Altered oxidative DNA damage repair systems in muscles of old mice: role in the age-related increase in muscle production of cytokines/chemokines.

在老年小鼠的肌肉中，氧化的DNA碱基损伤8-氧-7，8-二氢鸟嘌呤(8-oxoG)的存在增加。8-oxoG是通过8-oxoguanine DNA糖基酶-1(OGG1)启动的DNA碱基切除修复途径修复的，这一过程被认为是导致激活的B细胞核因子-kappa轻链增强因子(NF-kB)激活的过程。我们假设老年小鼠肌肉中的慢性DNA氧化损伤导致OGG1激活的核因子-kB的慢性增加，从而导致骨骼肌炎性细胞因子的产生，从而导致肌肉功能的增龄性恶化。检测肌肉细胞DNA损伤修复反应，包括8-oxoG、OGG1、PARP-1、核因子-kB的激活以及细胞因子/趋化因子基因的表达和释放。检测OGG1选择性抑制剂TH5487(5um)对核因子-kB活化和细胞因子产生的影响。从成年小鼠分离的肌肉纤维以类似于上述成肌细胞的方式处理，并检查细胞因子/趋化因子的释放。用Luminex多重分析方法检测OGG1在老年小鼠离体肌纤维慢性产生细胞因子中的作用。本实验观察了TH5487对成年小鼠成肌细胞分泌细胞因子和老年小鼠成肌细胞慢性释放细胞因子/趋化因子的影响。50 uM过氧化氢能维持成肌细胞的活力，但Ogg1基因表达水平显著升高，OGG1、PARP-1和裂解PARP-1蛋白表达水平显著升高。核因子-kB明显激活，并伴有大量促炎细胞因子的表达和释放(IL-6、TNF-α和CXCL1mRNA分别增加3.7倍、4.2倍和3倍)。当TH5487在过氧化氢处理细胞前处理细胞时，这些由过氧化氢引起的核因子-kB活性增加和细胞因子/趋化因子基因水平的增加被正常化。在用TH5487处理的肌肉纤维中，这种由过氧化氢介导的核因子-kB DNA结合活性的增加显著降低。与成年小鼠相比，老年小鼠分离的肌肉纤维显示细胞因子/趋化因子释放增加，而TH5487处理后细胞因子/趋化因子释放也减少。经H_2O_2处理后，成年小鼠纤维上清液中IL-6、CCL2、CXCL1、CCL7、GM-CSF、CX3CL1、CCL27、CXCL5、CCL11、CXCL16和CXCL-12均升高，TH5487可使IL-6、CCL2、CXCL1、CCL7、CCL27、CXCL5、CCL11、CXCL16和CXCL-12降至正常水平。老年小鼠离体肌纤维中IL-6、CXCL1、CCL2、CCL7的表达显著增加，经TH5487处理后均降低，提示OGG1通路可能是老年小鼠肌肉产生细胞因子/趋化因子增多的原因之一。还将提供成年和老年小鼠肌肉的蛋白质组学和mRNA分析数据，以寻找DNA修复途径随年龄变化的证据。一种DNA损伤修复的计算模型正在设计中，以确定DNA损伤和修复过程中的治疗靶点，以修改老年小鼠肌肉中核因子-kB的激活。MRC-关节炎研究英国肌肉骨骼老化综合研究中心

An increased presence of the oxidised DNA base lesion 8-oxo-7,8-dihydroguanine (8-oxoG) is seen in muscles of old mice. 8-oxoG is repaired by the 8-oxoguanine DNA glycosylase-1 (OGG1)-initiated DNA base excision repair pathway, a process proposed to lead to activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). We hypothesised that chronic oxidative DNA damage in muscles of old mice leads to the chronic increase in activation of NF-kB by OGG1 and the subsequent production of inflammatory cytokines by skeletal muscle, contributing to age-related deterioration of muscle function.An in vitro model of oxidative DNA damage was established by treating C2C12 myoblasts with H2O2. DNA damage repair responses including 8-oxoG, OGG1, PARP-1, NF-kB activation and cytokine/chemokine gene expression and release by muscle cells were determined. The effect of TH5487 (5uM), a selective OGG1 inhibitor on NF-kB activation and cytokine production was determined. Isolated muscle fibres from adult mice were treated in a similar manner to myoblasts described above and examined for the release of cytokines/chemokines. The role of OGG1 in the chronic production of cytokines by isolated muscle fibres of old mice was examined using Luminex multiplex analysis. The effect of TH5487 on H2O2-mediated cytokine release by fibres of adult mice and on chronic release of cytokines/chemokines by fibers of old mice was examined.Cell viability was maintained in myoblasts treated with 50uM H2O2 but Ogg1 mRNA levels were significantly increased as was acetylation of OGG1, PARP-1 and cleaved PARP-1 protein levels. Activation of NF-kB was evident and accompanied by an increased expression and release of a number of pro-inflammatory cytokines (3.7 fold, 4.2 fold, 3 fold increases in IL-6 TNF-a and CXCL1 mRNA respectively). These H2O2-mediated increase in NF-kB activation and increase in cytokine/chemokine mRNA levels were normalized when cells were pretreated with TH5487 prior to treatment with H2O2.Experiments using isolated muscle fibers from adult mice demonstrated an increase in NF-kB activation following treatment with H2O2. This H2O2-mediated increase in NF-kB DNA binding activity was significantly decreased in muscle fibers pretreated with TH5487. Isolated muscle fibers from old compared with adult mice showed an increase in cytokine/chemokine release which was also reduced by treatment with TH5487. Increases in IL-6, CCL2, CXCL1, CCL7, GM-CSF, CX3CL1,CCL27,CXCL5,CCL11, CXCL16 and CXCL-12 were seen in the media of fibres from adult mice following treatment with H2O2 and IL-6,CCL2, CXCL1,CCL7, CCL27, CXCL5,CCL11,CXCL16 and CXCL-12 were decreased to normal values by pretreatment with TH5487. Isolated muscle fibers from old mice showed a significant increase in IL-6, CXCL1, CCL2, CCL7, of which all were reduced following treatment with TH5487, suggesting that OGG1 pathway may be responsible, at least in part for the increased production of cytokines/chemokines by muscle of old mice. Data on proteomic and mRNA analyses of muscles from adult and old mice for evidence of changes in DNA repair pathways with age will also be presented. A computational model of DNA damage repair is being designed to identify DNA damage and repair process therapeutic targets to modify activation of NF-kB in muscles of old mice.MRC-Arthritis Research UK Centre for Integrated Research into Musculoskeletal Ageing (CIMA)