靶向性失调 CD38/NAD + 稳态抑制多器官纤维化
Targeting Dysregulated CD38/NAD+ Homeostasis Mitigates Multiple Organ Fibrosis
Background/Purpose: Persistent myofibroblast activation underlies unresolving multi-organ fibrosis in systemic sclerosis (SSc). We had shown that fibrosis is associated with reduced expression of NAD-dependent sirtuins (SIRTs) 1 and 3. The activity of SIRTs is determined by availability of NAD+, which in turn is regulated by CD38, the main NAD-consuming enzyme. We had shown that during chronological aging, up-regulation of CD38 drives cellular NAD+ consumption, resulting in reduced SIRT activity, mitochondrial dysfunction, cellular metabolic collapse and senescence. We sought to test the hypothesis that biological aging is accelerated in SSc, and causes NAD+ depletion and metabolic imbalance contributing to cellular senescence and fibrosis. We also hypothesize restoring NAD+ homeostasis will prevent fibrosis in mouse model.
背景/目的: 持续的肌成纤维细胞活化是系统性硬化症(SSc)多器官纤维化解决不了的基础。我们已经证实，纤维化与 nad- 依赖性 sirtuins (SIRTs)1和3的表达减少有关。SIRTs 的活性取决于 NAD + 的有效性，而 NAD + 又受到主要的 NAD- 消耗酶 CD38的调节。我们已经发现，在时间衰老过程中，CD38的上调促进了细胞 NAD + 的消耗，导致 SIRT 活性降低，线粒体功能障碍，细胞代谢崩溃和衰老。我们试图验证这一假设: 衰老(生物)在 SSc 中加速，导致 NAD + 损耗和代谢失衡，从而促进细胞衰老和纤维化。我们还假设恢复 NAD + 稳态可以防止小鼠模型的纤维化。
Methods: Expression and regulation of NAD+ metabolic pathway enzymes and fibrotic pathway genes was examined in human and mouse SSc transcriptomes, tissue biopsies, and explanted fibroblasts. Cellular senescence was investigated by immunostaining. Fibrosis and inflammation induced by chronic subcutaneous bleomycin were examined in aged mice treated with the orally available NAD+ precursor nicotinamide riboside (NR), and a novel potent and selective CD38 inhibitor (78c). <>Results: The number of p16-positive senescent cells was elevated in SSc skin biopsies. Moreover, levels of CD38 transcript and protein were increased in SSc skin biopsies as well as explanted skin fibroblasts compared to matched controls. CD38 expression was also elevated in skin from mice with fibrosis. Notably, CD38 levels showed strong correlation with the TGF-ß signature (p<0.0001, r=0.42) as well as Modified Rodnan skin score (p<0.003, r =0.34), while an anti-correlation with SIRT activity was observed. Overexpressing CD38 lowered cellular SIRT activity and augmented, while CD38 inhibition diminished, fibrotic responses in cultured fibroblasts. NR alone, or combined with the CD38 inhibitor 78c, abrogated TGF-ß-mediated fibrotic responses and Smad-dependent transcriptional activity in fibroblasts. Treatment of aged mice with 78c alone, or combined with NR supplementation, attenuated fibrosis and inflammation in the skin and lungs. These anti-fibrotic effects were correlated with increased tissue levels of NAD+.
方法: 在人和小鼠 SSc 转录组、组织活检和移植的成纤维细胞中检测 NAD + 代谢途径和纤维化途径基因的表达和调控。免疫组织化学染色观察细胞衰老。用口服 NAD + 烟酰胺前体核糖苷(NR)和新型高效选择性 CD38抑制剂(78c)治疗老龄小鼠，观察慢性皮下博莱霉素诱导的肝纤维化和炎症反应。结果: 在 SSc 皮肤活检组织中 p16阳性衰老细胞数明显增多。此外，与匹配对照组相比，移植皮肤成纤维细胞和 SSc 皮肤活检组织中 CD38转录体和蛋白水平增加。CD38在纤维化小鼠皮肤中的表达也升高。CD38水平与 tgf-β 信号及修饰的 Rodnan 皮肤评分呈显著正相关(p < 0.0001，r = 0.42) ，与 SIRT 活性呈显著负相关(p < 0.003，r = 0.34)。过度表达 CD38降低细胞 SIRT 活性并增强，而 CD38抑制作用减弱，成纤维细胞的纤维化反应。单独使用 NR 或与 CD38抑制剂78c 联合使用，可抑制 tgf-β 介导的纤维化反应和 smad 依赖的转录活性。单用78c 或联合补充硝酸还原酶治疗老年小鼠，可减轻皮肤和肺部的纤维化和炎症。这些抗纤维化作用与组织 NAD + 水平升高有关。
Conclusion: Expression of CD38 and other NAD-consuming enzymes is elevated in fibrotic skin. Elevated CD38 potentially causes cellular NAD+ depletionand reduced SIRT activity with protein hyperacetylation. SIRT dysfunction in turn contributes to persistent fibroblast activation and senescence underlying unresolving fibrosis (Fig.1). Restoring NAD+ homeostasis through CD38 inhibition and NR supplementation ameliorated fibrosis in mouse models and could represent a novel strategy for the treatment of SSc.
结论: 纤维化皮肤组织中 CD38及其他 nad- 消耗酶的表达增加。CD38升高可能导致细胞 NAD + 缺失和蛋白质高乙酰化导致 SIRT 活性降低。SIRT 功能障碍反过来促进持续的成纤维细胞激活和衰老的基础上解决纤维化(图1)。通过抑制 CD38和补充硝酸还原酶改善小鼠肝纤维化，恢复 NAD + 内环境稳定性，可能是治疗 SSc 的新策略。