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Abstract：
The progressive deterioration of tissue-tissue crosstalk with aging causes a striking impairment of tissue homeostasis and functionality, particularly in the musculoskeletal system. Rejuvenation of the systemic and local milieu via interventions such as heterochronic parabiosis and exercise has been reported to improve musculoskeletal homeostasis in aged organisms. We have shown that Ginkgolide B (GB), a small molecule from Ginkgo biloba, improves bone homeostasis in aged mice by restoring local and systemic communication, implying a potential for maintaining skeletal muscle homeostasis and enhancing regeneration. In this study, we investigated the therapeutic efficacy of GB on skeletal muscle regeneration in aged mice.
Muscle injury models were established by barium chloride induction into the hind limb of 20-month-old mice (aged mice) and into C2C12-derived myotubes. Therapeutic efficacy of daily administrated GB (12 mg/kg body weight) and osteocalcin (50 μg/kg body weight) on muscle regeneration was assessed by histochemical staining, gene expression, flow cytometry, ex vivo muscle function test and rotarod test. RNA sequencing was used to explore the mechanism of GB on muscle regeneration, with subsequent in vitro and in vivo experiments validating these findings.
GB administration in aged mice improved muscle regeneration (muscle mass, P = 0.0374; myofiber number/field, P = 0.0001; centre nucleus, embryonic myosin heavy chain-positive myofiber area, P = 0.0144), facilitated the recovery of muscle contractile properties (tetanic force, P = 0.0002; twitch force, P = 0.0005) and exercise performance (rotarod performance, P = 0.002), and reduced muscular fibrosis (collagen deposition, P < 0.0001) and inflammation (macrophage infiltration, P = 0.03). GB reversed the aging-related decrease in the expression of osteocalcin (P < 0.0001), an osteoblast-specific hormone, to promote muscle regeneration. Exogenous osteocalcin supplementation was sufficient to improve muscle regeneration (muscle mass, P = 0.0029; myofiber number/field, P < 0.0001), functional recovery (tetanic force, P = 0.0059; twitch force, P = 0.07; rotarod performance, P < 0.0001) and fibrosis (collagen deposition, P = 0.0316) in aged mice, without an increased risk of heterotopic ossification.
GB treatment restored the bone-to-muscle endocrine axis to reverse aging-related declines in muscle regeneration and thus represents an innovative and practicable approach to managing muscle injuries. Our results revealed the critical and novel role of osteocalcin-GPRC6A-mediated bone-to-muscle communication in muscle regeneration, which provides a promising therapeutic avenue in functional muscle regeneration.
Muscle injury models were established by barium chloride induction into the hind limb of 20-month-old mice (aged mice) and into C2C12-derived myotubes. Therapeutic efficacy of daily administrated GB (12 mg/kg body weight) and osteocalcin (50 μg/kg body weight) on muscle regeneration was assessed by histochemical staining, gene expression, flow cytometry, ex vivo muscle function test and rotarod test. RNA sequencing was used to explore the mechanism of GB on muscle regeneration, with subsequent in vitro and in vivo experiments validating these findings.
GB administration in aged mice improved muscle regeneration (muscle mass, P = 0.0374; myofiber number/field, P = 0.0001; centre nucleus, embryonic myosin heavy chain-positive myofiber area, P = 0.0144), facilitated the recovery of muscle contractile properties (tetanic force, P = 0.0002; twitch force, P = 0.0005) and exercise performance (rotarod performance, P = 0.002), and reduced muscular fibrosis (collagen deposition, P < 0.0001) and inflammation (macrophage infiltration, P = 0.03). GB reversed the aging-related decrease in the expression of osteocalcin (P < 0.0001), an osteoblast-specific hormone, to promote muscle regeneration. Exogenous osteocalcin supplementation was sufficient to improve muscle regeneration (muscle mass, P = 0.0029; myofiber number/field, P < 0.0001), functional recovery (tetanic force, P = 0.0059; twitch force, P = 0.07; rotarod performance, P < 0.0001) and fibrosis (collagen deposition, P = 0.0316) in aged mice, without an increased risk of heterotopic ossification.
GB treatment restored the bone-to-muscle endocrine axis to reverse aging-related declines in muscle regeneration and thus represents an innovative and practicable approach to managing muscle injuries. Our results revealed the critical and novel role of osteocalcin-GPRC6A-mediated bone-to-muscle communication in muscle regeneration, which provides a promising therapeutic avenue in functional muscle regeneration.
