Sestrin2对高迁移率族蛋白B1诱导树突细胞凋亡的保护作用

Protective effect of Sestrin2 on apoptosis of dendritic cells induced by high mobility group box-1 protein

目的探讨Sestrin2(SESN2)在高迁移率族蛋白B1(HMGB1)诱导树突细胞(DC)凋亡中的作用。方法将培养的小鼠树突细胞DC2.4分为正常对照组及HMGB1 8、24、48h组(n=4),HMGB1各亚组加入10ng/ml HMGB1培养相应时间;另将DC2.4细胞分为正常对照组及HMGB1组,HMGB1组设1、10、100ng/ml亚组(n=4),予以不同浓度HMGB1刺激48h。采用Western blotting检测各组细胞SESN2、cleaved-caspase-3、Bcl-2蛋白的表达水平,共聚焦激光显微镜检测SESN2在细胞中的定位及表达,流式细胞术检测各组细胞凋亡情况。将载有过表达空载体NC、沉默空载体NS、过表达病毒载体SESN2 LV-RNA及沉默病毒载体SESN2 si RNA转入DC2.4细胞中(n=4),以100ng/ml HMGB1刺激48h,采用流式细胞术检测细胞凋亡情况,并检测凋亡相关蛋白cleaved-caspase-3、Bcl-2的表达水平。结果与正常对照组比较,10ng/ml HMGB1刺激24、48h后,以及1、10、100ng/ml HMGB1刺激48h后,DC2.4细胞中SESN2的表达水平均显著上调,差异有统计学意义(P<0.05)。10、100ng/ml HMGB1刺激48h后,DC2.4细胞的凋亡率(分别为17.02%±4.85%、17.48%±4.04%)明显高于正常对照组(7.35%±1.33%,P<0.05,P<0.01),而10ng/ml HMGB1刺激8h后DC2.4细胞凋亡率与正常对照组比较差异无统计学意义(P>0.05)。在不同转染组中,100ng/ml HMGB1刺激48h后,SESN2 si RNA组DC2.4细胞凋亡率(65.96%±2.50%)明显高于空载体组(50.01%±2.07%),同时凋亡相关蛋白cleaved-caspase-3表达明显升高,抗凋亡蛋白Bcl-2表达明显下降,差异均有统计学意义(P<0.05);而SESN2 LV-RNA组细胞凋亡率(35.57%±1.69%)明显低于空载体组(49.04%±4.87%),cleaved-caspase-3表达明显下降,Bcl-2表达明显升高(P<0.05)。结论 SESN2在HMGB1诱导DC2.4细胞的凋亡过程中具有一定保护作用。

Objective To investigate the potential role of Sestrin2 （SESN2） in regulating the apoptosis of dendritic cells （DCs） induced by high mobility group box-1 protein （HMGB1）. Methods DCs （the routine DC cell line DC2.4） were cultured with or without HMGB1 stimulation （cultured with 10ng/ml HMGB1 for 8, 24 and 48 hours, or cultured with HMGB1 for 48 hours at different concentrations of 1, 10 and 100ng/ml, respectively, n=4）. The protein level of SESN2, cleaved-caspase-3 and Bci-2 were analyzed with Western blotting. Localization of SESN2 in cells was observed under confocal laser microscope （LSCM）. Cell apoptosis was analyzed with flow cytometry. In addition, DC2.4 cells were transfected with lentivirus containing SESN2 LV-RNA, SESN2 siRNA sequence expressing plasmids or blank vector （NC, NS, n=4）. These cells were then stimulated with HMGB1 （100ng/ml） for 48 hours, and the apoptosis was accessed as mentioned above. Results Compared with the control group, the expression of SESN2 was obviously up-regulated after HMGB1 （10 ng/ml） stimulation for 24 and 48 hours （P〈0.05）. In a dose-dependent response, the expression of SESN2 was markedly enhanced in treatment with 1, 10, 100ng/ml HMGB1 for 48 hours （P〈0.05）. Compared with the control group （7.35% ± 1.33%）, the percentage of apoptosis was significantly increased with 10, 100ng/ml HMGB1 for 48 hours [（17.02% ± 4.85%, 17.48% ± 4.04%, respectively, P〈0.05 or P〈0.01]. After transfection, compared with blank vector group, the apoptosis of SESN2 siRNA group obviously elevated [（65.96% ± 2.50%） vs. （50.01% ± 2.07%）, P〈0.05], and cleaved-caspase-3 expression significantly increased while Bcl-2 expression obviously decreased. In SESN2 LV-RNA group, the apoptosis significantly decreased [（35.57% ± 1.69%） vs. （49.04% ± 4.87%）, P〈0.05], and cleaved-caspase-3 expression decreased and Bcl-2 expression obviously increased compared with blank vector group （P〈0.05）. Conclusion SESN2 has a protective effect against HMGB1 induced apoptosis of DC2.4 cells.