Xi Jihao, Fu Danqing, Xu Dan, Shen Ruhan, Zhao Yan, Dai Haoqiang, Xia Chenjie, Zhou Peihong
Affiliated Cixi Hospital, Wenzhou Medical University, Ningbo, China.
Department of Orthopedic Surgery, The Affiliated Lihuili Hospital of Ningbo University, Ningbo, China.
Front Endocrinol (Lausanne). 2025 Jul 4;16:1570685. doi: 10.3389/fendo.2025.1570685. eCollection 2025.
Jinkui Shenqi Pill (JKSQP), a traditional Chinese herbal formula, is clinically utilized in China for managing bone disorders secondary to kidney deficiency, including osteoporotic fractures (OPFs). The present study aims to elucidate the pharmacological mechanism underlying JKSQP's therapeutic effects on OPF healing.
LC-MS/MS was employed to characterize the chemical constituents of JKSQP. Two-month-old female C57BL/6J mice underwent bilateral ovariectomy (OVX) followed by transverse tibial osteotomy to establish the OPF model. These OPF mice were randomly divided into the JKSQP group and OPF group, in which mice were gavaged with 1 g/kg/day JKSQP and equivalent volume of normal saline, respectively. At 4, 14, and 24 days post-fracture, biological specimens including serum, tibiae, dorsal root ganglion (DRG) and hypothalamus were collected for ELISA assay, μCT analysis and histopathology staining. Primary bone marrow stromal cells (BMSCs) were treated with the serum obtained from Sprague-Dawley rats administered with 1.5 g/kg/day JKSQP via oral gavage for three consecutive days. The conditioned medium derived from these JKSQP serum-treated BMSCs and the serum collected from the JKSQP-treated mice were applied to the DRG neurons. The levels of COX-2, PGE2, EP4 and CGRP were detected using qRT-PCR, western blot, ELISA and immunofluorescence (IF).
LC-MS/MS analysis identified 1872 chemical components in JKSQP. μCT evaluation demonstrated accelerated healing of OPF in JKSQP-treated mice. Histomorphometric analysis combined with Calcein double-labeling revealed enhanced bone formation within the fracture callus. Compared with OPF controls, mice in the JKSQP group exhibited elevated serum PGE2 levels, upregulated Osterix, COX-2 and EP4 expression in fracture callus, increased EP4 and CGRP in DRG, and enhanced p-CREB in hypothalamus. , JKSQP-containing serum increased both PGE2 secretion and COX-2 expression in BMSCs. Furthermore, qRT-PCR and IF analyses confirmed that both conditioned medium from JKSQP-treated BMSCs and serum from JKSQP-administered mice upregulated EP4 and CGRP expressions in DRG neurons.
Jinkui Shenqi Pill accelerates OPF healing by promoting bone formation possibly through activation of neurosensory PGE2/EP4/p-CREB axis.
金匮肾气丸（JKSQP）是一种传统中药配方，在中国临床上用于治疗肾虚继发的骨疾病，包括骨质疏松性骨折（OPF）。本研究旨在阐明金匮肾气丸对OPF愈合治疗作用的药理机制。
采用液相色谱-串联质谱（LC-MS/MS）对金匮肾气丸的化学成分进行表征。对2月龄雌性C57BL/6J小鼠进行双侧卵巢切除（OVX），然后进行胫骨横向截骨术以建立OPF模型。将这些OPF小鼠随机分为金匮肾气丸组和OPF组，分别给予1 g/kg/天的金匮肾气丸和等量生理盐水灌胃。在骨折后4天、14天和24天，收集血清、胫骨、背根神经节（DRG）和下丘脑等生物标本进行ELISA检测、μCT分析和组织病理学染色。用连续3天经口灌胃给予1.5 g/kg/天金匮肾气丸的Sprague-Dawley大鼠的血清处理原代骨髓间充质干细胞（BMSCs）。将这些经金匮肾气丸血清处理的BMSCs的条件培养基和经金匮肾气丸处理的小鼠收集的血清应用于DRG神经元。使用qRT-PCR、蛋白质免疫印迹、ELISA和免疫荧光（IF）检测COX-2、PGE2、EP4和CGRP的水平。
LC-MS/MS分析鉴定出金匮肾气丸中的1872种化学成分。μCT评估显示金匮肾气丸治疗的小鼠OPF愈合加速。组织形态计量学分析结合钙黄绿素双标记显示骨折痂内骨形成增强。与OPF对照组相比，金匮肾气丸组小鼠血清PGE2水平升高，骨折痂中Osterix、COX-2和EP4表达上调，DRG中EP4和CGRP增加，下丘脑p-CREB增强。含金匮肾气丸的血清增加了BMSCs中PGE2的分泌和COX-2的表达。此外，qRT-PCR和IF分析证实，经金匮肾气丸处理的BMSCs的条件培养基和经金匮肾气丸给药的小鼠的血清均上调了DRG神经元中EP4和CGRP的表达。
金匮肾气丸可能通过激活神经感觉PGE2/EP4/p-CREB轴促进骨形成从而加速OPF愈合。