摘要：
背景：随着年龄的增长，组织-组织串扰的逐渐恶化会导致组织稳态和功能的显着损害，特别是在肌肉骨骼系统中。据报道，通过异慢性共生和运动等干预措施使全身和局部环境恢复活力，可以改善老年生物的肌肉骨骼稳态。我们已经证明银杏内酯B(GB)，一个来自银杏的小分子,通过恢复局部和全身通讯改善老年小鼠的骨骼稳态，暗示着维持骨骼肌稳态和增强再生的潜力。在这项研究中,我们研究了GB对老年小鼠骨骼肌再生的治疗效果。
方法:通过将氯化钡诱导到20月龄小鼠(老年小鼠)的后肢和C2C12来源的肌管中,建立肌肉损伤模型。通过组织化学染色评估每日给药GB（12mg/kg体重）和骨钙蛋白（50μg/kg体重）对肌肉再生的治疗效果，基因表达，流式细胞术,离体肌肉功能试验和旋转试验。RNA测序用于探索GB对肌肉再生的作用机制。随后的体外和体内实验验证了这些发现。
结果：GB在老年小鼠中的给药改善了肌肉再生（肌肉质量，P=0.0374；肌纤维数/场，P=0.0001；中心核，胚胎肌球蛋白重链阳性肌纤维区，P=0.0144),促进肌肉收缩特性的恢复(强直力，P=0.0002;抽搐力,P=0.0005)和运动性能(旋转性能，P=0.002),和减少肌肉纤维化（胶原蛋白沉积，P<0.0001)和炎症(巨噬细胞浸润，P=0.03)。GB逆转了衰老相关的骨钙蛋白表达下降（P<0.0001），一种成骨细胞特异性激素,促进肌肉再生。外源性骨钙蛋白补充足以改善肌肉再生(肌肉质量，P=0.0029；肌纤维数/场，P<0.0001),功能恢复(强直力，P=0.0059;抽搐力,P=0.07;转杆性能,P<0.0001)和纤维化(胶原沉积，P=0.0316）在老年小鼠中，不会增加异位骨化的风险。
结论：GB治疗恢复了骨-肌肉内分泌轴，以逆转与衰老相关的肌肉再生下降，因此代表了一种管理肌肉损伤的创新和可行的方法。我们的结果揭示了骨钙蛋白-GPRC6A介导的骨-肌肉通讯在肌肉再生中的关键和新作用，这为功能性肌肉再生提供了有希望的治疗途径。
方法:通过将氯化钡诱导到20月龄小鼠(老年小鼠)的后肢和C2C12来源的肌管中,建立肌肉损伤模型。通过组织化学染色评估每日给药GB（12mg/kg体重）和骨钙蛋白（50μg/kg体重）对肌肉再生的治疗效果，基因表达，流式细胞术,离体肌肉功能试验和旋转试验。RNA测序用于探索GB对肌肉再生的作用机制。随后的体外和体内实验验证了这些发现。
结果：GB在老年小鼠中的给药改善了肌肉再生（肌肉质量，P=0.0374；肌纤维数/场，P=0.0001；中心核，胚胎肌球蛋白重链阳性肌纤维区，P=0.0144),促进肌肉收缩特性的恢复(强直力，P=0.0002;抽搐力,P=0.0005)和运动性能(旋转性能，P=0.002),和减少肌肉纤维化（胶原蛋白沉积，P<0.0001)和炎症(巨噬细胞浸润，P=0.03)。GB逆转了衰老相关的骨钙蛋白表达下降（P<0.0001），一种成骨细胞特异性激素,促进肌肉再生。外源性骨钙蛋白补充足以改善肌肉再生(肌肉质量，P=0.0029；肌纤维数/场，P<0.0001),功能恢复(强直力，P=0.0059;抽搐力,P=0.07;转杆性能,P<0.0001)和纤维化(胶原沉积，P=0.0316）在老年小鼠中，不会增加异位骨化的风险。
结论：GB治疗恢复了骨-肌肉内分泌轴，以逆转与衰老相关的肌肉再生下降，因此代表了一种管理肌肉损伤的创新和可行的方法。我们的结果揭示了骨钙蛋白-GPRC6A介导的骨-肌肉通讯在肌肉再生中的关键和新作用，这为功能性肌肉再生提供了有希望的治疗途径。
