基于蛋白组学体外分析益母草水煎液对补体和凝血级联通路相关活性因子的作用

doi:10.11843/j.issn.0366-6964.2022.06.027

摘要/Abstract

摘要： 旨在通过蛋白组学探索益母草水煎液对人真皮微血管内皮细胞(human dermal microvascular endothelial cells, HDMECs)凝血与抗凝血相关因子表达的调节作用。MTT法筛选益母草水煎液对HDMECs的安全浓度；使用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantification, iTRAQ)技术分析50和100 μg·mL^-1益母草水煎液作用24 h后HDMECs蛋白表达谱的变化，通过对比各组蛋白谱的变化筛选出差异显著的相关通路，分析该通路中筛选到的可信差异表达蛋白(differentially expressed proteins, DEPs)，并选取可信DEPs中与凝血与抗凝血相关的拮抗因子进行RT-PCR和ELISA验证。结果表明：1 mg·mL^-1以下益母草水煎液对HDMECs无毒副作用；益母草水煎液能够同时调节与凝血相关的血小板活化等过程和与抗凝血相关的肝素结合过程。对筛选出的补体和凝血级联通路中的5种可信DEPs分析显示，与空白组相比，50 μg·mL^-1益母草水煎液组凝血酶原(F2)、抗凝血酶-Ⅲ(AT-Ⅲ)、组织型纤溶酶原激活剂(t-PA)、凝血因子Ⅴ(F5)和激肽原(KNG)同时显著下调，100 μg·mL^-1益母草组F2、t-PA、AT-Ⅲ、KNG显著下调；与50 μg·mL^-1益母草水煎液组相比，100 μg·mL^-1益母草水煎液组F2、AT-Ⅲ、t-PA、F5和KNG等凝血级联相关因子均显著升高。结果提示，益母草水煎液可显著改变HDMECs蛋白表达谱，并可能通过调控补体和凝血级联通路相关因子F2、AT-Ⅲ、t-PA、F5、KNG蛋白的表达对凝血与抗凝血相关拮抗因子发挥双向调控作用。

关键词: 益母草水煎液, HDMECs, iTRAQ, 补体和凝血级联通路, 凝血, 抗凝血

Abstract: The present study was aimed to investigate the regulatory effect of Leonurus artemisia decoction on the expression of coagulation and anticoagulation cytokines in human dermal microvascular endothelial cells (HDMECs) by proteomics. The safe concentration of Leonurus artemisia decoction to HDMECs was evaluated by MTT method. The changes of protein expression of HDMECs treated with 50 and 100 μg·mL^-1 Leonurus artemisia decoction for 24 h were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) technology. By comparing the changes of protein profiles in each group, the significantly different related pathways were screened out, and the credible differentially expressed proteins (DEPs) screened in the corresponding pathway were analyzed. The antagonistic factors related to coagulation and anticoagulation in credible DEPs were selected for RT-PCR and ELISA validation. Results showed that Leonurus artemisia decoction less than 1 mg·mL^-1 had no toxic effect on HDMECs. Leonurus artemisia decoction could regulate both platelet activation related to coagulation, and heparin binding related to anticoagulation. The analysis of five credible DEPs in the screened complement and coagulation cascade pathways showed that compared with the control group, the levels of prothrombin (F2), antithrombin-Ⅲ (AT-Ⅲ), tissue plasminogen activator (t-PA), coagulation factor Ⅴ (F5) and kininogen (KNG) were significantly decreased in 50 μg·mL^-1 Leonurus artemisia decoction group. The levels of F2, AT-Ⅲ, t-PA, KNG were significantly decreased in 100 μg·mL^-1 Leonurus artemisia decoction group. Compared with 50 μg·mL^-1 Leonurus artemisia decoction group, the levels of F2, AT-Ⅲ, t-PA, KNG and F5 were significantly increased in 100 μg·mL^-1 Leonurus artemisia decoction group. This study indicate that Leonurus artemisia decoction incubation could significantly change the protein expression profile of HDMECs, and bidirectionally regulate antagonistic factors related to coagulation and anticoagulation by regulating the expression of complement and coagulation cascade-related factors F2, AT-Ⅲ, t-PA, F5 and KNG.

Key words: Leonurus artemisia decoction, HDMECs, iTRAQ, complement and coagulation cascade pathway, coagulation, anticoagulation

中图分类号:

S853.75

张蒙, 王伟然, 冯晨, 张依, 张倩, 穆祥. 基于蛋白组学体外分析益母草水煎液对补体和凝血级联通路相关活性因子的作用[J]. 畜牧兽医学报, 2022, 53(6): 1934-1944.

ZHANG Meng, WANG Weiran, FENG Chen, ZHANG Yi, ZHANG Qian, MU Xiang. The Effect of Leonurus artemisia Decoction on the Active Factors of Complement and Coagulation Cascade Pathway Based on Proteomics in vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1934-1944.

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