Feng Jing, Miao Hongyan, Xiao Zijun, Xu Lei, Wang Yide, Cao Xin, Wang Junhui, Wu Zijing, Xia Ping, Liu Wei
Department of Orthopedics, Traditional Chinese and Western Medicine Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
College of Acupuncture of Orthopedics, Hubei University of Chinese Medicine, Wuhan 430061, China.
Phytomedicine. 2025 Oct;146:157113. doi: 10.1016/j.phymed.2025.157113. Epub 2025 Jul 30.
Intervertebral disc degeneration (IVDD) is a major cause of spinal disorders, marked by nucleus pulposus cell (NPC) loss and extracellular matrix (ECM) degradation. Exosomes (EXOs), as nanoscale vesicles rich in bioactive molecules, have shown promise in treating degenerative diseases. Duhuo Jisheng decoction (DHJSD), a classical traditional Chinese herbal prescription, has been reported to alleviate IVDD, yet its underlying mechanisms remain unclear.
To explore whether DHJSD alleviates IVDD via exosome-mediated delivery of miRNAs and to clarify the associated molecular pathways.
Differential miRNA expression in IVDD patient nucleus pulposus tissues was identified by bioinformatics and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Active compounds in DHJSD were identified by high-performance liquid chromatography (HPLC). EXOs were isolated from human bone marrow mesenchymal stem cells (hBMSCs) treated with DHJSD-containing serum (5.4, 10.8, 21.6 g/kg) and characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting(WB). miRNA expression in EXOs was analyzed using RT-qPCR. An in vitro IVDD model was established using interleukin-1β (10 ng/ml)-treated human NPCs. Cell viability, apoptosis, mitochondrial function, ferroptosis, and ECM markers were assessed. The interaction between miR-19b-3p and ACSL4 was confirmed by dual-luciferase assay and gain/loss-of-function experiments. Wnt/β-catenin and Hippo signaling involvement was evaluated via WB, immunofluorescence (IF), and rescue assays. In the rat IVDD model, intradiscal injections of DHJSD-EXOs were administered at weeks 1 and 5, with magnetic resonance imaging and histological assessments conducted at week 9.
miR-19b-3p was downregulated in IVDD tissues. DHJSD promoted the secretion of hBMSC-derived EXO enriched in miR-19b-3p, which enhanced NPC proliferation, suppressed apoptosis, and inhibited ferroptosis by targeting ACSL4 and concurrently modulated Wnt/β-catenin (inhibition) and Hippo (activation) pathways. In vivo, DHJSD-EXOs elevated miR-19b-3p expression, reduced ACSL4 levels, and improved disc structure and degeneration grade.
椎间盘退变（IVDD）是脊柱疾病的主要原因，其特征是髓核细胞（NPC）丢失和细胞外基质（ECM）降解。外泌体（EXOs）作为富含生物活性分子的纳米级囊泡，在治疗退行性疾病方面显示出前景。独活寄生汤（DHJSD）是一种经典的传统中药方剂，据报道可缓解IVDD，但其潜在机制仍不清楚。
探讨DHJSD是否通过外泌体介导的miRNA传递来缓解IVDD，并阐明相关的分子途径。
通过生物信息学和逆转录定量聚合酶链反应（RT-qPCR）鉴定IVDD患者髓核组织中差异表达的miRNA。采用高效液相色谱（HPLC）鉴定DHJSD中的活性成分。从用含DHJSD血清（5.4、10.8、21.6 g/kg）处理的人骨髓间充质干细胞（hBMSCs）中分离EXOs，并通过透射电子显微镜、纳米颗粒跟踪分析和蛋白质免疫印迹（WB）进行表征。使用RT-qPCR分析EXOs中的miRNA表达。使用白细胞介素-1β（10 ng/ml）处理的人NPC建立体外IVDD模型。评估细胞活力、凋亡、线粒体功能、铁死亡和ECM标志物。通过双荧光素酶报告基因检测和功能获得/丧失实验证实miR-19b-3p与ACSL4之间的相互作用。通过WB、免疫荧光（IF）和挽救实验评估Wnt/β-连环蛋白和Hippo信号通路的参与情况。在大鼠IVDD模型中，在第1周和第5周进行椎间盘内注射DHJSD-EXOs，在第9周进行磁共振成像和组织学评估。
miR-19b-3p在IVDD组织中表达下调。DHJSD促进了富含miR-19b-3p的hBMSC来源EXO的分泌，通过靶向ACSL4增强了NPC增殖、抑制了凋亡并抑制了铁死亡，同时调节了Wnt/β-连环蛋白（抑制）和Hippo（激活）信号通路。在体内，DHJSD-EXOs提高了miR-19b-3p表达，降低了ACSL4水平，并改善了椎间盘结构和退变程度。
