice neural function improved.Conclusion: The cerebral ischemia hypoxic environment promotes microglia HIF-1αexpression, results in the microglia autophagy death and inflammatory response throughbeclin1pathways, and further aggravates the neurological damage. Interfere with theautophagy pathway can inhibit the microglia death and inflammation, provide an idealtherapeutical strategy for ischemic stroke and brain protection. |
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