Yao Baojin, Liu Jia, Xu Duoduo, Pan Daian, Zhang Mei, Zhao Daqing, Leng Xiangyang
1Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun, 130117 Jilin China.
2College of Pharmacy, Changchun University of Chinese Medicine, Changchun, 130117 Jilin China.
Chin Med. 2019 Aug 27;14:29. doi: 10.1186/s13020-019-0252-y. eCollection 2019.
Guzhi Zengsheng Zhitongwan (GZZSZTW) is an effective formula of traditional Chinese herbal medicine and has been widely applied in the treatment of joint diseases for many years. The aim of this study was to dissect the molecular targets and signaling pathways of Guzhi Zengsheng Zhitongwan based on the analysis of serum proteomics.
The Chinese herbs of GZZSZTW were immersed in 5 l distilled water and boiled with reflux extraction method. The extract was filtered, concentrated and freeze-dried. The chemical profile of GZZSZTW extract was determined by high-performance lipid chromatography (HPLC). The 7-week old Sprague-Dawley (SD) rats in GZZSZTW groups were received oral administration at doses of 0.8, 1.05, and 1.3 g/kg per day and the rats in blank group were fed with drinking water. Serum samples were collected from the jugular veins. Primary chondrocyte viability was evaluated by CCK-8 assay. A full spectrum of the molecular targets and signaling pathways of GZZSZTW were investigated by isobaric tags for relative and absolute quantitation (iTRAQ) analysis and a systematic bioinformatics analysis accompanied with parallel reaction monitoring (PRM) and siRNA validation.
GZZSZTW regulated a series of functional proteins and signaling pathways responsible for cartilage development, growth and repair. Functional classification analysis indicated that these proteins were mainly involved in the process of cell surface dynamics. Pathway analysis mapped these proteins into several signalling pathways involved in chondrogenesis, chondrocyte proliferation and differentiation, and cartilage repair, including hippo signaling pathway, cGMP-PKG signaling pathway, cell cycle and calcium signaling pathway. Protein-protein interaction analysis and siRNA knockdown assay identified an interaction network consisting of TGFB1, RHO GTPases, ILK, FLNA, LYN, DHX15, PKM, RAB15, RAB1B and GIPC1.
Our results suggest that the effects of GZZSZTW on treating joint diseases might be achieved through the TGFB1/RHO interaction network coupled with other proteins and signaling pathways responsible for cartilage development, growth and repair. Therefore, the present study has greatly expanded our knowledge and provided scientific support for the underlying therapeutic mechanisms of GZZSZTW on treating joint diseases. It also provided possible alternative strategies for the prevention and treatment for joint diseases by using traditional Chinese herbal formulas.
骨质增生止痛丸（GZZSZTW）是一种有效的中药配方，多年来已广泛应用于关节疾病的治疗。本研究的目的是基于血清蛋白质组学分析剖析骨质增生止痛丸的分子靶点和信号通路。
将骨质增生止痛丸的中药材浸入5升蒸馏水中，采用回流提取法煮沸。提取物经过过滤、浓缩和冻干。通过高效液相色谱（HPLC）测定骨质增生止痛丸提取物的化学图谱。骨质增生止痛丸组7周龄的Sprague-Dawley（SD）大鼠每天按0.8、1.05和1.3 g/kg的剂量口服给药，空白组大鼠饮用蒸馏水。从颈静脉采集血清样本。通过CCK-8法评估原代软骨细胞活力。通过相对和绝对定量等压标签（iTRAQ）分析以及伴随平行反应监测（PRM）和siRNA验证的系统生物信息学分析，研究骨质增生止痛丸的全部分子靶点和信号通路。
骨质增生止痛丸调节了一系列负责软骨发育、生长和修复的功能蛋白和信号通路。功能分类分析表明，这些蛋白主要参与细胞表面动力学过程。通路分析将这些蛋白映射到几个参与软骨形成、软骨细胞增殖和分化以及软骨修复的信号通路中，包括河马信号通路、cGMP-PKG信号通路、细胞周期和钙信号通路。蛋白质-蛋白质相互作用分析和siRNA敲低试验确定了一个由TGFB1、RHO GTPases、ILK、FLNA、LYN、DHX15、PKM、RAB15、RAB1B和GIPC1组成的相互作用网络。
我们的结果表明，骨质增生止痛丸治疗关节疾病的作用可能是通过TGFB1/RHO相互作用网络以及其他负责软骨发育、生长和修复的蛋白质和信号通路实现的。因此，本研究极大地扩展了我们的知识，并为骨质增生止痛丸治疗关节疾病的潜在治疗机制提供了科学支持。它还为使用中药配方预防和治疗关节疾病提供了可能的替代策略。
