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Supplementary Material for: Total flavonoids of Polygala fallax Hemsl induce apoptosis of human ectopic endometrial stromal cells through PI3K/AKT/Bcl-2 signaling pathway

posted on 2023-03-17, 04:36 authored by Zhong C., Ju G., Yang S., Zhao X., Chen J., Li N.
Abstract Objective: 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 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 (Fresh human ovarian endometriosis tissue was used for primary culture)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 bloting. Results: The network pharmacology predicts that PFHF treat 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 < 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 < 0.05). Conclusion: PFHF reduce 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.