Liuting Zeng1, Kailin Yang2, Liting Liu3, Tianqing Zhang4, Huiping Liu5, Zhanwang Tan6, Lei Lei7. 1. 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. Electronic address: 2057188405@qq.com. 2. Capital Medical University, Beijing, China; Beijing Anzhen Hospital, Capital Medical University, Beijing, China. Electronic address: yang.cardiology@ccmu.edu.cn. 3. Huazhong University of Science and Technology, Wuhan, Hubei Province, China. Electronic address: 3243901099@qq.com. 4. Graduate College, University of South China, Hengyang, Hunan Province, China; Department of Cardiology, The First Affiliated Hospital of University of South China, Hengyang, Hunan Province, China. Electronic address: 892821194@qq.com. 5. Hunan University of Chinese Medicine, Changsha, Hunan Province, China. Electronic address: 1074983953@qq.com. 6. Hunan University of Chinese Medicine, Changsha, Hunan Province, China. Electronic address: zhanwang910@sohu.com. 7. Hunan University of Chinese Medicine, Changsha, Hunan Province, China. Electronic address: leilei1398@outlook.com.
Abstract
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/J*BALB/C, and the RSA model was established by CBA/J*DBA/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 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/J*BALB/C, and the RSA model was established by CBA/J*DBA/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 RSAmice. (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 RSAmice, 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.