系统生物学和蛋白质组学策略探索寿胎丸治疗复发性自然流产的生物网络调控机制。
Systematic biological and proteomics strategies to explore the regulation mechanism of Shoutai Wan on recurrent spontaneous Abortion's biological network.
机构信息
Department of rheumatology and clinical immunology, Peking Union Medical college Hospital, Chinese Academy of Medical Sciences & Peking Union Medical college, Beijing, China; Key Laboratory of Rheumatology and Clinical Immunology, Ministry of Education, Beijing, China.
Capital Medical University, Beijing, China; Beijing Anzhen Hospital, Capital Medical University, Beijing, China.
出版信息
J Ethnopharmacol. 2020 Dec 5;263:113156. doi: 10.1016/j.jep.2020.113156. Epub 2020 Aug 5.
ETHNOPHARMACOLOGICAL RELEVANCE
Shoutai Wan (STW) is a classic herbal formula for the treatment of recurrent spontaneous abortion (RSA), and clinical studies have shown the effectiveness of STW on RSA. However, the molecular mechanism of STW treatment of RSA is still unclear.
METHODS
(1) Animal experiments: The normal pregnancy model was established with CBA/JBALB/C, and the RSA model was established by CBA/JDBA/2. The RSA model CBA/J*DBA/2 pregnant mice were randomly divided into four groups (RSA model group, STW low, medium and high dose groups) according to the order of pregnancy, respectively. The drug administration starts from the first day of pregnancy to the 14th day of pregnancy. The embryo loss rate (ELR) of each group was calculated. (2) Proteomic analysis of decidual tissue: The total protein of decidual tissue of each group was isolated by solid phase pH gradient 2-DE technique. The differentially expressed protein spots were analyzed and identified by PDQuest images; the peptide quality fingerprinting (PMF) was obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Then, the proteins were identified by Mascot software searching, their functions were identified by bioinformatics strategy. (3) The expression of HSP27, α-enolase and Transferrin was detected by Western blotting and the expression of Annexin A2 and Transferrin was detected by immunohistochemistry. (4) The differential proteins and potential targets were analyzed by systematic biological strategy.
RESULTS
(1) Compared with the normal group, the ELR in the RSA model group was significantly higher (P < 0.01). Compared with the model group, the ELR in the STW high, medium dose groups was lower (P < 0.01). (2) A 2-DE map of the decidual tissue of the RSA model group, normal pregnancy group, STW low, medium and high dose groups was established. Thirty proteins were identified. (3) The results of western blot showed that the expression of HSP27 and a-enolase in the RSA model group was higher than that in the normal group, and the expression of transferrin was lower (P < 0.01). Compared with the model group, the expression of HSP27 and a-enolase in STW high, medium dose groups was decreased (P < 0.01); Compared with the model group, the expression of Transferrin in the STW high dose group was increased (P < 0.01). (5) A lot of RSA treatment-related targets, biological processes and pathways were found after the systematic biological analysis.
CONCLUSION
(1) STW may reduce the ELR of the RSA mice. (2) The results of proteomics suggest that RSA is a complex process involving multiple proteins. STW can regulate the expression of various proteins in the decidual tissue of RSA mice, suggesting that it can act on multiple targets. (3) The results of western blotting of HSP27, a-enolase, transferrin were consistent with the results of proteomic analysis. (4) STW may achieve therapeutic effects by interfering with the targets, biological processes and signaling pathways discovered in this study.
ETHNOPHARMACOLOGICAL 相关性：寿胎丸（STW）是一种经典的治疗复发性自然流产（RSA）的草药配方，临床研究表明 STW 对 RSA 有效。然而，STW 治疗 RSA 的分子机制仍不清楚。
方法
（1）动物实验：采用 CBA/JBALB/C 建立正常妊娠模型，采用 CBA/JDBA/2 建立 RSA 模型。将 RSA 模型 CBA/J*DBA/2 孕鼠按妊娠顺序随机分为四组（RSA 模型组、STW 低、中、高剂量组），分别于妊娠第 1 天至第 14 天给药。计算各组胚胎丢失率（ELR）。（2）蜕膜组织蛋白质组学分析：采用固相 pH 梯度 2-DE 技术分离各组蜕膜组织总蛋白，采用 PDQuest 图像分析差异表达蛋白点，采用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）获得肽质量指纹图谱（PMF），然后利用 Mascot 软件检索鉴定蛋白质，采用生物信息学策略鉴定其功能。（3）Western blot 检测 HSP27、α-烯醇酶和转铁蛋白的表达，免疫组化检测 Annexin A2 和转铁蛋白的表达。（4）采用系统生物学策略分析差异蛋白和潜在靶点。
结果
（1）与正常组相比，RSA 模型组 ELR 明显升高（P<0.01）；与模型组相比，STW 高、中剂量组 ELR 降低（P<0.01）。（2）建立 RSA 模型组、正常妊娠组、STW 低、中、高剂量组蜕膜组织 2-DE 图谱，鉴定 30 种蛋白。（3）Western blot 结果显示，RSA 模型组 HSP27 和α-烯醇酶表达高于正常组，转铁蛋白表达降低（P<0.01）；与模型组相比，STW 高、中剂量组 HSP27 和α-烯醇酶表达降低（P<0.01）；与模型组相比，STW 高剂量组转铁蛋白表达升高（P<0.01）。（4）系统生物学分析后发现了大量与 RSA 治疗相关的靶点、生物过程和途径。
结论
（1）STW 可降低 RSA 小鼠的 ELR。（2）蛋白质组学研究结果表明，RSA 是一个涉及多种蛋白的复杂过程，STW 可调节 RSA 小鼠蜕膜组织中多种蛋白的表达，提示其可作用于多个靶点。（3）HSP27、α-烯醇酶、转铁蛋白 Western blot 结果与蛋白质组学分析结果一致。（4）STW 可能通过干扰本研究发现的靶点、生物过程和信号通路发挥治疗作用。
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