| Background:Subarachnoid hemorrhage(SAH)is a disease with high mortality and morbidity,survivors may also suffer from different degrees of neurological dysfunction.Previous studies have shown that mechanism of secondary brain injury after SAH is complicated,even with the most effective treatment,outcomes of patients are still critical.Natural killer cells(NK cells)play an important role in innate immune recognition and response,involved in infection,anti-tumor and immune regulation.In non-infectious disease of central nervous system,NK also plays a considerable part.Objectives:To explore the molecular mechanism of subarachnoid hemorrhage in the perspective of immune pathogenesis.To introduce NK cells of importance in innate immune response to research of hemorrhagic disease of the central nervous system.The study is designed to combine clinical retrospective study and laboratory research,with an intention to elaborate important values of NK cells in subarachnoid hemorrhage.Methods:Preliminary study included immunohistochemical staining of tissues from glioma tumor patients to analyse NK cell surface receptor NKp30 ligand B7-H6 expression and its correlation with clinical parameters.Down-regulation of B7-H6 was performed by using RNA interference method in human glioma cell lines to obeserve impact of B7-H6 on proliferation,migration,invasion,apoptosis and cell cycle of glioma cell lines,suggesting effect of NK cells and their surface proteins in non-infectious diseases of central nervous system.Secondly we collected clinical data and peripheral blood specimens from subarachnoid hemorrhage patients.Flow cytometry was performed to evaluate changes of NK cells and its subsets percentage over time.Survival and Logistics regression analyzes were conducted to examine clinical value of NK cells.Then a subarachnoid hemorrhage model of rats was established.By ways of immunohistochemical stain,Western blot,and real-time quantitative RNA validation,infiltration of NK cells in brains after SAH was identified,so was the relative time phase and comparability with clinical results.In the last step,subarachnoid hemorrhage model of mice was build.NK cells were inhibited by intraperitoneal injection of antibody.The influence of NK cells was validated along with the impact of Wnt pathways in the brain,by neurological assessment,immunohistochemistry,and Western Blot.Results:High expression of NK cell receptor NKp30 ligand B7-H6 in human brain glioma tissues,is statiscally correlated with pathological types(Z/x2=3.935,P<0.001),WHO classification(Z/x2=3.008,P<0.01),and tissue types(Z/x2=13.908,P<0.01).By using the RNA interference technology,we successfully decreased B7-H6 expression in human glioma cell lines.CCK-8 cell proliferation experiment showed that in 72 h,cell proliferation levels were significantly lower than the control groups,the differences were statistically significant(P<0.001).In wound healing assay,the cell-free areas of the B7-H6 interference groups were significantly wider than that of control groups,after 24 h(P<0.05).In Transwell invasion assay glioma cells from B7-H6 interference groups showed significantly reduced number of cells invading through the matrigel,as compared to cells from control groups(P<0.01).Cell cycle analysis results showed cells from the B7-H6 interference groups,displayed increased percentage of cells in the G1-phase and decreased percentage in G2/M phases,in comparison to cells from control groups.Clinical study showed changes of NK cells and subsets after subarachnoid hemorrhage in peripheral blood in different time phase.On day 3-6,the percentage of NK cells decreased obviously,the difference was statistically significant(F=7.77,P<0.01);NK cells subsets propotion(CD16+CD56bright subset vs CD16+CD56dim subset)also decreased obviously,the difference was statistically significant(F=8.57,P<0.01).According to the NK cells subset data on day 3-6,patients were divided into two groups,Hunt-Hess had a significant difference between groups(F=10.33,P<0.01).Patient deaths had obvious differences between groups(x2=7.19,P=7.19),significant difference of modified Rankin scale was shown(F= 10.46,P<0.01),along with survival(x2=7.198,P=7.198).Subarachnoid hemorrhage animal model study results:NK cell infiltration were shown in rats in 24 h after SAH(F= 42.62,P<0.0001).During 24h,expression of NK cell surface receptor CXCR3 in brain tissue was significantly increased(F= 10.62,P<0.05),also increased after 48h(F=6.13,P<0.05).KLRC3 significantly increased at 24h(F=5.98,P<0.05).Expression of NK cell recruitment related protein IP-10 mRNA increased in 6h after SAH(F = 16.19,P<0.05).IFN-y mRNA expression decreased,after 12h with statistically significant differences(F=13.90,P<0.05).Prf-1 mRNA expression increased in 12h and 24h,and differences were statistically significant(F=10.70,P<0.05;F=14.58,P<0.05).Mice subarachnoid hemorrhage animal model results:neurological function scores in NK cells depletion group declined compared with the control group(F=5.18,P<0.05).24h after SAH,Wnt signal pathway was suppressed with APC protein expression increases(F=10.84,P<0.01);β-catenin protein decreased(F=8.59,P<0.05);phosphorylated β-catenin protein increased(F=25.01,P<0.01);Teriminal protein Survivin expression decreased(F= 19.50,P<0.01),all with statistically significant differences.Conclusion:NK cells and their surface proteins may play an important role in non-infectious diseases of central nervous system.Changes of NK cells and subsets in peripheral blood of subarachnoid hemorrhage patients may have clinical values in outcome predicts.As shown in animal model study,NK cells infiltrates in brain tissues after SAH,may have an effect of neuroprotection through Wnt signal pathway. |
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