cantly decreased (Figure the protein δ Opioid Receptor/DOR Synonyms levels the protein levels of Cdk1, and substantially decreased (Figure 1C). Similarly, 1C). Similarly, of these cell cycle regulators have been also decreased (Figure 1D,E). These findings account the decreased these cell cycle regulators were also decreased (Figure 1D,E). These findings account the expression of cell cycle regulators Ccna2, Ccnb1, Cdk1, and Cdk2 for the inhibition of sperreduced expression of cell cycle regulators Ccna2, Ccnb1, Cdk1, and Cdk2 for the inhibition matogonial proliferation.of spermatogonial proliferation.Figure 1. Effects of different concentrations of apigenin remedy around the proliferation and apoptosis ofFigure 1. Effects of distinctive concentrations was determined by CCK-8proliferation and apoptosis spermatogonia. (A) Cell proliferation of apigenin remedy around the assay immediately after different concentrations of spermatogonia. (A) Cell proliferation was determined by CCK-8 assay immediately after unique concentraof apigenin therapy for 48 h. (B) Cell cycle was analyzed by flow cytometry right after applying distinctive tions of apigenin treatment for 48 h. (B) Cell cycle was analyzed by flow cytometry right after applying concentrations of apigenin remedy for for (C) (C) Relative mRNA expression Ccna2, Ccnb1, Cdk1, various concentrations of apigenin treatment48 h.48 h. Relative mRNA expression of of Ccna2, Ccnb1, Cdk2 and Cdk2 examined by RT-qPCR in MEK5 Purity & Documentation spermatogonia treated with 0and 20 M apigenin for 48 h. and Cdk1, examined by RT-qPCR in spermatogonia treated with 0 and apigenin for 48 h.The protein levels of CCNA2, CCNB1 (D), and CDK1 at the same time as CDK2 (E) had been de(D,E) (D,E) The protein levels of CCNA2, CCNB1 (D), and CDK1 too as CDK2 (E) have been detected by tected by Western blotting in spermatogonia treated with 0 and 20 M apigenin for 48 h. (F) Cell Western blotting in spermatogonia treated with 0 and 20 apigenin for 48 h. (F) Cell apoptosis apoptosis was analyzed by flow cytometry immediately after applying diverse concentrations of apigenin forwas analyzed by flow cytometry soon after applying distinctive concentrations of apigenin for 48 h. (G) Relative mRNA expression of Bcl2, Bax, and P53 examined by RT-qPCR in spermatogonia treated with 0 and 20 apigenin for 48 h. (H)The protein levels of BCL2, BAX, P53 have been detected by Western blotting in spermatogonia treated with 0 and 20 apigenin for 48 h. Data from 3 independent experiments are expressed as mean SEM. indicates statistical significance. p 0.05, p 0.01, and p 0.001.2.2. Apigenin Promotes Apoptosis of Spermatogonia Next for the inhibitory impact of apigenin on the proliferation of spermatogonia, we examined its effect on apoptosis, making use of Annexin V-PI staining followed by flow cytometryInt. J. Mol. Sci. 2021, 22,four ofanalysis. In comparison to control, the apoptosis rate of spermatogonia treated with five, 10, and 20 apigenin elevated inside a dose-dependent manner: from no effect of 5 apigenin, to slightly enhanced apoptosis price with ten apigenin, to drastically improved apoptosis with 20 apigenin (Figure 1D). For that reason, the expression of apoptosis regulators in spermatogonia was additional examined in the presence or absence of 20 apigenin. At this concentration Bcl2 expression decreased when Bax and P53 expression improved (Figure 1E,F), indicating that apigenin impacted the expression of apoptosis regulators and thereby enhanced the apoptotic death of spermatogonia. two.three. Apigenin Reduces the Expression of Prmt7 and Akt3 in Spermato