The Protective Mechanism Study In Treg Transformation After Subarachnoid Hemorrhage In Mice By Suppressing Nerve Inflammation

ObjectiveSubarachnoid hemorrhage (SAH), with high incidence, high mortality, high morbidity, is a kind of common hemorrhagic cerebrovascular disease, survival often living in low quality of life due to different degree of nerve function defect. Studies confirm that early brain injury after SAH and the severity of cerebral vasospasm directly affects the prognosis of patients. Therefore, it is necessary to develop new drugs to control the early inflammatory immune response after SAH, reduce the pathogenic probability of EBI and CVS, relieve the suffering of the patients. The study found that regulatory T cell(CD4+CD25+regulatory T cell, Treg) plays an important effect in immune defense and immune tolerance, regulatory T cell can inhibit cerebral infarction reperfusion in mice brain damage, playing an endogenous protection role.The same as intracranial ischemia, SAH also is induced by anoxic injury,dose regulatory T cell can play the same regulatory role in inflammatory immune response?To simulate brain ischemia anoxic process of inflammatory reaction after SAH, We use lipopolysaccharide induce BV2 microglia activation model, extract regulatory T cell from the mice spleen and lymph nodes in vitro, and amplify Treg. Verify the protective role of microglia attributed to Treg by the cocultivation of regulatory T cell and BV2 microglia.To study the effect of brain protection attributed to intravenous transplantation of regulatory T cell, we use line pin prick the legal system for SAH model in mice, extracted Treg in vitro from the mice spleen, lymph nodes, and amplify Treg, femoral intravenous transplantation to SAH mice.To test and verify the conclusion of experiment in vitro cell could be replicated in animal models, and further explore the regulatory T cell transplantation by intravenous infusion of microglia and SAH in mice brain protection mechanism principle, we respectively from cell culture in vitro and in vivo experiments on animals in both directions, from the perspective of organization, the protein and mRNA level, comprehensive discusse the protective mechanism in microglia and in SAH mice brain.MethodsWe use immune magnetic beads double choose method to extract the mice spleen and lymph nodes of CD4+CD25+T cell, sorting and amplification in vitro. By the cocultivation of regulatory T cell and lipopolysaccharide induce BV2 microglia in vitro, study the polarization state regulated by Treg. MTT method is used to detect the activity of microglia, Nitrate reductive enzymatic detecting the content of NO in cell cultures, ELISA method was used to detect cell cultures of inflammatory cytokine TNF-a, IL-6, IL-10; Swallowed up the Nile red microspheres experiment to validate Treg of LPS induced microglia phagocytosis.Internal carotid artery line pin prick the legal system for SAH model in Male C57BL/6 j mice, mice were randomly divided into Sham group, the SAH+PBS group, SAH+SP (Splenocyte, spleen cells) group, the SAH+Treg group, femoral intravenous transplantation of CD4+CD25+Treg,48h after building success, record cerebral blood flow and each animal behavior index; Prepare paraffin and frozen brain tissue section respectively, to observe the morphology change of cerebral vascular by HE staining, to observe the distribution of positive cells in brain by TUNEL method, to validate the protection of mice brain tissue contributed by the transplantation of Treg.Using immunofluorescence staining method to detect the expression of M1, M2 phase positive cells in vitro microglia and in mice brain tissue after SAH; Using RT-PCR method to detect the expression of mRNA level of Ml, M2 phase positive cells in vitro microglia and in mice brain tissue after SAH; Using Western blot method to detect the signal pathway expression of TLR4/p-NFκB and p-P38/p-ERK1/2 in vitro microglia and in mice brain tissue after SAH.ResultsBy the cocultivation of regulatory T cell and LPS induced BV2 microglia, the inflammatory factor in cell cultures such as NO, TNF-a and IL-6 significantly reduced, inflammatory inhibition factor IL-10 increases at the same time, Treg significantly enhance phagocytosis of LPS induced BV2 microglia,with obvious protective effect.Treg intravenous transplantation can reduce the mortality in mice after SAH, obviously improve the vital signs and neural function, improve nerve function study score, improve the brain blood flow, release brain edema, reduce the degree of CVS and EBI, reduce vascular endothelial cell damage degree, reduce the apoptosis in mice hippocampus and cortex cells, reduce the neurons damage degree.Immunofluo rescence staining showed that by Treg transplantation, M1 phase marker expression is reduced and the M2 phase marker is increased in vitro and SAH in mice brain tissue Rt-pcr results showed that by Treg transplantation, mRNA level of M1 phase marker expression is reduced and mRNA level of M2 phase marker is increased in vitro and SAH in mice brain tissue.Western blot results show that by Treg transplantation, inflammatory signaling pathway TLR4/p-NF-κB and p-P38/p-ERK1/2 dramatically reduce the amount of protein expression in microglia in vitro and SAH in the brain tissue of mice.Conclusion:By the cocultivation of regulatory T cell and LPS induced BV2 microglia, Treg can reduce the content of inflammatory cytokines, increase microglia phagocytosis. By Treg transplantation, the degree of CVS and EBI is reduced in mice after SAH. The protective effect attributed by Treg is probably by suppressing the signaling pathway of TLR4/p-NF-κB and p-P38/p-ERK1/2.This experiment innovation in the applications of regulatory T cells to subarachnoid hemorrhage in the mice model, by the experiment of in vitro cell level and in vivo animal models of experiment, the combination of animal behavior observation and the level of cell and molecular mechanism research, the combination of various angles fully discusses the protective role of regulatory T cells for tissue. The experiment proves that the regulatory T cells has obvious protective effect in microglia and subarachnoid hemorrhage mice, however, there is large distance between animal model level and clinical application, being a kind of biological active substances, cells are not like chemical stability, there are many problems existing in the aspects such as the preservation and transportation is an important bottleneck in clinical application. In addition, the transplanting of human living cells also is an ethical issues to be solved.