IL-2 Improves Neurological Functions After Subarachnoid Hemorrhage By Promoting The Expansion Of Tregs And Its Anti-inflammatory Mechanism Study

Subarachnoid hemorrhage（SAH）is the third acute cerebrovascular disease whose incidence is second only to cerebral infarction and cerebral hemorrhage.It is an acute clinical disease and the main cause is the rupture of cerebral aneurysm into the subar-achnoid space.The pathological process of SAH experienced two stages:early brain injury（EBI）and delayed brain injury（DBI）.EBI occurs at day 0-3 after hemorrhage,the main pathological mechanisms are elevated intracranial pressure,changes in ion concentration,destruction of blood-brain barrier,neuroinflammation,apoptosis and oxidative stress;DBI occurs at day 3-14 after hemorrhage,the main pathological mechanisms are cerebral vasospasm,cerebral microcirculation disorder,cerebral microthrombosis,neuroinflammat-ion and so on^[1].Numerous studies have shown that neuroinflammation plays an important role in the pathological process after SAH.If it can effectively regulate the neuroinflammatory response after SAH,it can largely inhibit brain injury and reduce neurobehavioral and cognitive impairment.Recent studies suggest that adoptive regulatory T-cell（Tregs）therapy protects against several cerebral vascular diseases inclusive of subarachnoid hemorrhage（SAH）by suppressing peripheral and central inflammation.But the clinical transformation of Tregs therapy requires further research.Furthermore,the effect of Tregs on the microglia polarization remains undefined.In our preliminary studies,we have obtained that exciting results showing that:（1）Adoptive Treg therapy could preserve blood-brain barrier（BBB）integrity and alleviate brain edema by suppressing SAH induced cerebral inflammation;（2）IL-2 could greatly increase the number of Tregs in the periphery after SAH.In this study,we used SD（Sprague-Dawley）rats to make SAH model,to explore whether IL-2 can promote the proliferation of Treg cells to achieve the following goals:（1）IL-2 therapy could alleviate cell apoptosis by suppressing cerebral inflammation after SAH;（2）IL-2 therapy could promote microglia M2 polarization,and then enhance its abilities of phagocytosis and repair.The success of this project will propel the protective researches of Treg cells,and promote the clinical transformation of Treg cell therapy by IL-2.Experiment purpose:（1）To prove the change of Tregs in vivo after SAH.（2）By intraperitoneal injection of different doses of IL-2 into SAH model,we studied whether IL-2 could proliferate Tregs after SAH,and determined the minimum dose of IL-2,which maximized the proliferation of Tregs,was the optimal dose.（3）The best dose of IL-2 was used to treat SAH rats to study whether IL-2 therapy could improve neurological function and behavior after SAH.（4）The best dose of IL-2 was used to treat SAH rats to study whether IL-2 therapy could alleviate neuronal apoptosis by suppressing cerebral inflammation after SAH,and ultimately provide protective effect.（5）The best dose of IL-2 was used to treat SAH rats to study whether IL-2 therapy could promote microglia M2 polarization,and then enhance its abilities of phagocytosis and repair.Research methods:（1）The SAH model we chose was the endovascular puncture model,select the adult male SD rats with a weight of 300g-320g for SAH model.When proving the changes of Tregs in vivo after SAH,rats were randomly divided into the following two groups:sham group,SAH group,sham group and SAH group without any drugs.When studying whether IL-2 could proliferate Tregs after SAH,the SAH model was divided into four groups:vehicle group,IL2-1μg group,IL2-2μg group and IL2-4μg group.The different doses of IL-2 were dissolved in 200μl PBS respectively,then the drugs were given to IL-2group three times by intraperitoneal injection within 1 hour to 2 days after operation,and vehicle group was given the same dose of PBS.After determining the optimal dose,the rats were divided into sham group,vehicle group and IL-2 group,then the best dose of drugs were given to IL-2 group three times by intraperitoneal injection within 1 hour to 2days after operation,and vehicle group was given the same dose of PBS.Finally,the optimal dose was selected for further study.（2）Flow cytometer was used to detect the number of Tregs in peripheral blood,lymph nodes and spleen.（3）In this study,the modified Garcia scoring system was used to evaluate neurological deficits.Morris water maze test was used to assess long-term cognitive function.Corner test and Rotarod test were used to evaluate sensorimotor function.（4）Immunofluorescence staining was used to detect neuronal apoptosis,neutrophile granulocyte,microglia,M2 microglia in basal cortex and hippocampal CA1 region.（5）The expression of inflammatory cytokines such as IL-1β,IL-6 and TNF-αin plasma was detected by Elisa kit and Real-time PCR.Results（1）The changes of Tregs in vivo after subarachnoid hemorrhage are as follows:decreased-increased-decreased.（2）IL-2 therapy could proliferate Tregs in vivo after subarachnoid hemorrhage,and the number of Tregs is the largest in IL2-2ug group.（3）IL-2 therapy could improve neurological function score and behavior after SAH.（4）IL-2 therapy could reduce the expression of neutrophils in basal cortex.（5）IL-2 therapy could promote microglia M2 polarization.（6）IL-2 therapy could alleviate neuronal apoptosis.（7）IL-2 therapy could reduce the levels of IL-1β,IL-6 and TNF-αin plasma.Conclusion（1）IL-2 therapy could suppress the expression of neutrophils and pro-inflammatory factors such as IL-1β,IL-6 and TNF-αby promoting the proliferation of endogenous Tregs after SAH,thus alleviating neuronal apoptosis and ultimately playing a neuroprotective role.（2）IL-2 therapy could promote microglia M2 polarization by promoting the proliferation of endogenous Tregs after SAH,and then enhance its abilities of phagocytosis and repair and ultimately playing a neuroprotective role.