Arginine Biosynthesis Mediates Wulingzhi Extract Resistance to Busulfan-Induced Male Reproductive Toxicity

Abstract

Busulfan, an indispensable medicine in cancer treatment, can cause serious reproductive system damage to males as a side effect of its otherwise excellent therapeutic results. Its widespread use has also caused its accumulation in the environment and subsequent ecotoxicology effects. As a Chinese medicine, Wulingzhi (WLZ) has the effects of promoting blood circulation and improving female reproductive function. However, the potential effects of WLZ in male reproduction and in counteracting busulfan-induced testis damage, as well as its probable mechanisms, are still ambiguous. In this study, busulfan was introduced in a mouse model to evaluate its production of the testicular damage. The components of different WLZ extracts were compared using an untargeted metabolome to select extracts with greater efficacy, which were further confirmed in vivo. Here, we demonstrate abnormal spermatogenesis and low sperm quality in busulfan-injured testes. The WLZ extracts showed a strong potential to rehabilitate the male reproductive system; this effect was more prominent in room-temperature extracts. Additionally, both water and ethanol WLZ extracts at room temperature alleviated various busulfan-induced adverse effects. In particular, WLZ recovered spermatogenesis, re-activated arginine biosynthesis, and alleviated the increased oxidative stress and inflammation in the testis, ultimately reversing the busulfan-induced testicular injury. Collectively, these results suggest a promising approach to protecting the male reproductive system from busulfan-induced adverse side effects, as well as those of other similar anti-cancer drugs.

Keywords: Wulingzhi; busulfan; inflammation; oxidative stress; testicular damage.

Figure 1

Effect of busulfan on testes of mice. (A) Schematic diagram of busulfan treatment during the test period. (B) Bright-field diagram of testicular size in control and busulfan injection groups. (C) Average testis weight. (D) Ratio of testis weight/body weight. (E) Average epididymis weight. (F) Ratio of epididymis weight/body weight. (G) Representative images of H&E staining in testis sections, with the 2nd row showing magnifications of the boxed regions in the 1st row; scale bar = 100 μm (1st row) and 10 μm (2nd row). (H) The percentage of normal spermatogenesis tubules in testicular samples. (I) Sperm concentration in epididymides. (J,K) Motility (J) and progressive motility (K) of sperm were assessed by computer-assisted semen analysis. n = 6 for each group. All data are presented as means ± SEM. Statistical significance was determined by the unpaired Student’s t-test. *** p < 0.001.

Figure 2

The composition changes in different WLZ extracts. (A) The untargeted metabolome profiles generated on different WLZ extract samples using an LC-MS (Liquid Chromatograph–Mass Spectrometer). WLZ (w) RT, the WLZ water extract at room temperature; WLZ (e) RT, the WLZ ethanol extract at room temperature; WLZ (w) H, the WLZ water extract at 85 °C; WLZ (e) H, the WLZ ethanol extract at 85 °C. (B–E) The components found in different WLZ extracts. (F,G) The Venn diagrams generated to describe the common and unique metabolites in the WLZ water extract (F) and WLZ ethanol extract (G) at different temperatures. (H–K) The components of the unique metabolites in WLZ (w) RT (H), WLZ (e) RT (I), WLZ (w) H (J), and WLZ (e) H (K). n = 3 for each group.

Figure 3

The metabolome changes in the WLZ water extract and WLZ ethanol extract at different temperatures. (A–E) The metabolomic changes in WLZ (w) RT and WLZ (w) H; (A) the 3D principal component analysis (PCA) score plot; (B) volcano plot showing the changes in metabolites; (C) heatmap of the top 50 differential metabolites; (D,E) the KEGG enrichment analysis of differential metabolites in WLZ (w) RT and WLZ (w) H. (F–J) The metabolomics changes in WLZ (e) RT and WLZ (e) H; (F) the 3D principal component analysis (PCA) score plot; (G) volcano plot showing the changes in metabolites; (H) heatmap of the top 50 differential metabolites; (I,J) the KEGG enrichment analysis of differential metabolites in WLZ (e) RT and WLZ (e) H. (K,L) The bar plot based on the differential metabolites which were enriched in the reproduction-related classes. n = 3 for each group.

Figure 4

The metabolome changes in WLZ water and ethanol extracts at room temperature. (A–C) The Venn diagram was generated to describe the common and unique metabolites in the WLZ water extract and WLZ ethanol extract at room temperature, and the components of the unique metabolites. (D) The 3D principal component analysis (PCA) score plot. (E) Volcano plot showing the changes in metabolites. (F) Heatmap of the top 50 differential metabolites. (G,H) The pathway enrichment analysis of differential metabolites in WLZ (w) RT and WLZ (e) RT. The darker the color and larger the shape of the circle, the stronger the pathway influence. (I,J) The KEGG enrichment analysis of differential metabolites in WLZ (w) RT and WLZ (e) RT. n = 3 for each group.

Figure 5

Effects of different WLZ extracts on testis injury caused by busulfan. (A) Schematic diagram of experiment. (B) Bright-field diagram of testicular size. (C) Average testis weight. (D) Ratio of testis weight/body weight. (E) Representative images of H&E staining in testis sections, with the 2nd row showing magnifications of the boxed regions in the 1st row; scale bar = 100 μm (1st row) and 10 μm (2nd row). (F) The percentage of normal spermatogenesis tubules in testicular samples. (G–I) The tubular diameter (G), epithelium height (H), and lumen width (I) of the testis. n = 6 for each group. All data are presented as means ± SEM. Statistical analysis was conducted using one-way ANOVA followed by Sidak’s multiple comparisons test. ** p < 0.01, *** p < 0.001.

Figure 6

Changes in sperm parameters and marker genes involving spermatogenesis after WLZ treatment. (A) Statistical analysis of sperm concentration. (B,C) Motility and progressive motility of sperm. (D–R) Real-time PCR analysis of the mRNA expression of late-spermatogenic-event-related markers (D–H), spermatogonia-related markers (I–M), spermatocyte-related markers (N,O), and spermatid-related markers (P,R). n = 6 for each group. All data are presented as means ± SEM. Statistical analysis was performed using one-way ANOVA followed by Sidak’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

WLZ regulated arginine biosynthesis to affect the oxidative stress and inflammation in the testes. (A) Real-time PCR analysis of the mRNA expression of arginine biosynthesis-related markers. (B) Gene expression in arginine biosynthesis pathway. Green represents down-regulated genes, while red represents up-regulated genes in the Bus group. (C,D) Real-time PCR analysis of the mRNA expression of oxidative stress-related markers (C) and inflammation-related markers (D). (E) Sperman’s correlation between testis-related indicators and arginine-related indicators. (F) The correlation network of significant markers in (E). n = 6 for each group. All data are presented as means ± SEM. Statistical analysis was conducted using one-way ANOVA followed by Sidak’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 8

Schematic diagram for the hypothetical molecular mechanism of arginine biosynthesis-mediated WLZ resistance to busulfan-induced testicular dysfunction. Busulfan exposure led to impaired testicular function, mainly manifesting as oxidative stress and inflammation of the testes, abnormal spermatogenesis, and decreased sperm parameters, while WLZ supplementation induced arginine biosynthesis, which then relieved the increased oxidative stress and inflammation in the testes and finally rescued the spermatogenesis and testis function injured by busulfan.