Total Flavonoids of Polygala fallax Hemsl Induce Apoptosis of Human Ectopic Endometrial Stromal Cells through PI3K/AKT/Bcl-2 Signaling Pathway
Authors: Zhong Chuanmei , Ju Gongchenhao , Yang Sufang , Zhao xiangpei , Chen Jixiang , Li Ning 
Published: March 2023

Objective: The objective of this study was to explore the inhibitory effect of total flavonoids of Polygala fallax Hemsl (PFHF) on human ectopic endometrial stromal cells (HEcESCs) and its mechanism. Design: The apoptosis, cell cycle, migration, and invasion ability of HEcESCs (Fresh human ovarian endometriosis tissue was used for primary culture) after PFHF treatment were detected, and the mechanism of action was explored. Materials: The Polygala fallax Hemsl (PFH), RPMI 1640 culture medium, Dulbecco’s modified Eagle’s medium (DMEM)/F-12, fetal bovine serum, penicillin/streptomycin, cell counting kit-8 (CCK-8) kit, trypsin, phenylmethylsulfonyl fluoride, radioimmunoprecipitation assay tissue/cell lysate, bicinchoninic acid protein concentration detection kits, protein loading buffer, the apoptosis and cell cycle extraction kits, the matrix glue, TRIzol Universal Reagent, the reverse transcription kit, AB HS Green qPCR Mix, the ECL chromogenic solution, enzyme labeling instrument, flow cytometry, automatic real-time fluorescence quantitative PCR instrument, Goat anti-rabbit, rabbit anti-β-actin, vimentin, phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), B-cell lymphoma-2 (Bcl-2), Bcl-extra long (Bcl-xl), Bcl-2 associated death promoter (Bad) antibody, Alexa Fluor 594-labeled secondary antibody, the inverted microscope, the constant temperature carbon dioxide cell incubator. Setting: Five parts included introduction, materials and methods, results, discussion, and conclusion. Methods: The potential targets and pathways of PFHF in the treatment of endometriosis were predicted by network pharmacology. The effect of PFHF on the proliferation, apoptosis and cell cycle, migration, and invasion of HEcESCs was detected by CCK-8 method, flow cytometry, and Transwell chamber experiment. Label-free quantitative proteomics based on mass spectrometry was used to analyze the protein mass spectrum of differential expression of HEcESCs before and after PFHF, and the biological information was analyzed. The effects of PFHF on the mRNA and protein expression of pathway-related genes predicted in HEcESCs were detected by reverse transcription-quantitative polymerase chain reaction and Western blotting. Results: The network pharmacology predicts that PFHF treats endometriosis through PI3K/AKT signaling pathway. Compared with control group (DMEM/F-12 medium alone), the high dose PFHF can significantly reduce the viability, migration, and invasion of HEcESCs, increase the apoptosis rate of HEcESCs, and make the HEcESCs accumulated in G0/G1 phase in a time- and dose-dependent manner (p &lt; 0.05). The analysis of label-free quantitative proteomics indicated that PFHF flavonoids may induce apoptosis of EESCs through PI3K/AKT signaling pathway. The results of RT-qPCR and Western blotting showed that the expressions of PI3K, AKT, Bcl-2, and Bcl-xl were significantly downregulated, while the bad expression was upregulated in HEcESCs treated with PFHF (p &lt; 0.05). Limitations: This research investigated the effects of PFHF on the stromal endometriotic cells only. So it is unknown how PFHF can affect the entire endometriotic lesion. And the research is carried out in vitro, which gives no impression about the bioavailability of the flavonoids. Conclusion: PFHF reduces the expression of PI3K, AKT, Bcl-2, and Bcl-xl through the PI3K/AKT/Bcl-2 signaling pathway to inhibit HEcESCs proliferation, migration, and invasion and promote their apoptosis.