摘要
目的基于网络药理学、分子对接和动物实验阐释填精荣胞方治疗复发性流产(RSA)合并血栓前状态(PTS)的作用机制。方法①液质联用技术检测填精荣胞方的成分。②网络药理学预测填精荣胞方治疗RSA的作用靶点和信号通路。③分子对接初步评估关键成分与关键靶点的结合性能。④动物实验验证填精荣胞方的作用机制。将36只CBA/J雌鼠随机分为6组,每组6只。空白组和模型组给予蒸馏水灌胃,阿司匹林组给予阿司匹林11.38 mg/kg灌胃,填精荣胞方低、中、高剂量组分别给予填精荣胞方6.27 g/kg、18.81 g/kg、56.42 g/kg灌胃,均1次/d。连续灌胃14 d后,空白组CBA/J雌鼠与BALB/c雄鼠合笼建立正常妊娠小鼠模型,其余组CBA/J雌鼠与DBA/2雄鼠合笼建立RSA合并PTS小鼠模型。妊娠第1~14天,每组继续原来方法灌胃。妊娠第15天取材,计算胚胎丢失比例,检测血清雌二醇(E_(2))、孕酮(P)、D-二聚体(D-D)水平,HE染色观察胎盘组织病理形态,免疫组化法检测胎盘组织中血管内皮生长因子(VEGF)、血管内皮生长因子受体1(VEGFR1)、血管内皮生长因子受体2(VEGFR2)表达情况,Western blot法检测胎盘组织中白血病抑制因子(LIF)、Janus激酶1(JAK1)、信号转导和转录激活因子3(STAT3)、VEGF、VEGFR1、VEGFR2蛋白表达情况。结果筛选出填精荣胞方中相对丰度值由高到低排序的前20种成分,预测其作用于RSA的153个靶点,其中TP53、AKT1、STAT3、TNF和SRC可能是关键靶点,JAK-STAT可能是关键通路。分子对接结果显示关键成分与关键靶点结合性能较好。动物实验结果显示:各药物组胚胎丢失比例和血清D-D水平均明显低于模型组(P均<0.05);阿司匹林组和填精荣胞方高剂量组血清E_(2)、P水平及填精荣胞方中剂量组血清E_(2)水平、填精荣胞方低剂量组血清P水平均明显高于模型组(P均<0.05);各药物组胎盘迷路区血管数量增加,胎盘出血和淤血减少;各药物组胎盘组织中LIF和STAT3蛋白相对表达量、填精荣胞方低剂量组JAK1和VEGF蛋白相对表达量、填精荣胞方中剂量组VEGF蛋白相对表达量、填精荣胞方高剂量组JAK1和VEGFR2蛋白相对表达量均明显高于模型组(P均<0.05)。结论填精荣胞方通过多成分、多靶点、多通路对RSA合并PTS起作用,调控JAK-STAT信号通路,上调胎盘中LIF、JAK1、STAT3、VEGF、VEGFR2表达可能是其作用机制之一。
Objective It is to elucidate the mechanism of Tianjing Rongbao Formula(TJRBF)in the treatment of recurrent spontaneous abortion(RSA)with prethrombotic state(PTS)based on network pharmacology,molecular docking and animal experiment.Methods①The TJRBF’s ingredients were detected by liquid chromatography-mass spectrometry technology.②The target molecules and signaling pathways involved in the therapeutic effects of TJRBF on RSA were predicted by network pharmacology.③The binding affinity of key active ingredients with key targets was preliminarily evaluated by molecular docking.④The mechanism of TJRBF was confirmed by animal experiments.Thirty-six female CBA/J mice were randomly divided into 6 groups,with 6 mice in each group.The blank and model groups received distilled water via gavage,the aspirin group received aspirin 11.38 mg/kg via gavage,the low-,medium-,and high-dose groups of TJRBF respectively received TJRBF 6.27 g/kg,18.81 g/kg and 56.42 g/kg via gavage,all once daily.After 14 days of continuous administration,the female CBA/J mice of the control group were co-housed with male BALB/c mice to establish models of normal pregnancy,the female CBA/J mice from the remaining groups were co-housed with male DBA/2 mice to establish models of RSA with PTS.From the 1st to 14 days of gestation,each group was continuously treated with the original administration regimen.On the 15th of gestation,the samples were collected,embryo loss rate was calculated,serum levels of estradiol(E_(2)),progesterone(P)and D-dimer(D-D)were measured,the placental histopathology was observed by HE staining,the expressions of vascular endothelial growth factor(VEGF),vascular endothelial growth factor receptor 1(VEGFR1),VEGF receptor 2(VEGFR2)in placental tissue were detected by immunohistochemistry,the protein expressions of leukemia inhibitory factor(LIF),Janus kinase 1(JAK1),signal transducer and activator of transcription 3(STAT3),VEGF,VEGFR1 and VEGFR2 were detected by Western blot.Results The top 20 components in TJRBF were screened out based on their relative abundance values from highest to lowest.These components were predicted to act on 153 targets in RAS,inwhich TP53,AKT1,STAT3,TNF and SRC might be the key targets,and JAK-STAT pathway might be the key pathway.The molecular docking results showed that the key ingredients could bind well with the key targets.Animal experimental results showed that the embryo loss rate and serum level of D-D in all medicated groups were significantly lower than those in the model group(all P<0.05);the serum levels of E_(2) and P in the aspirin group and high-dose TJRBF group,serum level of E_(2) in the medium-dose TJRBF group,and serum level of P in the low-dose TJRBF group were significantly higher than those in the model group(all P<0.05);the number of blood vessels in placental labyrinth were increased,while placental hemorrhage and congestion were decreased in each medicated group;the relative protein expressions of LIF and STAT3 in placental tissue in each medicated group,the relative protein expressions of JAK1 and VEGF in the low-dose TJRBF group,the relative protein expression of VEGF in the medium-dose TJRBF group,and the relative protein expressions of JAK1 and VEGFR2 in the high-dose TJRBF group were all significantly higher than those in the model group(all P<0.05).Conclusion TJRBF had a therapeutic effect on RSA with PTS through multiple ingredients,targets and pathways,among which regulating JAK-STAT signaling pathway and up-regulating the expressions of LIF,JAK1,STAT3,VEGF and VEGFR2 in placentas may be one of the mechanisms.
作者
朱友华
黄欲晓
赵嘉静
杨亚楠
方梦鑫
卜梦雅
张雨晴
孙佳琦
ZHU Youhua;HUANG Yuxiao;ZHAO Jiajing;YANG Yanan;FANG Mengxin;BU Mengya;ZHANG Yuqing;SUN Jiaqi(Xiyuan Hospital,China Academy of Chinese Medical Sciences,Beijing 100091,China)
出处
《现代中西医结合杂志》
2025年第21期2921-2933,共13页
Modern Journal of Integrated Traditional Chinese and Western Medicine
基金
中国中医科学院科技创新工程项目(CI2021A02411)
国家自然科学基金培育项目(XY20-09)。
关键词
复发性流产
血栓前状态
填精荣胞方
网络药理学
分子对接
JAK-STAT信号通路
recurrent spontaneous abortion
prethrombotic state
Tianjing Rongbao Formula
network pharmacology
molecular docking
JAK-STAT signaling pathway
