CD137信号调控P53/P21通路对血管平滑肌细胞衰老的机制研究

Mechanism of CD137 signal regulating P53/P21 pathway to promote senescence of vascular smooth muscle cells

目的探讨CD137信号调控P53/P21通路对血管平滑肌细胞(VSMC)衰老的机制。方法选择8周龄雄性C57BL/6J小鼠30只,随机分为年轻组(8周龄)和衰老组(80周龄),每组15只。饲养对应时间后处死小鼠并分离血浆及主动脉血管。细胞实验中将正常VSMC分为空白对照组、博来霉素(BLM)组、联合激动剂组和联合抑制剂组。采用细胞衰老β-半乳糖苷酶染色试剂盒染色检测VSMC衰老水平。Western blot和聚合酶链反应检测组织及细胞中衰老相关蛋白,酶联免疫吸附测定衰老相关炎性因子表达。结果衰老组小鼠主动脉中CD137、组蛋白h2ax(γ-H2AX)表达明显高于年轻组,增殖细胞核抗原(PCNA)表达明显低于年轻组(P<0.05)。衰老组小鼠血浆CD137水平明显高于年轻组[(154.0±4.1)pg/ml vs(98.0±2.3)pg/ml,P<0.05]。与空白对照组比较,BLM组衰老VSMC明显增加(P<0.05);与BLM组比较,联合激动剂组衰老VSMC明显增加(P<0.05);与联合激动剂组比较,联合抑制剂组衰老VSMC明显减少,差异有统计学意义(P<0.05)。与空白对照组比较,BLM组肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-6和IL-1β水平明显增高(P<0.05);与BLM组比较,联合激动剂组TNF-α、IL-6和IL-1β水平明显增加(P<0.05);与联合激动剂组比较,联合抑制剂组TNF-α、IL-6和IL-1β水平明显减少,差异有统计学意义(P<0.05)。与空白对照组比较,BLM组、联合激动剂组和联合抑制剂组B淋巴细胞瘤2基因、γ-H2AX、P53和P21表达明显增加,PCNA表达明显减少(P<0.05);与BLM组比较,联合激动剂组P53和P21表达明显增加(P<0.05);与联合激动剂组比较,联合抑制剂组P53表达明显减少,差异有统计学意义(P<0.05)。结论CD137信号调控P53/P21通路促进VSMC衰老。

Objective To explore the mechanism by which CD137 signal regulates the aging of vascular smooth muscle cells(VSMCs).Methods Thirty 8-week-old male C57BL/6J mice were randomly divided into a young group(8 weeks old)and an aged group(80 weeks old),with 30 mice in each group.After corresponding periods of feeding,the mice were euthanized,and the plasma and aortic blood vessels were isolated.In the cell experiments,normal VSMCs were divided into a control group,bleomycin(BLM)group,combined agonist group,and combined inhibitor group.The cellular senescence level of VSMCs was assessed using a cellular senescenceβ-galactosidase staining kit.Western blotting and PCR were employed to examine the expression of senescence-related proteins in tissues and cells,while ELISA was utilized to measure the expression of senescence-related inflammatory factors.Results The expression of CD137 andγ-H2AX in the aorta was significantly higher,while that of PCNA was obviously lower in the aged group than the young group(P<0.05).The plasma level of CD137 was notably higher in the aged group than the young group(154.0±4.1 pg/ml vs 98.0±2.3 pg/ml,P<0.05).Compared with the normal control group,there were significantly more aged VSMCs in the BLM group(P<0.05).While,treatment of combined agonist resulted in larger amount of aged VSMCs when compared with the BLM group(P<0.05),which was reversed by combined inhibitor treatment(P<0.05).The levels of TNF-α,IL-6 and IL-1βwere significantly elevated in the BLM group than the normal control group(P<0.05).The combined agonist group had even higher levels of TNF-α,IL-6,and IL-1βthan the BLM group(P<0.05),but the levels were decreased in the combined inhibitor group(P<0.05).Compared with the normal control group,the expression of Bcl-2,γ-H2AX,P53,and P21 were significantly increased in the BLM group,combined agonist group,and combined inhibitor group,while that of PCNA was significantly decreased(P<0.05).Compared with the BLM group,the expression of P53 and P21 in the combined agonist group showed an increase(P<0.05),and the expression of P53 was significantly decreased in the combined inhibitor group(P<0.05).Conclusion CD137 signal regulates the P53/P21 pathway to promote VSMC aging.