目的 基于生物信息学结合动物实验探究“养血活血安胎”法指导下当归-白芍通过调节磷脂酰肌醇3-激酶(phosphatidylinositol 3 kinase,PI3K)/磷酸化丝氨酸/苏氨酸激酶(serine/threonine kinase,AKT)改善复发性流产(RSA)母胎界面血管生成的作用机制。方法 基于基因表达综合数据库(gene expression omnibus,GEO)获取RSA相关基因芯片,并通过R软件limma包进行差异表达分析。通过人类基因数据库(GeneCards)得到血管生成相关基因,并通过Venn在线制图工具将其与得到的差异表达基因(differentially expressed genes,DEGs)取交集。应用基因富集分析在线工具进行京都基因和基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)信号通路分析。建立正常妊娠小鼠模型和Clark经典RSA模型,将正常妊娠小鼠模型作为正常对照组(n=10),将RSA小鼠随机分为模型组、黄体酮组、高剂量、中剂量和低剂量当归-白芍水煎剂组,并于妊娠第1天开始给予相应药物灌胃,每日1次,共给药15 d;正常对照组给予等体积等频次生理盐水灌胃,于末次给药后12 h处死小鼠并取材。采用免疫组织化学法测定各组小鼠蜕膜组织中内皮抑素(endostain,ES)和微血管密度(micro vessel density,MVD)标记物白细胞分化抗原34(cluster of differentiation,CD34)的阳性表达,免疫印迹(Western blotting)法检测蜕膜组织中PI3K、AKT、ES和CD34的水平。结果 芯片GSE113790被纳入本研究,从中共获得704个RSA相关的差异表达基因。此外,通过Genecards数据库共获得1 258个血管生成相关基因。两者交集共有65个基因,此即为RSA血管生成异常相关DEGs。富集分析结果显示65个DEGs共参与45个信号通路的调控(P<0.05),其中PI3K/AKT富集最为显著。免疫组化结果表明,与对照组相比,模型组ES水平显著升高(P<0.01);与模型组相比,当归-白芍中剂量、高剂量组CD34水平显著升高,ES水平显著下降(P<0.05)。Western blotting结果表明,模型组ES水平显著升高,CD34、PI3K、pAKT/AKT水平显著下降(P<0.05),而黄体酮组及当归-白芍各剂量组ES水平均显著下降,pAKT/AKT水平显著上升;黄体酮组CD34水平显著上升;黄体酮组及当归-白芍高剂量组PI3K水平显著上升。结论 RSA母胎界面存在血管生成障碍,在“养血活血安胎”法指导下,当归-白芍药对可促进RSA小鼠母胎界面血管生成,并与激活PI3K/AKT通路相关。
Abstract
OBJECTIVE To investigate the mechanism of the effect of Angelica Sinensis-Radix Paeoniae Alba on improving angiogenesis at the maternal-fetal interface in recurrent spontaneous abortion (RSA) through regulating PI3K/AKT under the guidance of “nourishing blood, invigorating blood and calming the fetus” method based on bioinformatics and animal experiments. METHODS Microarray involving RSA was obtained based on gene expression omnibus (GEO) and differential expression analysis was conducted by limma package of R software. Genes related to angiogenesis were obtained from the Human Gene Database (GeneCards) and then intersected with differentially expressed genes (DEGs) by Venn online mapping tool. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis on intersected genes was conducted using Web-based Gene Set Analysis Toolkit (Webgestalt). Normal pregnant mouse model and Clark classical RSA model were established, in which RSA mice were randomly divided into model group, progesterone group, high dose, medium dose and low dose of Angelica sinensis-Radix Paeoniae groups (n=10 for each group). And the mice were given corresponding drugs by gavage on the first day of pregnancy once a day for 15 days. The normal control group was given the same volume of normal saline by gavage at the same frequency. All the mice were sacrificed 12 hours after the last dose and sampled. The positive expressions of endostain (ES) and micro vessel density (MVD) marker CD34 in decidual tissues of mice in each group were determined by immunohistochemistry. The levels of phosphatidylinositol 3 kinase (PI3K) and the ratio of phosphorylated serine/threonine kinase and serine/threonine kinase in decidua were detected by Western blotting. RESULTS The microarray GSE113790 was included in this study, and a total of 704 RSA-related DEGs were obtained. In addition, 1 258 genes related to angiogenesis were obtained from GeneCards database. And a total of 65 overlapped genes were obtained, which were taken as the DEGs associated with angiogenesis abnormality in RSA. Enrichment analysis showed that 65 DEGs were involved in the regulation of 45 signaling pathways (P<0.05), among which PI3K/AKT signaling pathway was the most significant. The results of immunohistochemistry showed that the level of ES in the model group was significantly higher than that in the control group (P<0.01). Compared with the model group, CD34 level was significantly increased, and ES levels were significantly decreased in the medium-dose and high-dose groups of Angelica Sinensis-Radix Paeoniae Alba (P<0.05). Western blotting results showed that compared with the control group, the level of ES in the model group was significantly increased, while the levels of CD34, PI3K and pAKT/AKT were significantly decreased (P<0.05). Compared with the model group, the levels of ES in progesterone group and all dose groups of Angelica Sinensis-Radix Paeoniae Alba were significantly decreased, the level of CD34 in progesterone group was significantly increased, the levels of PI3K in progesterone group and high-dose Angelica sinensis-Radix Paeoniae group were significantly increased, and the levels of pAKT/AKT in progesterone group and all dose groups of Angelica sinensis-Radix Paeoniae were significantly increased (P<0.05). CONCLUSION Angiogenesis is impaired at the maternal-fetal interface in RSA. Under the guidance of “nourishing blood, invigorating blood, and calming the fetus” treatment, the Angelica Sinensis-Radix Paeoniae Alba pair can promote angiogenesis at the maternal-fetal interface of RSA mice, which is associated with activation of PI3K/Akt pathway.
关键词
复发性流产 /
母胎界面 /
血管生成 /
养血活血安胎 /
磷脂酰肌醇3-激酶 /
丝氨酸/苏氨酸激酶 /
当归-白芍
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Key words
recurrent spontaneous abortion /
maternal-fetal interface /
angiogenesis /
nourishing and promoting blood for miscarriage prevention /
phosphatidylinositol 3-kinase /
serine/threonine kinase /
Angelica sinensis-Radix Paeoniae Alba
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脚注
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基金
宁波市自然科学基金项目资助(202003N4208);宁波市医学科技计划项目资助(2018A18)
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