Abstract：
BACKGROUND: Tongluo Shenggu Capsule (TLSGC) is a product of Traditional Chinese patent medicine that has been effective in glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) clinically for many years. It is made from water extracts of a well-used herbal and dietary supplement-pigeon pea leaves. Nevertheless, the material basis and pharmacological mechanisms of TLSGC ameliorating GIONFH needed to be better defined.
OBJECTIVE: To investigate the material basis and pharmacological mechanisms of TLSGC to ameliorate GIONFH.
METHODS: The chemical compositions in TLSGC were characterized using the LC-MS system. Based on integrating the relevant targets of TLSGC in MedChem Studio software and GIONFH-related genes in our previous work, a \"drug targets-disease genes\" interaction network was constructed. The candidate targets of TLSGC ameliorating GIONFH were filtrated by topological characteristic parameters and further experimental validated based on methylprednisolone-induced rat model and dexamethasone-inhibited human umbilical vein endothelial cells (HUVECs).
RESULTS: A total of 33 chemical compositions were characterized in TLSGC. Based on these compositions and GIONFH-related genes, 122 hub genes were selected according to topological parameters calculation. Biological functions were mainly enriched in four over-expressed modules of vascular damage, inflammation and apoptosis, bone metabolism and energy metabolism. The hub genes had the maximum enrichment degree in the VEGF-VEGFR2-PKC-Raf1-MEK-ERK signaling axis of the VEGF pathway. Experimentally, the therapeutic effects of TLSGC against GIONFH in rats were proved by micro-CT and pathological examination. Then, the protective effects of TLSGC on vascular damage were determined using angiography, CD31 immunohistochemistry, vascular function indicators in vivo, aortic ring test ex vivo, and the HUVECs activities in vitro including migration, invasion and tube formation. Mechanically, TLSGC effectively suppressed the downregulation of VEGF and VEGFR2 and their downstream targets, including Raf-1, PKC, p-MEK, and p-ERK proteins both in vivo and in vitro.
CONCLUSIONS: TLSGC could promote angiogenesis by upregulating the VEGF-VEGFR2-PKC-Raf-1-MEK-ERK signaling axis, thereby exerting an apparent curative effect on GIONFH.
OBJECTIVE: To investigate the material basis and pharmacological mechanisms of TLSGC to ameliorate GIONFH.
METHODS: The chemical compositions in TLSGC were characterized using the LC-MS system. Based on integrating the relevant targets of TLSGC in MedChem Studio software and GIONFH-related genes in our previous work, a \"drug targets-disease genes\" interaction network was constructed. The candidate targets of TLSGC ameliorating GIONFH were filtrated by topological characteristic parameters and further experimental validated based on methylprednisolone-induced rat model and dexamethasone-inhibited human umbilical vein endothelial cells (HUVECs).
RESULTS: A total of 33 chemical compositions were characterized in TLSGC. Based on these compositions and GIONFH-related genes, 122 hub genes were selected according to topological parameters calculation. Biological functions were mainly enriched in four over-expressed modules of vascular damage, inflammation and apoptosis, bone metabolism and energy metabolism. The hub genes had the maximum enrichment degree in the VEGF-VEGFR2-PKC-Raf1-MEK-ERK signaling axis of the VEGF pathway. Experimentally, the therapeutic effects of TLSGC against GIONFH in rats were proved by micro-CT and pathological examination. Then, the protective effects of TLSGC on vascular damage were determined using angiography, CD31 immunohistochemistry, vascular function indicators in vivo, aortic ring test ex vivo, and the HUVECs activities in vitro including migration, invasion and tube formation. Mechanically, TLSGC effectively suppressed the downregulation of VEGF and VEGFR2 and their downstream targets, including Raf-1, PKC, p-MEK, and p-ERK proteins both in vivo and in vitro.
CONCLUSIONS: TLSGC could promote angiogenesis by upregulating the VEGF-VEGFR2-PKC-Raf-1-MEK-ERK signaling axis, thereby exerting an apparent curative effect on GIONFH.
摘要：
背景：通络生骨胶囊（TLSGC）是中成药产品，多年来在临床上有效治疗糖皮质激素性股骨头坏死（GIONFH）。它是由一种常用的草药和膳食补充剂-木豆叶的水提取物制成的。然而，需要更好地确定TLSGC改善GIONFH的物质基础和药理机制。
目的：探讨TLSGC改善GIONFH的物质基础和药理机制。
方法:使用LC-MS系统表征TLSGC中的化学组成。在整合MedChemStudio软件中TLSGC相关靶点和GIONFH相关基因的基础上,构建了“药物靶标-疾病基因”相互作用网络。通过拓扑特征参数筛选TLSGC改善GIONFH的候选靶标，并基于甲基强的松龙诱导的大鼠模型和地塞米松抑制的人脐静脉内皮细胞（HUVECs）进行进一步的实验验证。
结果：在TLSGC中表征了总共33种化学组成。基于这些组成和GIONFH相关基因，根据拓扑参数计算选择122个hub基因。生物学功能主要富集在4个血管损伤过表达模块中,炎症和细胞凋亡，骨代谢和能量代谢。hub基因在VEGF通路的VEGF-VEGFR2-PKC-Raf1-MEK-ERK信号轴中富集程度最大。实验上,通过显微CT和病理检查证实了TLSGC对大鼠GIONFH的治疗作用。然后，使用血管造影确定TLSGC对血管损伤的保护作用，CD31免疫组织化学，体内血管功能指标，离体主动脉环试验，和HUVECs在体外的活动，包括迁移，入侵和管形成。机械上,TLSGC有效抑制VEGF和VEGFR2及其下游靶点的下调,包括Raf-1,PKC,p-MEK,和p-ERK蛋白在体内和体外。
结论：TLSGC可通过上调VEGF-VEGFR2-PKC-Raf-1-MEK-ERK信号轴促进血管生成，从而对GIONFH产生明显的疗效。
目的：探讨TLSGC改善GIONFH的物质基础和药理机制。
方法:使用LC-MS系统表征TLSGC中的化学组成。在整合MedChemStudio软件中TLSGC相关靶点和GIONFH相关基因的基础上,构建了“药物靶标-疾病基因”相互作用网络。通过拓扑特征参数筛选TLSGC改善GIONFH的候选靶标，并基于甲基强的松龙诱导的大鼠模型和地塞米松抑制的人脐静脉内皮细胞（HUVECs）进行进一步的实验验证。
结果：在TLSGC中表征了总共33种化学组成。基于这些组成和GIONFH相关基因，根据拓扑参数计算选择122个hub基因。生物学功能主要富集在4个血管损伤过表达模块中,炎症和细胞凋亡，骨代谢和能量代谢。hub基因在VEGF通路的VEGF-VEGFR2-PKC-Raf1-MEK-ERK信号轴中富集程度最大。实验上,通过显微CT和病理检查证实了TLSGC对大鼠GIONFH的治疗作用。然后，使用血管造影确定TLSGC对血管损伤的保护作用，CD31免疫组织化学，体内血管功能指标，离体主动脉环试验，和HUVECs在体外的活动，包括迁移，入侵和管形成。机械上,TLSGC有效抑制VEGF和VEGFR2及其下游靶点的下调,包括Raf-1,PKC,p-MEK,和p-ERK蛋白在体内和体外。
结论：TLSGC可通过上调VEGF-VEGFR2-PKC-Raf-1-MEK-ERK信号轴促进血管生成，从而对GIONFH产生明显的疗效。
