ARC-18 Improved Motor Performance Through Inhibiting ACLY-Mediated Smad2/3 Acetylation in a Model of Duchenne Muscular Dystrophy

IF 9.1 1区医学 Q1 GERIATRICS & GERONTOLOGY

Journal of Cachexia Sarcopenia and Muscle Pub Date : 2025-10-07 DOI:10.1002/jcsm.70081

Chongyang Chen, Bingge Zhang, Chao Yang, Jing Wang, Ye He, Haitao Yu, Jianjun Liu, Yongmei Xie, Xifei Yang, Gong-Ping Liu

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引用次数: 0

Abstract

Background

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle weakness, with inflammation and fibrosis contributing to its pathogenesis. Despite advancements in genetic disease-modifying treatment, there is currently no effective pharmacological treatment for DMD.

Methods

New compound ARC-18, a derivative of Arctigenin known for its anti-inflammatory activity, was designed and synthesized in our lab and administered prophylactically to 2-month-old mdx mice for 60 days. The motor performance was investigated by rotarod test, climbing-pole test, grip strength test, hanging endurance test, treadmill endurance test and gait analysis. Afterwards, molecular biological experiments, including proteomics, immunohistochemistry, immunofluorescence, western blots, gene transfection and immunoprecipitation, were employed to investigate the molecular mechanism of ARC-18 in the treatment of mdx.

Results

ARC-18 significantly ameliorated the motor performance of DMD mice (rotating time +65.9%, p < 0.01; hanging time +59.7%, p < 0.05; grip strength +32.1%, p < 0.0001; climbing time −29.0%, p < 0.0001; numbers of electric shocks −69.3%, p < 0.01) by up-regulating the expression of dystrophin-associated proteins (dystrophin, p < 0.01; α-dystroglycan, p < 0.01) and down-regulating the expression of muscle satellite/stem cell proteins (Pax7, p < 0.05; Myod, p < 0.05; Myog, p < 0.05; α-SMA, p < 0.01; fibronectin, p < 0.001; collagen I, p < 0.05). ARC-18 prevented the progression of muscle fibrosis, reduced inflammatory factors transforming growth factor (TGF) β1 (p < 0.05), IL-1β (p < 0.05) and TNF-α (p < 0.05) levels, and promoted the structural integrity of gastrocnemius and triceps muscles. Proteomics analysis demonstrated that ARC-18 treatment reversed the protein expression pattern of DMD model mice, with ATP-citrate synthase (ACLY) enriched in the TCA cycle pathway, showing a significant correlation with DMD expression levels (R = −0.72, p = 0.00031). Further investigations revealed that ARC-18 directly bound with ACLY (EC₅₀ = 120.2 nM) to promote its degradation by the proteasome system and suppressed the ACLY-mediated acetylation of Smad2/3 (p < 0.01) to reduce its nuclear localization (p < 0.05) to inhibit fibrosis.

Conclusions

Our study indicated that oral ARC-18 treatment decelerated the progression of neuromuscular disease in a reliable DMD animal model, suggesting its potential as a promising drug for DMD.

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ARC‐18通过抑制ACLY‐介导的Smad2/3乙酰化改善杜氏肌营养不良模型的运动表现

杜氏肌营养不良症（DMD）是一种以进行性肌肉无力为特征的遗传性疾病，其发病机制与炎症和纤维化有关。尽管遗传疾病修饰治疗取得了进展，但目前尚无有效的药物治疗DMD。方法在实验室设计合成了具有抗炎活性的牛蒡子苷元衍生物ARC - 18，并对2月龄mdx小鼠进行了60天的预防性治疗。通过旋转杆试验、爬杆试验、握力试验、悬挂耐力试验、跑步机耐力试验和步态分析对运动性能进行了研究。随后，通过蛋白质组学、免疫组织化学、免疫荧光、免疫印迹、基因转染和免疫沉淀等分子生物学实验，探讨ARC‐18治疗mdx的分子机制。ResultsARC 18显著改善电机性能的DMD应承担的老鼠(旋转时间+ 65.9%,p & lt; 0.01;挂时间+ 59.7%,p & lt; 0.05;握力+ 32.1%,p & lt; 0.0001;登山时间−29.0%,p & lt; 0.0001;数字电击−69.3%,p & lt; 0.01)的量调节肌营养不良蛋白的表达量相关蛋白(肌营养不良蛋白p & lt; 0.01;α高dystroglycan p & lt; 0.01),向下调节肌肉卫星/干细胞的表达蛋白(Pax7 p & lt; 0.05; Myod, p & lt; 0.05;Myog, p < 0.05；α‐SMA, p < 0.01；纤维连接蛋白，p < 0.001；I型胶原蛋白，p < 0.05)。ARC‐18可以阻止肌肉纤维化的进展，降低炎症因子转化生长因子(TGF) β1 （p < 0.05）、IL‐1β (p < 0.05)和TNF‐α (p < 0.05)水平，促进腓肠肌和三头肌的结构完整性。蛋白质组学分析表明，ARC‐18处理逆转了DMD模型小鼠的蛋白质表达模式，TCA循环途径中ATP‐柠檬酸合成酶（ACLY）富集，与DMD表达水平显著相关（R = - 0.72, p = 0.00031）。进一步研究发现，ARC‐18直接与ACLY结合（EC50 = 120.2 nM），促进其被蛋白酶体系统降解，并抑制ACLY介导的Smad2/3乙酰化（p < 0.01），减少其核定位（p < 0.05），从而抑制纤维化。我们的研究表明，在可靠的DMD动物模型中，口服ARC‐18治疗可以减缓神经肌肉疾病的进展，这表明它有潜力成为一种有前景的DMD药物。

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来源期刊

Journal of Cachexia Sarcopenia and Muscle MEDICINE, GENERAL & INTERNAL-

CiteScore

13.30

自引率

12.40%

发文量

234

审稿时长

16 weeks

期刊介绍： The Journal of Cachexia, Sarcopenia and Muscle is a peer-reviewed international journal dedicated to publishing materials related to cachexia and sarcopenia, as well as body composition and its physiological and pathophysiological changes across the lifespan and in response to various illnesses from all fields of life sciences. The journal aims to provide a reliable resource for professionals interested in related research or involved in the clinical care of affected patients, such as those suffering from AIDS, cancer, chronic heart failure, chronic lung disease, liver cirrhosis, chronic kidney failure, rheumatoid arthritis, or sepsis.