Cao Lang, Deng Chang-Qing
Molecular Pathology Laboratory, Key Laboratory of Hunan Province for Prevention and Treatment of Integrated Traditional Chinese and Western Medicine on Cardio-cerebral Diseases, Key Laboratory of Hunan Universities for Cell biology and Molecular Techniques, Hunan University of Chinese Medicine, Changsha 410208, China.
Zhongguo Zhong Yao Za Zhi. 2016 May;41(10):1889-1897. doi: 10.4268/cjcmm20161022.
This paper was aimed to explore the effects of glycosides, the effective component of Buyang Huanwu decoction, and its main active components such as astragaloside Ⅳ, amygdalin, peoniflorin and their combinations on vascular smooth muscle cells (VSMC) proliferation, clarify the major active materials of anti-VSMC proliferation and investigate the mechanisms via the signal transduction pathway. Plasma containing drug was prepared via oral administration in rats. VSMCs of rats aorta were cultured, and then VSMC proliferation was stimulated by using platelet derived growth factor (PDGF).The plasma containing drug was added to detect the activity of cell proliferation, cell cycle and related protein expressions of signaling pathway such as extracellular signal-regulated kinase (ERK), phos-phatidylinositol-3-kinase/protein kinase B (PI3K/Akt) and Janus kinase/signal transducer and activator of transcription (JAK/STAT). After being stimulated by PDGF, the proliferation activity of VSMC was strengthened (P<0.01), G₀/G₁ phase cells were decreased (P<0.01), S/M phase cells were increased (P<0.01), and PcNA, cyclin D1 protein expressions related to cell cycle were up-regulated (P<0.01). Glycosides, astragaloside Ⅳ, amygdalin, peoniflorin and their combinations could inhibit the cell proliferation (P<0.05 or P<0.01) in a dose-effect relationship and time-effect relationship. They could increase G₀/G₁ phase cells (P<0.01), decrease S/M phase cells (P<0.01), and down-regulate the protein expressions of PCNA, cyclin D1 (P<0.01); and the effects of the combinations were greater than those of single active component (P<0.05). After VSMC proliferation was induced by PDGF, p-ERK1/2 expression was increased (P<0.01), PI3K expression was down-regulated while p-PI3K expression was up-regulated (all P<0.01), and STAT3expression was reduced while p-STAT3 expression was increased (all P<0.01). Glycosides, astragaloside Ⅳ, amygdalin, peoniflorin and the combinations of these active components could reduce p-ERK1/2 expression (P<0.05), increase PI3K expression (P<0.01), decreasep-PI3K expression (P<0.05 or P<0.01), increase STAT3 expression (P<0.01), and decrease p-STAT3 expression (P<0.05 or P<0.01). These results suggested that PDGF could induce the cell cycle conversion of VSMC, leading to VSMC proliferation. The mechanism was related to the activation of ERK, PI3K/Akt and JAK/STAT signaling pathways. Glycosides and its main active components such as astragaloside Ⅳ, amygdalin, peoniflorin and their combinations can inhibit the cell cycle conversion of VSMC, with the effect against VSMC proliferation, and the mechanisms may be associated with the inhibition of PI3K/Akt, mitogen-activated protein kinase (MAPK) and JAK/STAT signaling pathways. astragaloside Ⅳ, amygdalin and peoniflorin were the major active materials of anti-VSMC proliferation, and their combination showed enhanced effect.
本研究旨在探讨补阳还五汤有效成分糖苷及其主要活性成分黄芪甲苷Ⅳ、苦杏仁苷、芍药苷及其配伍对血管平滑肌细胞（VSMC）增殖的影响，明确抗VSMC增殖的主要活性物质，并通过信号转导通路探讨其作用机制。通过大鼠灌胃给药制备含药血浆。培养大鼠主动脉VSMCs，然后用血小板衍生生长因子（PDGF）刺激VSMC增殖。加入含药血浆检测细胞增殖活性、细胞周期及细胞外信号调节激酶（ERK）、磷脂酰肌醇-3激酶/蛋白激酶B（PI3K/Akt）和Janus激酶/信号转导子和转录激活子（JAK/STAT）等信号通路相关蛋白表达。PDGF刺激后，VSMC增殖活性增强（P<0.01），G₀/G₁期细胞减少（P<0.01），S/M期细胞增多（P<0.01），与细胞周期相关的增殖细胞核抗原（PCNA）、细胞周期蛋白D1蛋白表达上调（P<0.01）。糖苷、黄芪甲苷Ⅳ、苦杏仁苷、芍药苷及其配伍可呈剂量效应和时间效应关系抑制细胞增殖（P<0.05或P<0.01）。它们可增加G₀/G₁期细胞（P<0.01），减少S/M期细胞（P<0.01），下调PCNA、细胞周期蛋白D1蛋白表达（P<0.01）；且配伍组作用大于单一活性成分组（P<0.05）。PDGF诱导VSMC增殖后，p-ERK1/2表达增加（P<0.01），PI3K表达下调而p-PI3K表达上调（均P<0.01），STAT3表达降低而p-STAT3表达增加（均P<0.01）。糖苷、黄芪甲苷Ⅳ、苦杏仁苷、芍药苷及这些活性成分的配伍可降低p-ERK1/2表达（P<0.05），增加PI3K表达（P<0.01），降低p-PI3K表达（P<0.05或P<0.01），增加STAT3表达（P<0.01），降低p-STAT3表达（P<0.05或P<0.01）。这些结果提示，PDGF可诱导VSMC细胞周期转化，导致VSMC增殖。其机制与ERK、PI3K/Akt和JAK/STAT信号通路激活有关。糖苷及其主要活性成分黄芪甲苷Ⅳ、苦杏仁苷、芍药苷及其配伍可抑制VSMC细胞周期转化，具有抗VSMC增殖作用，其机制可能与抑制PI3K/Akt、丝裂原活化蛋白激酶（MAPK）和JAK/STAT信号通路有关。黄芪甲苷Ⅳ、苦杏仁苷和芍药苷是抗VSMC增殖的主要活性物质，其配伍显示出增强作用。
