Molecular Mechanism of Endometrial Cell Senescence During Window of Implantation in Unexplained Recurrent Spontaneous Abortion and Intervention Effect of Bushen Huoxue Method
10.13422/j.cnki.syfjx.20231619
- VernacularTitle:不明原因复发性流产着床窗口期子宫内膜细胞衰老的分子机制及补肾活血法的干预作用
- Author:
Xiaoxuan ZHAO
1
;
Jing MA
1
;
Xinyi DING
2
;
Hongli ZHAO
1
Author Information
1. Hangzhou Traditional Chinese Medicine Hospital Affiliated to Zhejiang Chinese Medical University，Hangzhou 310007，China
2. The Third Clinical School of Zhejiang Chinese Medical University，Hangzhou 310053，China
- Publication Type:Journal Article
- Keywords:
unexplained recurrent spontaneous abortion;
window of implantation;
cell senescence;
Bushen Huoxue method;
machine learning
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2024;30(19):106-115
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveThis study aims to explore the molecular mechanism of endometrial cell senescence during the window of implantation （WOI） in unexplained recurrent spontaneous abortion （URSA） and the intervention effect of Bushen Huoxue method through the integrated research strategy of bioinformatics， machine learning， and animal experiments. MethodThe microarray gene sets of the endometrium of healthy women of childbearing age and patients with URSA during WOI were retrieved through the gene expression omnibus （GEO） database. The differentially expressed genes （DEGs） of URSA were screened by using the limma package of R language. Weighted gene co-expression network analysis （WGCNA） was used to obtain the most relevant module genes of URSA， and the gene ontology （GO） enrichment analysis and Kyoto encyclopedia of genes and genomes （KEGG） enrichment analysis were performed. Gene sets related to cell senescence were obtained by the Human Gene Database （GeneCards） and Online Mendelian Inheritance in Man （OMIM）. The Venn online mapping software was used to screen the intersection genes of DEGs of URSA， the most relevant module genes， and the genes related to cell senescence. Then， the search tool for the retrieval of interacting genes/proteins （STRING） was used to analyze the protein-protein interaction （PPI） network of the interacting genes， and the hub genes were screened through Cytohubba. In addition， the least absolute selection and shrinkage operator （LASSO） and random forest algorithm were used to screen diagnostic genes related to cell senescence of URSA. Finally， mouse models with URSA were established and randomly divided into the model group， Bushen Huoxue group， and aspirin group， with six mice in each group， and six normal pregnant mice were selected as a blank group. The Bushen Huoxue group was given Shoutai pill combined with Danggui powder formula granular liquid （12.35 g·kg-1） by gavage， and the aspirin group was gavaged with aspirin at a dose of 0.011 mL·g-1. The blank group and model group were given the same volume of distilled water by gavage. Drug administration began on the first day after the discovery of vaginal thrombus （recorded as the first day of gestation， i.e. GD1）， and the mice were sacrificed 12 hours after administration at GD5. The endometrium samples were collected. Quantitative real-time polymerase chain reaction （Real-time PCR） and immunofluorescence were used to detect the expression of cell senescence-related diagnostic genes in the endometrium tissue of URSA mice during WOI and the intervention effect of the Bushen Huoxue method. ResultThe gene set of the GSE165004 microarray was included for bioinformatics analysis. According to the “limma” package of R language， 585 DEGs of URSA were selected （P<0.05）. WGCNA results suggested that the gene in the magenta module was the most closely related to URSA （r=0.32， P<0.05）. The results of the KEGG analysis of the magenta module suggested that the genes were mainly enriched in cell senescence， Hippo， and NF-κB signaling pathways （P<0.05）. In addition， 2 138 genes related to cell senescence were obtained from Genecards and OMIM websites. The Venn online mapping tool was used to obtain 27 intersecting genes of DEGs of URSA， the magenta module genes， and cell senescence-related genes， namely， cell senescence-related DEGs in endometrium tissue during WOI of URSA. PPI analysis showed that synuclein alpha （SNCA）， heme oxygenase 1 （HOMX1）， and matrix metallopeptidase 1 （MMP1） were hub genes. Besides， four diagnostic genes were identified by LASSO regression and random forest， including gelsolin （GSN）， cyclin2 （CCND2）， RB binding protein 8 （RBBP8）， and lysophosphatidic acid receptor 1 （LPAR1）. Animal experiments confirmed that the mRNA level and fluorescence intensity of GSN were significantly increased （P<0.05）， while the mRNA and fluorescence intensity of CCND2， LPAR1， and RBBP8 were significantly decreased （P<0.05） in the model group when compared with the blank group. The expression level of GSN was decreased （P<0.05）， while the expression levels of CCND2， LPAR1， and RBBP8 were remarkably increased （P<0.05） in the Bushen Huoxue group when compared with the model group. ConclusionThe senescence of endometrial cells during WOI of URSA is related to multiple genes. Bushen Huoxue method can act on multiple targets that are related to cell senescence， which may be the key mechanism of improving endometrial receptivity in URSA.
