肢体缺血后处理对手术绝经大鼠全脑缺血后海马CA1区星形胶质细胞极化的调控

REGULATION OF LIMB ISCHEMIA POSTCONDITIONING ON POLARIZATION OF ASTROCYTES AFTER GLOBAL CEREBRAL ISCHEMIA IN HIPPOCAMPAL CA1 REGION OF OVARIECTOMIZED-RATS

目的 研究肢体缺血后处理对手术绝经大鼠全脑缺血后海马CA1区星形胶质细胞的影响,并探索其对星形胶质细胞亚型的调控。方法 3月龄SPF级SD大鼠行双侧卵巢切除制备手术绝经模型,并随机分为假手术组(Sham)、肢体缺血后处理假手术组(Sham+LIP)、全脑缺血再灌注组(IR)和肢体缺血后处理组(IR+LIP);采用四动脉结扎法制备全脑缺血模型(GCI)、结扎近心端后肢股动脉行肢体缺血后处理(LIP);GFAP、C3和S100A10免疫荧光染色观察星形胶质细胞形态及表达;Western blot技术检测海马CA1区星形胶质细胞标志蛋白GFAP、A1型(S100A10)和A2型(C3)星形胶质细胞标志蛋白水平。结果①Western blot结果显示,与Sham组比较,IR 7d组大鼠海马CA1区GFAP与C3蛋白表达显著增加,同时伴有S100A10蛋白表达降低;而肢体缺血后处理显著降低了GFAP和C3的蛋白表达,并显著上调了S100A10的蛋白表达。②免疫荧光染色结果可见IR 7d组大鼠海马CA1区GFAP和C3免疫荧光强度较Sham组和IR 7d+LIP组显著增强,而S100A10免疫荧光强度较Sham组和IR 7d+LIP组显著降低,且IR 7d组可见少量S100A10与GFAP免疫共定位和较强的GFAP与C3免疫共定位;而肢体缺血后处理增强了S100A10与GFAP免疫共定位,同时降低了GFAP与C3免疫共定位。结论 肢体缺血后处理能降低反应性星形胶质细胞表达;并通过促进星形胶质细胞向保护型A2型分化,而抑制其向损伤型A1型分化来调控星形胶质细胞的分型。

Objective To investigate the effect of limb ischemia postconditioning(LIP) on astrocytes in hippocampal CA1 region after global cerebral ischemia in ovariectomized-rats and to explore its regulation on astrocyte subtypes.Methods 3-moth-old female Sprague-Dawley rats were bilaterally ovariectomized to induce the surgical menopause model and were randomly divided into sham operation group, sham control group(Sham+LIP),ischemia reperfusion group(IR),and LIP group(IR+LIP).Global cerebral ischemia(GCI)was induced by four-artery ligation and LIP was induced by ligation of the proximal posterior femoral artery.Immunofluorescence staining was used to detect morphology and protein expression of astrocyte markers(GFAP),type A1(C3) and A2(S100 A10),and in the hippocampal CA1 region.Western blot analysis was performed further to confirm the results of immunofluorescence staining.Results① Immunofluorescence staining showed that compared with Sham and IR 7 d +LIP groups, IR 7 d significantly increasedthe fluoscenceintensity of GFAP and C3 in the hippocampal CA1 region, while S100 A10 intensity was significantly decreased, and a small amount of S100 A10 and GFAP co-localization and a strong GFAP and C3 co-localization were observed in IR 7 d group.However, ILP markedly enhanced the co-localization of S100 A10 and GFAP,while decreased the co-localization of GFAP with C3.② Western blot analysis showed that the expression of GFAP and C3 protein in hippocampal CA1 region of IR 7 d rats was significantly increased compared to Sham group, and the expression of S100 A10 protein was decreased.However, ILP significantly decreased the protein expressions of GFAP and C3,and significantly upregulated the S100 A10 protein expression.Conclusion LIP can induce reactive astrocytes decreased, and LIP regulates astrocytes subtype by promoting the differentiation of A1 astrocyte to the favorable A2 type and inhibiting the differentiation to the harmful A1 type.