cantly decreased (ROCK2 medchemexpress Figure the protein levels the protein levels of Cdk1, and drastically decreased (Figure 1C). Similarly, 1C). Similarly, of those cell cycle regulators were also decreased (Figure 1D,E). These findings account the lowered these cell cycle regulators had been 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 diverse concentrations of apigenin therapy on the proliferation and apoptosis ofFigure 1. Effects of distinctive concentrations was determined by CCK-8proliferation and apoptosis spermatogonia. (A) Cell proliferation of apigenin remedy on the assay immediately after distinctive concentrations of spermatogonia. (A) Cell proliferation was determined by CCK-8 assay following distinct concentraof apigenin remedy for 48 h. (B) Cell cycle was analyzed by flow cytometry after applying unique tions of apigenin therapy for 48 h. (B) Cell cycle was analyzed by flow cytometry after applying concentrations of apigenin therapy for for (C) (C) Relative mRNA expression Ccna2, Ccnb1, Cdk1, distinct concentrations of apigenin treatment48 h.48 h. Relative mRNA expression of of Ccna2, Ccnb1, Cdk2 and Cdk2 examined by P2X1 Receptor Gene ID RT-qPCR in 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 as well as CDK2 (E) had been de(D,E) (D,E) The protein levels of CCNA2, CCNB1 (D), and CDK1 at the same time 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 various concentrations of apigenin forwas analyzed by flow cytometry immediately after applying different 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 three independent experiments are expressed as imply SEM. indicates statistical significance. p 0.05, p 0.01, and p 0.001.2.two. Apigenin Promotes Apoptosis of Spermatogonia Next to the inhibitory impact of apigenin around the proliferation of spermatogonia, we examined its effect on apoptosis, employing Annexin V-PI staining followed by flow cytometryInt. J. Mol. Sci. 2021, 22,4 ofanalysis. In comparison to handle, the apoptosis price of spermatogonia treated with 5, 10, and 20 apigenin increased inside a dose-dependent manner: from no impact of five apigenin, to slightly improved apoptosis rate with 10 apigenin, to considerably elevated apoptosis with 20 apigenin (Figure 1D). As a result, the expression of apoptosis regulators in spermatogonia was additional examined inside the presence or absence of 20 apigenin. At this concentration Bcl2 expression decreased while 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. 2.3. Apigenin Reduces the Expression of Prmt7 and Akt3 in Spermato