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琥珀安神汤通过激活PI3K/AKT通路加速创伤性脑损伤大鼠模型的骨折愈合。

Hu'po Anshen Decoction Accelerated Fracture-Healing in a Rat Model of Traumatic Brain Injury Through Activation of PI3K/AKT Pathway.

作者信息

Shen Jing, Li Yan-Ze, Yao Sai, Zhu Zhou-Wei, Wang Xiang, Sun Hui-Hui, Ji Wei-Feng

机构信息

Department of Orthopedics, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China.

Department of Neurology, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China.

出版信息

Front Pharmacol. 2022 Jul 18;13:952696. doi: 10.3389/fphar.2022.952696. eCollection 2022.

DOI:10.3389/fphar.2022.952696
PMID:35924045
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9341486/
Abstract

Hu'po Anshen decoction (HPASD) is a traditional Chinese medicine formula comprising five herbal medicines for the treatment of concussion and fracture healing, but its pharmacological mechanism is still unclear. Ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC/Q-TOF MS) was used to analyze the main active components of HPASD. Rats were randomly assigned to fracture group, fracture combined with traumatic brain injury (TBI) group (FBI) and FBI combined with HPASD treatment group (FBIH). Rats in the FBIH group were given oral doses of HPASD (2.4 g/kg, 4.8 g/kg and 9.6 g/kg) for 14 or 21 consecutive days. The fracture callus formation and fracture sites were determined by radiographic analysis and micron-scale computed tomography (micro-CT) analysis. Hematoxylin and eosin (H&E) staining and a three-point bending test were applied to assess histological lesions and biomechanical properties, respectively. The levels of cytokines-/protein-related to bone formation and differentiation as well as PI3K/AKT pathway-related proteins were determined by Enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription-polymerase chain reaction (qRT-PCR), or western blot assays, respectively. UPLC-Q/TOF-MS-based serum metabolomic analysis was also performed to investigate the therapeutic effects of HPASD in the treatment of FBI. UPLC/Q-TOF MS analysis showed the chemical components in HPASD, including flavonoids, amino acids, saponins, and phenylpropanoid constituents, etc. HPASD dose-dependently promoted callus formation, increased bone density, improved mechanical parameters and morphological scores, and facilitated the expressions of VEGF, PDGF, bFGF, VEGFA, CoL1A1, RUNX2, BMP2, and Aggrecan, inhibited the expression of MMP13, and activated PI3K/AKT pathway. Metabolomics analysis revealed abnormalities of malate-aspartate shuttle and glucose-alanine. HPASD accelerates fracture healing by promoting bone formation and regulating the malate-aspartate shuttle and glucose-alanine cycle, which might be associated with the activation of the PI3K/AKT pathway.

摘要

琥珀安神汤（HPASD）是一种由五味草药组成的中药方剂，用于治疗脑震荡和促进骨折愈合，但其药理机制尚不清楚。采用超高效液相色谱-四极杆飞行时间质谱联用技术（UPLC/Q-TOF MS）分析HPASD的主要活性成分。将大鼠随机分为骨折组、骨折合并创伤性脑损伤（TBI）组（FBI）和FBI合并HPASD治疗组（FBIH）。FBIH组大鼠连续14天或21天口服HPASD（2.4 g/kg、4.8 g/kg和9.6 g/kg）。通过X线分析和微米级计算机断层扫描（micro-CT）分析确定骨折痂形成和骨折部位。分别采用苏木精-伊红（H&E）染色和三点弯曲试验评估组织学损伤和生物力学性能。分别通过酶联免疫吸附测定（ELISA）、定量逆转录-聚合酶链反应（qRT-PCR）或蛋白质免疫印迹法检测与骨形成和分化相关的细胞因子/蛋白质以及PI3K/AKT通路相关蛋白的水平。还进行了基于UPLC-Q/TOF-MS的血清代谢组学分析，以研究HPASD对FBI的治疗作用。UPLC/Q-TOF MS分析显示HPASD中的化学成分，包括黄酮类、氨基酸、皂苷和苯丙素类成分等。HPASD剂量依赖性地促进骨痂形成，增加骨密度，改善力学参数和形态学评分，并促进VEGF、PDGF、bFGF、VEGFA、CoL1A1、RUNX2、BMP2和聚集蛋白聚糖的表达，抑制MMP13的表达，并激活PI3K/AKT通路。代谢组学分析揭示了苹果酸-天冬氨酸穿梭和葡萄糖-丙氨酸的异常。HPASD通过促进骨形成和调节苹果酸-天冬氨酸穿梭和葡萄糖-丙氨酸循环来加速骨折愈合，这可能与PI3K/AKT通路的激活有关。

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