摘要
目的:观察通络生骨胶囊(TLSGC)对糖皮质激素致血管内皮细胞功能损伤的影响,并从丝裂原活化蛋白激酶激酶(MEK)/细胞外调节蛋白激酶(ERK)信号通路初步探索其作用机制。方法:取正常SD大鼠胸主动脉环,用甲泼尼龙琥珀酸钠(MPS,0.04 g·L^(-1))和(或)血管内皮细胞生长因子(VEGF,20μg·L^(-1))体外干预,加入TLSGC(12.5,25,50μg·L^(-1))连续作用5 d,观察血管环芽出的微生血管数目、长度和面积;此外,以甲泼尼龙琥珀酸钠(MPS,0.04μg·L^(-1))加入由VEGF(20μg·L^(-1))诱导的人脐静脉内皮细胞(HUVEC),再加入TLSGC(12.5,25,50μg·L^(-1)),然后分别采用transwell迁移,transwell侵袭及管腔形成实验检测HUVEC的迁移、侵袭及管腔形成能力,并以硝酸还原酶法检测细胞上清中一氧化氮(NO)含量,干粉法检测细胞上清中内皮素-1(ET-1)含量,蛋白免疫印迹法检测细胞中血管内皮细胞生长因子受体2(VEGFR2),ERK,磷酸化(p)-ERK,MEK和p-MEK蛋白表达量。结果:与正常组比较,MPS能明显抑制由VEGF诱导的大鼠胸主动脉环微血管数目、长度和面积以及HUVEC细胞迁移、侵袭和管腔形成能力,降低NO并升高ET-1含量,MPS还能明显减少由VEGF诱导的VEGFR2,p-MEK和p-ERK在HUVEC中的蛋白含量(P<0.05,P<0.01);与模型组比较,TLSGC能剂量依赖地改善由MPS降低的大鼠胸主动脉环微血管数目、长度和面积以及HUVEC细胞迁移、侵袭和管腔形成能力,提高HUVEC中的NO,VEGFR2,p-MEK和p-ERK蛋白含量并降低ET-1含量(P<0.05,P<0.01)。结论:TLSGC对糖皮质激素所致血管内皮细胞血管生成和分泌功能的损伤具有保护作用,其机制可能与活化MEK/ERK信号通路有关。
Objective:To observe the effect of Tongluo Shenggu capsule(TLSGC)on glucocorticoidinduced vascular endothelial cell functional damage,and to preliminally explore the mechanism of action through MEK-ERK signaling pathway. Method:The blood vessel of aorta rings of normal SD rats were induced in vitro intervention with methylprednisolone sodium succinate(MPS,0.04 g·L^(-1))and/or vascular endothelial growth factor(VEGF,20 μg·L^(-1)),and were treated with TLSGC(12.5,25,50 μg·L^(-1))continuously for 5 days to observe the number, length and area of microvascular ring buds. In addition, human umbilical vein endothelial cells(HUVEC)induced by VEGF(20 μg·L^(-1))were added into MPS(0.04 g·L^(-1))and TLSGC(12.5,25,50 μg·L^(-1))were added. Then,Transwell migration,Transwell invasion and lumen formation experiments were used to detect the migration,invasion and lumen formation ability of HUVEC,respectively.The content of nitric oxide(NO)in the cell supernatant was detected by nitrate reductase method,the content of endothelin 1(ET-1)in the cell supernatant was detected by dry powder method. Moreover,the protein contents of vascular endothelial growth factor receptor 2(VEGFR2),extracellular signal-regulated kinase(ERK),phospho-extracellular signal-regulated kinase(p-ERK), mitogen extracellular kinase1(MEK) and phosphorylated mitogen extracellular kinase1(p-MEK)in the cells were determined by Western blot. Result:Compared with the normal group,MPS could significantly inhibit the number,length and area of VEGFinduced rat thoracic aortic ring microvessels,HUVEC cell migration,invasion and lumen formation ability. It could reduce NO content and increase ET-1 content. MPS could also significantly reduce the protein content of VEGF-induced VEGFR2,p-MEK and p-ERK in HUVEC(P<0.05,P<0.01). Compared with the model group,TLSGC could dose-dependently increase the number,length and area of MPS-induced abnormally reduced rat thoracic aortic ring microvessels,promote MPS-induced abnormally decreased HUVEC cell migration,invasion and lumen formation ability. It could increase the protein contents of NO,VEGFR2,p-MEK and p-ERK in HUVEC, and reduce abnormally increased ET-1 content(P<0.05,P<0.01). Conclusion: TLSGC has a protective effect on the damage of angiogenesis and secretion of vascular endothelial cells induced by glucocorticoid,and the mechanism may be related to the activation of MEK/ERK signaling pathway.
作者
王金霞
贾可欣
明瑞蕊
徐腾腾
刘春芳
林娜
WANG Jin-xia;JIA Ke-xin;MING Rui-rui;XU Teng-teng;LIU Chun-fang;LIN Na(Chengde Medical University,Chengde 067000,China;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2021年第9期48-55,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家“重大新药创制”科技重大专项(2019ZX09731002)
中国中医科学院中药研究所技术研发项目(20181218)。
关键词
通络生骨胶囊
甲泼尼龙琥珀酸钠(MPS)
血管内皮细胞
细胞功能
血管生成
Tongluo Shenggu capsule
methylprednisolone sodium succinate(MPS)
human umbilical vein endothelial cells
cell function
angiogenesis
