Objective: To investigate the role of Orai1-mediated store-operated calcium entry in the immune damage of CD4+ T cells in septic mice. Methods: Sepsis mouse model was established by cecal ligation and puncture(CLP). Balb/c mice of clean grade were sacrificed 1, 3, and 5 days after operation. Spleen samples were harvested at given intervals. Splenic CD4+ T cells were selected by immunomagnetic beads and the expression of Orai1 protein was detected by western blotting, the storage operated calcium entry (SOCE) was detected by flow cytometry, the apoptosis of CD4+ T cells was detected by flow cytometry, the proliferation of CD4+ T cells was detected by CCK-8, and the IFN-γ and IL-4 were detected by enzyme-linked immunosorbent assay (ELISA). Then the expression of Orai1 protein was regulated to further detect the SOCE and immune function of splenic CD4+ T cells in mice. The experiment was divided into 4 groups, sham group, CLP3 group, Orai1 down group (Orai1-down group) and Orai1 up regulation group (Orai1-up group). Results: The relative expression of Orai1 protein in splenic CD4+ T cells in sham group was 1.03±0.16. Compared with sham group, Orai1 protein levels in CLP Group were all significantly lower (F=19.64, P=0.000 5). The increased value of splenic CD4+ T cells fluorescence intensity in sham group was 494±41. Compared with sham group, the levels of SOCE in CLP Group were all lower (F=30.01, P=0.001). The ratio of early and late apoptosis of CD4+ T cells in sham group was 8.7%±1.5%. Compared with sham group, the early and late apoptosis rates of CLP Group were significantly higher (F=32.29, P=0.000 1). The OD of sham group was 0.81±0.10 at 450 nm. Compared with sham group, the proliferation ability of splenic CD4+ T cells in CLP Group were significantly decreased (F=7.26, P=0.001 8). Compared with sham group, the secretion of IFN-γ and IL-4 by CD4+ T cells and the ratio of IFN-γ/IL-4 in CLP Group were all significantly decreased (F=19.690, 6.183, 11.230, all P<0.05). Compared with CLP3 group, the increased value of fluorescence intensity of CD4+ T cells was significantly decreased, the early and late apoptosis ratio of CD4+ T cells was significantly increased, the OD450 nm value of CD4+ T cells was decreased, the multiplication capacity of splenic CD4+ T cells were decreased, the level of IFN-γ and IL-4 secreted by T cells were decreased, and the value of IFN-γ/IL-4 in orai1-down group was decreased (t=4.819, 7.952, 2.988, 28.760, 3.140, 7.670, all P<0.05). However, Orail-up group showed the opposite trend. Conclusion: Orai1-mediated store-operated calcium entry can alleviate the immune dysfunction of CD4+ T cells in septic mice.
目的： 探讨Orai1介导钙池操纵的钙内流（SOCE）在脓毒症小鼠CD4+ T细胞免疫功能损害中的作用。 方法： 采用清洁级雄性Balb/c小鼠建立盲肠结扎穿孔（CLP）脓毒症模型，分别于术后1、3、5 d 处死小鼠，无菌留取脾脏，免疫磁珠法分选CD4+ T细胞，Western印迹法检测各组脾脏CD4+ T细胞Orai1蛋白表达，流式细胞术检测SOCE，流式细胞术检测CD4+ T细胞凋亡，CCK-8细胞计数试剂盒检测CD4+ T细胞增殖，酶联免疫吸附法（ELISA）检测干扰素（IFN）-γ和白细胞介素（IL）-4水平。然后调节Orai1蛋白的表达，实验分为4组，sham组、CLP3组、Orai1下调组（Orai1-down组）、Orai1上调组（Orai1-up组），进一步检测小鼠脾脏CD4+ T细胞SOCE和免疫功能。 结果： sham组脾脏CD4+ T细胞Orai1蛋白的相对表达量为1.03±0.16，与sham组相比，CLP组的Orai1蛋白水平明显降低（F=19.64，P=0.000 5）。与sham组相比，CLP组SOCE水平明显降低（F=30.01，P=0.001）。sham组的CD4+ T细胞早期和晚期凋亡比率为8.7%±1.5%，与sham组相比，CLP组明显升高（F=32.29，P=0.000 1）。与sham组相比，CLP组小鼠脾脏CD4+ T细胞增殖能力明显降低，差异有统计学意义（F=7.26，P=0.001 8）。与sham组相比，CLP组小鼠脾脏CD4+ T细胞分泌IFN-γ和IL-4的能力均明显降低，差异均有统计学意义（F=19.690、6.183，均P<0.05）。与sham组相比，CLP组IFN-γ/IL-4比值变小（F=11.23，P=0.003 1）。与CLP3组相比，Orai1-down组CD4+ T细胞SOCE的水平明显下降，早期和晚期凋亡比率明显升高，脾脏CD4+ T细胞增殖能力显著下降，分泌IFN-γ和IL-4下降，IFN-γ/IL-4值下降，差异均有统计学意义（t=4.819、7.952、2.988、28.760、3.140、7.670，均P<0.05）；而Orai1-up组则均获得相反的结果，差异亦均有统计学意义（t=2.983、6.796、9.390、19.670、3.692、15.870，均P<0.05）。 结论： Orai1介导SOCE减轻脓毒症小鼠CD4+ T细胞免疫功能障碍。.