Huang Xiaofei, Li Sijia, Wang Huna, Zhao Lei, Li Xihua, Fan Shusheng, Hu Wanting, Tong Haowei, Guo Guangyao, Xu Dengqiu, Zhang Luyong, Jiang Zhenzhou, Yu Qinwei
New Drug Screening and Pharmacodynamics Evaluation Center, China Pharmaceutical University, Nanjing, China.
Department of Neurology Children's, Hospital of Fudan University, Shanghai, China.
J Cachexia Sarcopenia Muscle. 2025 Apr;16(2):e13807. doi: 10.1002/jcsm.13807.
Oestrogen receptor alpha (ERα) plays an important role in maintaining mitochondrial function and regulating metabolism in skeletal muscle. However, its alterations and potential mechanisms in Duchenne muscular dystrophy (DMD) remain incompletely understood. In this study, we demonstrated the protective role of ERα in myocyte for skeletal muscle regeneration in mdx mice and explored the therapeutic effects of oestrogen receptor modulators on DMD.
DMD patients' biopsies were obtained for histological analysis to explore the expression of ERα. The phenotype of muscle was analysed by histology and molecular biology. The therapeutical effect of different oestrogen receptor modulators was examined in mdx mice treated with fulvestrant (FVT, 20 mg/kg once a week) or oestradiol (E2, 1 mg/kg per day) for 4 weeks. The protective effect of ERα was performed on mdx mice after conditional knockout of ERα in skeletal muscle (ERα mdx mice). Evidence of activation of ERα/oestrogen-related receptor alpha (ERRα)/myogenic differentiation 1 (MyoD) signalling pathway was inspected in the primary myoblasts isolated from mice, and C2C12 cells received intervention with E2/FVT/Esr1-siRNA/Esrra overexpression plasmid.
The ERα expression was increased in DMD patients' triceps (p < 0.05) and mdx mice muscles (p < 0.05). FVT reduced ERα levels in the mdx mice muscles (p < 0.01) but had no significant effect on skeletal muscle regeneration on mdx mice. Compared with mdx mice, E2 reduced the levels of creatine kinase (CK) and lactic dehydrogenase (LDH) (p < 0.001) in serum, enhanced skeletal muscle function, alleviated skeletal muscle atrophy and fibre loss and upregulated the expression of ERα in GAS (p < 0.001) and TA (p < 0.05). The myogenic factors such as myosin heavy chain (MyHC, p < 0.001), myogenin (MyoG, p < 0.05), MyoD (p < 0.05) and ERRα (p < 0.001) were increased in mdx mice GAS with E2. But E2 had no effect on ERα mdx mice. The primary myoblasts and C2C12 were treated with E2 displayed an increased-on myocyte fusion index (p < 0.05), ERα MyoD and ERRα expressions (p < 0.05). The myocytes' fusion index (p < 0.05) and ERα, MyoD and ERRα expression (p < 0.05) were decreased in si-Esr1-transfected C2C12 cells and increased in OE-Esrra-transfected C2C12 cells.
We demonstrated that ERα in myocyte exerted a protective effect on skeletal muscle regeneration in DMD patients and mdx mice through the ERα-ERRα-MyoD pathway, which has potential implications for DMD therapy strategies.
雌激素受体α（ERα）在维持线粒体功能和调节骨骼肌代谢中起重要作用。然而，其在杜氏肌营养不良症（DMD）中的改变及潜在机制仍未完全阐明。在本研究中，我们证实了ERα在肌细胞中对mdx小鼠骨骼肌再生的保护作用，并探讨了雌激素受体调节剂对DMD的治疗效果。
获取DMD患者的活检组织进行组织学分析，以探究ERα的表达。通过组织学和分子生物学方法分析肌肉表型。在用氟维司群（FVT，20mg/kg，每周一次）或雌二醇（E2，1mg/kg，每日一次）治疗4周的mdx小鼠中检测不同雌激素受体调节剂的治疗效果。在骨骼肌中条件性敲除ERα后的mdx小鼠（ERα mdx小鼠）上进行ERα的保护作用研究。在从小鼠分离的原代成肌细胞中检测ERα/雌激素相关受体α（ERRα）/肌源性分化1（MyoD）信号通路激活的证据，并且C2C12细胞接受E2/FVT/Esr1-siRNA/Esrra过表达质粒干预。
DMD患者肱三头肌（p<0.05）和mdx小鼠肌肉（p<0.05）中ERα表达增加。FVT降低了mdx小鼠肌肉中的ERα水平（p<0.01），但对mdx小鼠的骨骼肌再生无显著影响。与mdx小鼠相比，E2降低了血清中肌酸激酶（CK）和乳酸脱氢酶（LDH）的水平（p<0.001），增强了骨骼肌功能，减轻了骨骼肌萎缩和纤维丢失，并上调了腓肠肌（GAS，p<0.001）和胫前肌（TA，p<0.05）中ERα的表达。E2处理的mdx小鼠GAS中肌球蛋白重链（MyHC，p<0.001）、肌细胞生成素（MyoG，p<0.05）、MyoD（p<0.05）和ERRα（p<0.001）等肌源性因子增加。但E2对ERα mdx小鼠无影响。用E2处理的原代成肌细胞和C2C12细胞的肌细胞融合指数增加（p<0.05），ERα、MyoD和ERRα表达增加（p<0.05）。在转染si-Esr1的C2C1细胞中，肌细胞融合指数（p<0.05）以及ERα、MyoD和ERRα表达（p<0.05）降低，而在转染OE-Esrra的C2C12细胞中则增加。
我们证实肌细胞中的ERα通过ERα-ERRα-MyoD途径对DMD患者和mdx小鼠的骨骼肌再生发挥保护作用，这对DMD治疗策略具有潜在意义。
