The Effect And Protective Mechanism Of SLT On The Function Of Astrocyte By Ischemic Hypoxic Damage

Background:Stroke is the primary reason for mortality worldwide,is responsible for approximately 6 million deaths annually and is considered the key reason for long-term disability.Acute cerebral ischemia is caused in the great majority of stroke cases by the occlusion of a supplying arterial vessel.Inflammation and immune responses are considered essential for the development of strokes.The immune system is closely related to crucial events determining the fate of the ischemic brain and the survival of stroke patients.Inflammation may ultimately lead to neuronal cell death by inducing the entry of leukocytes,the release of inflammatory cytokines,and apoptosis.Some reagents that ameliorate ischemia-induced inflammation have a protective effect,suggesting that inflammation may be the crucial mechanism responsible for ischemic brain injury.Astrogliosis has the potential to lead to harmful effects,namely,neuroinflammation,and to interfere with synapse sprouting.Previous studies have suggested that Lipocalin-2(LCN2)acts as a key target in regulating the neuroinflammatory response.However,the underlying molecular mechanisms are still unknown.In the present study,we examined the anti-inflammatory and neuroprotective effects of Ginkgo biloba extract(EGB),a well-known compound with potential immunoregulatory properties in the nervous system.Methods:The first part,the SLT in treatment of acute phase of the mechanism of action of multiple cerebral infarction in the rat study,using the method of rat microspheres to multiple cerebral infarction experimental model,first through metabonomics preliminary evaluation after 24 hours within the SLT and its effective components of ischemia cerebral cortex metabolism;Secondly,the ultrastructure of astrocytes and HE staining were observed by neurobehavioral scoring and transmission electron microscopy.On this basis,according to the organization and the expression of inflammatory cytokines in serum,and qPCR,immunofluorescence,western blot LCN2 observe the astrocyte activation,the specific protein and its induced JAK2/STAT3 signaling pathway,to examine after cerebral ischemia injury of astrocyte activation and inflammatory reaction and the relationship between SLT cortex astrocytes function to ischemic injury.SLT recovery treatment,the second component of the mechanism of action of multiple cerebral infarction in the rat study,using the method of rat microspheres to multiple cerebral infarction experimental model,according to preliminary results,first through evaluation of mirnas genomics convalescence of cerebral infarction in the rat brain tissue ischemia side of mirnas in change,according to the results of early acute stage looking for related target genes,related pathways and SLT possible targets;Secondly,the effect of SLT on spatial memory dysfunction of rats in the recovery period was evaluated by the Morris water maze experiment.On this basis,on this basis,according to the organization and the expression of inflammatory cytokines in serum,and qPCR,immunofluorescence,western blot observation of astrocyte activation of LCN2,specific proteins and induce JAK2/STAT3 signaling pathway of change,to study of astrocyte activation after cerebral ischemia injury and the relationship between inflammatory reaction and the SLT cortex astrocytes function to ischemic injury.The third part,SLT and the mechanism of the main active ingredient anti-ha cell anoxia damage mechanism,selected SLT and the main active ingredient ginkgo biloba extract for cell level study.First,the Human Astrocyte cell culture system was established,and the influence of the two on the vitality of normal cultured HA cells was investigated through CCK8 method.On this basis,according to preliminary basis and reference to establish the HA cell sugar oxygen deprivation(oxygen and glucose deprivation,OGD)damage model,and USES the lentivirus LCN2 express purpose gene transfection method,to detect the contents of cells secrete inflammatory factors change,immunofluorescence,protein immunoblot observation of astrocyte activation of LCN2,specific proteins and induce JAK2/STAT3 signaling pathway,astrocytes SLT after injury of effective components on its influence.Results:In the first part of the experiment,we observed the following results:compared with model group,SLT can significantly reduced cerebral ischemia rats neurobehavioral score score,ginkgo biloba extract can reduce cerebral ischemia rats neurobehavioral score score,but slightly higher score in the SLT group;SLT can effectively reduce the activation and proliferation of astrocyte,SLT group lateral ischemic lesions,a small amount of cortex neurons degeneration,the nucleus pycnosis and karyolysis can alleviate neuronal apoptosis and necrosis is restrained,the more obviously improve brain cerebral ischemia injury in the rat cortex around half dark stripe neurons and synaptic ultrastructure damage;SLT inhibits lateral ischemic cortex and the expression of serum inflammatory cytokines and chemokines in,according to the result of immunofluorescence and western blot,SLT may inhibit the expression of JAK2/STAT3 signaling pathways induced by LCN2 to suppress the inflammatory reaction.In the second part of the experiment,we first found that for rats with cerebral ischemia convalescence,SLT can restore the mirna difference of ischemic side tissues,and can be predicted to be related to LCN2-JAK2/STAT3 pathway.Second,SLT and ginkgo biloba extract can significantly reduce cerebral ischemia of rats caused by spatial memory,cognitive dysfunction and acute phase results similar to that of SLT can effectively reduce the activation and proliferation of astrocyte,SLT group lateral ischemic lesions,a small amount of cortex neurons degeneration,the nucleus pycnosis and karyolysis can alleviate neuronal apoptosis and necrosis is restrained,the more obviously improve brain cerebral ischemia injury in the rat cortex around half dark stripe neurons and synaptic ultrastructure damage;SLT inhibits lateral ischemic cortex and the expression of serum inflammatory cytokines and chemokines,according to the result of immunofluorescence and western blot,SLT may inhibit the expression of JAK2/STAT3 signaling pathways induced by LCN2 to suppress the inflammatory reaction.In the third part of the experiment,SLT and EGB blocked OGD-induced STAT3 activation and the generation of pro-inflammatory cytokines in human astrocytes,and these effects were significantly enhanced by LCN2 overexpression.SLT and EGB downregulated these effects enhanced by LCN2 overexpression.Conclusions:This study demonstrates that SLT and EGB activates astrocytic LCN2 and thereby suppresses neuroinflammation via the JAK2/STAT3 pathway,providing insight into a promising therapeutic strategy for ischemic stroke.