Radix Bupleuri Chinensis Extract Ameliorates Doxorubicin-Induced Spermatogenic Dysfunction: Involvement of the GnRHR Signaling Pathway and Taurine Biosynthesis

Figures

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  Figure 1. CH confers protection against DOX-induced testicular dysfunction in mice. (A) Representative H&E-stained sections of mouse testicular tissue across experimental groups. Scale bar = 50 μm. (B) Testicular index of mice in different treatment groups. (C,D) Sperm motility and density analyses in experimental mice. (E,F) Serum concentrations of gonadotropin-releasing hormone (GnRH) and testosterone in the treatment groups. Data are presented as mean ± SD; * p < 0.05, ** p < 0.01 vs. NC group; # p < 0.05, ## p < 0.01 vs. M group.

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  Figure 2. Effects of CH on GnRHR and meiosis-related indicators in the testis of DOX-treated mice. Western blot analysis of GnRHR, SCP3, and REC8 expression in the testicular tissues of DOX-treated mice. Data are presented as mean ± SD. from three independent experiments. ** p < 0.01 vs. NC group; # p < 0.05, ## p < 0.01 vs. M group.

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  Figure 3. Effects of CH on improving spermatogenic dysfunction are closely related to the GnRH signaling pathway. (A) Representative H&E-stained sections of mouse testicular tissues across the experimental groups. Scale bar = 50 μm. (B) Testicular index of mice in different treatment groups. (C,D) Sperm motility and density analyses in experimental mice. (E,F) Serum concentrations of gonadotropin-releasing hormone (GnRH) and testosterone in the treatment groups. After administering cetrorelix, the ameliorative effect of CH on DOX-induced spermatogenic dysfunction was attenuated. Data are presented as mean ± SD; * p < 0.05 and ** p < 0.01 versus the NC group; # p < 0.05 and ## p < 0.01 versus the M group; and p < 0.05 and && p < 0.01 versus the CH group, & indicates the difference between CH + CE and CH groups.

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  Figure 4. CH counters testicular oxidative stress through the GnRH signaling pathway. The levels of (A,B) ROS, (C) SOD, (D) MDA, and (E) CAT in the testicular tissues of mice in each group. Data are presented as mean ± SD; ** p < 0.01 vs. NC group; ## p < 0.01 vs. M group; && p < 0.01 vs. the CH group.

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  Figure 5. GnRHR signaling critically mediates CH’s regulation of taurine synthesis and meiotic proteins. (A–E) WB analysis for FMO1, CDO1, REC8 and SCP3 protein expression in testicular tissues from DOX-treated mice. (F,G) qRT-PCR analysis of meiotic gene expression (REC8, SCP3, DAZ1, DDX4, and SMC1B) and steroidogenic acute regulatory protein (STAR) mRNA levels in testicular tissues from DOX-treated mice. (H–J) Immunofluorescence (IF) detection of the SCP3 and CDO1 proteins in mouse testicular sections. Scale bar = 50 μm. Data are presented as mean ± SD. ** p < 0.01 vs. NC group; ## p < 0.01 vs. M group; && p < 0.01 vs. the CH group.

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  Figure 6. CH ameliorates spermatogenic dysfunction through the GnRHR signaling pathway. (A–E) Western blot analysis of the protein expression of GnRHR, GNAS, PKA, and p-CREB/CREB in testicular tissues from DOX-treated mice. (F) qRT-PCR analysis for GnRHR, GNAS, and PKA expression in testicular tissues from DOX-treated mice. (G–I) Immunofluorescence (IF) detection of the GnRHR and ADCY3 proteins in mouse testicular sections. Scale bar = 50 μm. Data are presented as mean ± SD. ** p < 0.01 vs. NC group; # p < 0.05, ## p < 0.01 vs. M group; && p < 0.01 vs. the CH group, & indicates the difference between CH + CE and CH groups.

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  Figure 7. SSD attenuates DOX−induced injury in GC-1 cells. (A–D) Apoptosis and ROS levels. (E,F) TNF-α and IL-6 mRNA levels in GC-1 cells. Data are presented as mean ± SD. from three independent experiments. ** p < 0.01 vs. NC group; # p < 0.05, ## p < 0.01 vs. M group.

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  Figure 8. SSD modulates GnRHR signaling and taurine synthesis in GC-1 cells. (A–E) Protein levels of GnRHR, GNAS, SCP3, and CDO1 in GC-1 cells across treatment groups were detected using high-content imaging analysis. Scale bar = 100 μm. (F–K) Western blotting analysis of the protein expression levels of GnRHR, GNAS, ADCY3, SCP3, and CDO1 in GC-1 cells from each group. (L–O) qRT-PCR revealed SSd-induced upregulation of mRNA for meiosis-related genes REC8, DAZ1, DDX4 and CDO1. Data are presented as mean ± SD. ** p < 0.01 vs. NC group; # p < 0.05, ## p < 0.01 vs. M group.