Meng Shilong, Zhang Xu, Yu Yang, Tong Minghao, Yuan Yifeng, Cao Yanguang, Zhang Wei, Shi Xiaolin, Liu Kang
The Second Clinical School, Zhejiang Chinese Medical University, Zhejiang, Hangzhou, 310053, China.
The Second Affiliated Hospital, Zhejiang Chinese Medical University, Zhejiang, Hangzhou, 310005, China.
Adv Biol (Weinh). 2025 Mar;9(3):e2400166. doi: 10.1002/adbi.202400166. Epub 2024 Jun 27.
New-QiangGuYin (N-QGY), the addition of sea buckthorn on the basis of QGY formula, is herbal formula widely used clinically in China for the treatment of osteoporosis (OP), but its mechanism warrants further exploration. The mechanisms of QGY and N-QGY in the treatment of OP are probed from the perspective of osteoclast-osteoblast balance. Thirty Sprague-Dawley rats are randomly divided into N-QGY group, QGY group, and Control group. Beyond control rats that orally took normal saline, other rats are orally administered with isometric N-QGY or QGY twice every day for 3 days. The drug-containing serum and control serum are prepared and their effects on osteoclast-derived exosome secretion are determined by bicinchoninic acid assay (BCA), nanoparticle tracking analysis, and Western blot. GW4869 and Interleukin-1β (IL-1β) are adopted as the exosome inhibitor and inducer, respectively. Exosome uptake, cell counting kit-8, alkaline phosphatase (ALP) staining, alizarin red staining, enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, and Western blot are performed to examine the effects of altered osteoclast exosome content on osteogenic differentiation of mesenchymal stem cells (MSCs). N-QGY, QGY, and GW4869 inhibit osteoclast-derived exosome secretion and exosome uptake by MSCs, whereas IL-1β exerted the opposite effects (p < 0.05). Different from IL-1β, N-QGY, QGY, and GW4869 partially elevated MSC viability, osteocalcin secretion, ALP, RUNX Family Transcription Factor 2 (RUNX2) and Osteopontin (OPN) expressions, and calcium deposition in the osteoclast-MSCs coculture system (p < 0.05). Mechanically, osteoclasts increased Notum protein level but decreased β-catenin level, which is enhanced by IL-1β but is reversed by GW4869, QGY, and N-QGY (p < 0.05). And the effect of N-QGY is more conspicuous than that of QGY (P<0.05). N-QGY-containing serum inhibits exosome levels in osteoclasts, thereby enhancing osteogenic differentiation of MSCs via inhibition of Notum protein and promotion of β-catenin protein.
新强骨饮（N-QGY）是在强骨饮（QGY）配方基础上添加了沙棘的中药配方，在中国临床上广泛用于治疗骨质疏松症（OP），但其作用机制有待进一步探索。从破骨细胞-成骨细胞平衡的角度探讨QGY和N-QGY治疗OP的机制。将30只Sprague-Dawley大鼠随机分为N-QGY组、QGY组和对照组。除口服生理盐水的对照大鼠外，其他大鼠每天口服等剂量的N-QGY或QGY两次，共3天。制备含药血清和对照血清，采用二喹啉甲酸法（BCA）、纳米颗粒跟踪分析和蛋白质免疫印迹法测定其对破骨细胞来源外泌体分泌的影响。分别采用GW4869和白细胞介素-1β（IL-1β）作为外泌体抑制剂和诱导剂。进行外泌体摄取、细胞计数试剂盒-8、碱性磷酸酶（ALP）染色、茜素红染色、酶联免疫吸附测定、定量实时聚合酶链反应和蛋白质免疫印迹法，以检测破骨细胞外泌体含量改变对间充质干细胞（MSCs）成骨分化的影响。N-QGY、QGY和GW4869抑制破骨细胞来源的外泌体分泌以及MSCs对外泌体的摄取，而IL-1β则产生相反的作用（p<0.用05）。与IL-1β不同，N-QGY、QGY和GW4869在破骨细胞-MSCs共培养系统中部分提高了MSCs的活力、骨钙素分泌、ALP、RUNX家族转录因子2（RUNX2）和骨桥蛋白（OPN）的表达以及钙沉积（p<0.05）。机制上，破骨细胞增加了Notum蛋白水平，但降低了β-连环蛋白水平，IL-1β增强了这种作用，而GW4869、QGY和N-QGY则使其逆转（p<0.05）。并且N-QGY的作用比QGY更显著（P<0.05）。含N-QGY的血清抑制破骨细胞中的外泌体水平，从而通过抑制Notum蛋白和促进β-连环蛋白蛋白来增强MSCs的成骨分化。
