目的 探讨巴戟天Morinda officinalis中环烯醚萜类部位对衰老性肌少症的治疗作用及机制。方法 以水晶兰苷和去乙酰基车叶草苷酸为指标，考察不同体积分数乙醇对巴戟天中环烯醚萜类部位加热回流提取效率的影响，提取液经减压浓缩后，采用大孔吸附树脂富集纯化，获得总环烯醚萜类部位。通过比较不同极性洗脱溶剂的洗脱效果，优化纯化工艺参数，建立并验证高效液相色谱（high performance liquid chromatography，HPLC）分析方法后，对有效部位中主要环烯醚萜类成分进行含量测定，所得纯化部位用于后续药效学评价。利用C2C12细胞分化模型及D-半乳糖（D-galactose，D-gal）诱导的C2C12肌管细胞萎缩模型，以肌球蛋白重链（myosin heavy chain，MYHC）蛋白表达为指标，评价环烯醚萜类部位促进肌管分化及改善C2C12细胞肌管萎缩的作用。采用D-gal诱导的小鼠加速衰老模型，测量小鼠拉力及后肢肌肉质量，苏木素-伊红（hematoxylin-eosin，HE）染色观察胫骨前肌及腓肠肌病理变化，研究环烯醚萜类部位的治疗效果。利用转录组测序对各组小鼠腓肠肌进行差异基因分析，并综合网络药理学分析提示，进一步通过Western blotting及qRT-PCR验证转录组测序结果中相关差异基因表达变化。结果 60%乙醇加热回流提取巴戟天，环烯醚萜类部位含量最高。纯化工艺中，H103大孔吸附树脂吸附性能最佳，且以水为洗脱溶剂时目标成分洗脱率最高。巴戟天环烯醚萜类部位能显著改善D-gal诱导的C2C12细胞萎缩（P＜0.001），并促进肌管细胞分化（P＜0.05、0.01、0.001）。环烯醚萜类部位可显著提高加速衰老小鼠的四肢拉力和后肢肌肉质量与体质量比值（P＜0.05、0.01、0.001），显著增加衰老小鼠胫骨前肌和腓肠肌肌肉横截面积（P＜0.01、0.001），进而改善衰老相关的肌少症表型。进一步机制研究发现，环烯醚萜类部位可上调磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）/蛋白激酶B（protein kinase B，Akt）/哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）信号通路关键蛋白的表达水平（P＜0.05、0.01、0.001），并显著上调磷脂酰肌醇-4,5-二磷酸3激酶催化亚基α（phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha，PIK3CA）、丝氨酸/苏氨酸激酶1（serine/threonine kinase 1，AKT1）、mTOR基因表达水平（P＜0.05、0.01、0.001），且PI3K抑制剂LY294002显著抑制环烯醚萜类部位对PI3K/Akt/mTOR信号通路相关蛋白表达的上调作用（P＜0.05）。结论 巴戟天中环烯醚萜类部位可显著改善衰老引起的肌少症，其作用机制与调控PI3K/Akt/mTOR信号通路有关。

Objective To investigate the therapeutic effect and mechanism of iridoid fraction from Morinda officinalis on aging-related sarcopenia. Methods Using monotropein and deacetylasperulosidic acid as analytical markers, effects of different ethanol concentrations on extraction efficiency of active iridoids fraction from M. officinalis via heated reflux extraction was investigated. The extract was concentrated under reduced pressure and subsequently enriched and purified using macroporous adsorption resin to obtain the total active iridoid fraction. By comparing the elution efficiencies of solvents with varying polarities, purification parameters were optimized. Following the establishment and validation of a high performance liquid chromatography (HPLC) analytical method, the content of major iridoid components in active fraction was quantified. The purified fraction was then used for subsequent pharmacodynamic evaluations. The C2C12 cell differentiation model and a D-galactose (D-gal)-induced C2C12 myotube atrophy model were employed to assess the effects of iridoid fraction on promoting myotube differentiation and ameliorating myotube atrophy, with MYHC protein expression serving as the indicator. A D-gal-induced accelerated aging mouse model was utilized to evaluate the therapeutic effects of iridoid fraction through grip strength tests, hindlimb muscle mass measurements, and hematoxylin-eosin (HE) staining of tibialis anterior and gastrocnemius muscles. Differentially expressed genes in gastrocnemius muscle of mice in each group was analyzed by transcriptome sequencing, and the results were further verified by Western blotting and qRT-PCR for the expression changes of related differentially expressed genes indicated by transcriptome sequencing, in combination with network pharmacology analysis. Results The extraction of iridoid components from M. officinalis was optimized using a 60% ethanol solution under heated reflux conditions, resulting in the highest iridoid content. In the purification process, H103 macroporous adsorption resin demonstrated superior adsorption performance, with water as the eluent resulting in the highest elution rate of target constituents. The iridoid fraction from M. officinalis significantly ameliorated D-gal-induced atrophy in C2C12 myotubes (P < 0.001), and promoted myotube differentiation (P < 0.05, 0.01, 0.001). The iridoid fraction could significantly enhance the limb pulling force and ratio of hind limb muscle mass to body mass of accelerated aging mice (P < 0.05, 0.01, 0.001), significantly increase the cross-sectional area of the tibialis anterior muscle and gastrocnemius muscle in aging mice (P < 0.01, 0.001), thereby improving the phenotype of age-related sarcopenia. Further mechanism studies revealed that iridoid fraction could up-regulate the expression levels of key proteins in phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway (P < 0.05, 0.01, 0.001), and significantly up-regulate the expression levels of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), serine/threonine kinase 1 (AKT1) and mTOR genes (P < 0.05, 0.01, 0.001). And PI3K inhibitor LY294002 significantly inhibited the upregulation of PI3K/Akt/mTOR signaling pathway related protein expressions by iridoid fraction (P < 0.05). Conclusion The iridoid fraction of M. officinalis can significantly improve sarcopenia caused by aging, and its mechanism is related to the regulation of PI3K/Akt/mTOR signaling pathway.

R285.5

江苏省医院中药制剂融合创新中心项目（2024YYZYZJRCZX）；江苏省老年健康项目（LKM2025007）；南京中医药大学自然科学基金重点项目（2025039）