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可以看到氧化DNA碱基损伤8-氧-7,8-二氢鸟嘌呤（8-oxoG）的增加。8-oxoG通过8-氧鸟嘌呤DNA糖基化酶-1 （OGG1）启动的DNA碱基切除修复途径修复，这一过程被认为会导致活化B细胞（NF-kB）的核因子kappa-轻链增强子的激活。我们假设老年小鼠肌肉中的慢性氧化DNA损伤导致OGG1激活NF-kB的慢性增加，随后骨骼肌产生炎症细胞因子，从而导致与年龄相关的肌肉功能恶化。用H2O2处理C2C12成肌细胞，建立体外DNA氧化损伤模型。检测DNA损伤修复反应，包括8-oxoG、OGG1、PARP-1、NF-kB活化和细胞因子/趋化因子基因的表达和释放。研究了选择性OGG1抑制剂TH5487 （5uM）对NF-kB活化和细胞因子产生的影响。用与上述成肌细胞类似的方法处理成年小鼠分离的肌纤维，并检测细胞因子/趋化因子的释放。使用Luminex多重分析检测了OGG1在老年小鼠分离肌纤维慢性产生细胞因子中的作用。观察TH5487对h2o2介导的成年小鼠纤维细胞因子释放和老年小鼠纤维细胞因子/趋化因子慢性释放的影响。50uM H2O2处理的成肌细胞维持了细胞活力，但Ogg1 mRNA水平显著升高，Ogg1、PARP-1和裂解的PARP-1蛋白水平乙酰化。NF-kB的激活是明显的，并伴随着许多促炎细胞因子的表达和释放增加（IL-6 TNF-a和CXCL1 mRNA分别增加3.7倍，4.2倍和3倍）。当细胞在H2O2处理之前用TH5487预处理时，H2O2介导的NF-kB活化和细胞因子/趋化因子mRNA水平的增加是正常的。使用成年小鼠分离肌纤维进行的实验表明，H2O2处理后NF-kB活化增加。这种h2o2介导的NF-kB DNA结合活性的增加在TH5487预处理的肌纤维中显著降低。与成年小鼠相比，来自老年小鼠的分离肌纤维显示细胞因子/趋化因子释放增加，TH5487也减少了这种释放。H2O2和IL-6处理后，成年小鼠纤维介质中IL-6、CCL2、CXCL1、CCL7、GM-CSF、CX3CL1、CCL27、CXCL5、CCL11、CXCL16和cxcl12均升高，TH5487预处理后，CCL2、CXCL1、CCL7、CCL27、CXCL5、CCL11、CXCL16和cxcl12降至正常水平。老年小鼠的分离肌纤维显示IL-6、CXCL1、CCL2、CCL7显著增加，其中TH5487处理后均降低，提示OGG1途径可能至少部分地导致老年小鼠肌肉细胞因子/趋化因子的产生增加。还将介绍成年和老年小鼠肌肉的蛋白质组学和mRNA分析数据，以证明DNA修复途径随年龄的变化。一个DNA损伤修复的计算模型正在设计，以确定DNA损伤和修复过程的治疗靶点，以改变NF-kB在老年小鼠肌肉中的激活。英国关节炎研究中心肌肉骨骼老化综合研究中心（CIMA）

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)