CD137信号通过抑制自噬小体与溶酶体融合促进载脂蛋白E-/-小鼠斑块钙化形成

CD137 signaling promotes the formation of plaque calcification via inhibiting the fusion of autophagy and lysosomal in Apo E-/- mice

目的探讨CD137信号是否通过抑制自噬小体与溶酶体融合促进动脉粥样斑块钙化的形成。方法（1）体内实验：分别采用激动型或抑制型CD137抗体激动或抑制CD137信号，将15只6~8周龄载脂蛋白（Apo）E-/-小鼠分为3组（每组5只）：对照组（腹腔注射IgG2b 200 μg），CD137激动组（腹腔注射激动型CD137抗体200 μg），CD137抑制组（腹腔注射抑制型CD137抗体200 μg，24 h后注射激动型CD137抗体200 μg）。（2）细胞实验：采用组织块贴壁法原代培养小鼠血管平滑肌细胞，实验分为3组：对照组，CD137激动组（激动型CD137抗体10 μg/ml），CD137抑制组（预孵抑制型CD137抗体10 μg/ml 60 min后＋激动型CD137抗体10 μg/ml）。细胞及斑块钙化采用von Kossa染色，免疫组织化学染色检测斑块中LC3B、Beclin 1及p62蛋白表达水平。采用Western blot检测骨形成蛋白-2（BMP-2）、Runx2、LC3Ⅱ/Ⅰ及p62蛋白表达水平，荧光显微镜观察自噬流，透射电镜观察自噬小体形成。结果（1）体内实验结果：CD137激动组Apo E-/-小鼠斑块钙化面积比率大于对照组[（3.01±0.45）%比（0.27±0.06）%，P〈0.01]，而CD137抑制组斑块钙化面积比率少于CD137激动组[（1.23±0.39）%比（3.01±0.45）%，P〈0.05]。免疫组织化学染色结果显示，CD137激动组及抑制组自噬早期标记蛋白LC3B、Beclin 1表达水平均高于对照组，激动组更高（P〈0.05）；同样CD137激动组晚期标志蛋白p62表达水平高于CD137抑制组（P〈0.05）。（2）细胞实验结果：CD137激动组和抑制组自噬相关蛋白LC3Ⅱ/Ⅰ比值及p62表达水平高于对照组（P〈0.01），激动组p62蛋白表达水平高于抑制组（P〈0.05）。细胞钙化诱导实验中，CD137激动组BMP-2及Runx2蛋白表达水平明显高于对照组（P〈0.01），而CD137抑制组BMP-2、Runx2蛋白表达水平低于CD137激动组（P〈0.05）。结论激动CD137信号能抑制自噬小体与溶酶体融合从而促进Apo E-/-小鼠动脉粥样斑块钙化形成。

ObjectiveTo investigate whether CD137 signaling promoted the formation of atherosclerotic plaque calcification by inhibiting the fusion of autophagosome and lysosome.Methods（1） In vivo, CD137 agonist antibody and anti-CD137 antibody were used to stimulate and inhibit the CD137 signaling, respectively. Fifteen Apo E-/- mice were randomly divided into three groups： control group （intraperitoneal injection of IgG2b 200 μg） , CD137 agonist group （intraperitoneal injection of CD137 agonist antibody 200 μg） , anti-CD137 group （pretreatment with 200 μg anti-CD137 antibody for 24 hours, then injection of CD137 agonist antibody） . （2） In vitro, primary culture of mouse aortic VSMCs obtained through adherence methods for tissues explants. The cells was divided into three groups： control group, agonist-CD137 group （CD137 agonist antibody 10 μg/ml） , and anti-CD137 group （pretreatment with 10 μg/ml anti-CD137 antibody for 60 minutes, then incubated with 10 μg/ml CD137 agonist antibody） . Von kossa staining was used to detect the calcification in the cell and plaque. Immunohistochemical staining was used to observe the expression of LC3B, Beclin 1 and p62 which are associated with autophagy. The levels of autophagy related protein （LC3） , Beclin 1, p62, and the expression of Runx2 and bone morphogenetic protein 2, which is associated with osteogenic differentiation in the VSMCs, were determined by Western blot. The autophagy flow of each group was detected by fluorescence microscopy. The autophagy was observed by transmission electron microscope in vivo and in vitro.Results（1） In vivo, the calcified plaque area in CD137 agonist group was significantly larger than that in the control group （3.01%±0.45% vs. 0.27%±0.06%, P〈0.01） , and calcified plaque area in anti-CD137 group was significantly smaller compared with that in the CD137 agonist group （1.23%±0.39% vs. 3.01%±0.45%, P〈0.05） . Immunohistochemical staining showed that the expression of early autophagy marker protein LC3B and Beclin 1 were significantly upregulated in CD137 agonist group and anti-CD137 group than in control group, and the highest expression was observed in CD137 agonist group （P〈0.05） . The expression of advanced autophagy marker protein p62 was higher in the CD137 agonist group than in the anti-CD137 group （P〈0.05） . （2） In vitro, the ratio of autophagy related protein LC3 Ⅱ/Ⅰ and p62 protein expression were significantly higher in CD137 agonist group and anti-CD137 group than in control group （P〈0.01） , while the expression of p62 protein was significantly higher in CD137 agonist group than that in anti-CD137 group （P〈0.05） . In the cell calcification inducing experiment, the expression of BMP-2 and Runx2 protein was significantly higher in CD137 agonist group than that in control group （P〈0.01） , but the levels of BMP-2 and Runx2 protein were lower in anti-CD137 group than in CD137 agonist group （P〈0.05） .ConclusionOur results indicate that activation of CD137 signaling can promote the formation of atherosclerotic plaque calcification by inhibiting the fusion of autophagosome and lysosome.