Agmatine Abrogates Tacrolimus-Induced Testicular Injury in Rats

Abstract

Background/Objectives: Tacrolimus is an immunosuppressant drug widely used to prevent organ transplant rejection. Preclinical and clinical studies report that tacrolimus has destructive impacts on the male reproductive system owing to the induction of oxidative stress and inflammation. This study aimed at examining defensive impacts of agmatine against tacrolimus-induced testicular toxicity in rats. Methods: Male Wistar rats were randomly divided into six groups and treated based on the experimental design for 14 days. By the end of this study, blood samples were obtained to measure testosterone and luteinizing hormone. Also, both testes were removed for molecular analysis and histopathological examinations. Results: Agmatine administration increased serum levels of testosterone and luteinizing hormone and ameliorated all histopathological and toxicological changes induced by tacrolimus. Agmatine administration attenuated tacrolimus-induced oxidative stress as evidenced by the reduction of malondialdehyde content and inducible nitric oxide synthase expression and the elevation of reduced glutathione. This was parallel to the restoration of nuclear factor erythroid 2-related factor2 and hemeoxygenase-1 expression. Moreover, agmatine decreased the expressions of nuclear factor kappa B and interleukin-17. Agmatine also decreased the cell death revealed by decreased caspase-3 expression and increased expression of the antiapoptotic marker Bcl-2 in a dose-dependent manner. The antioxidant, anti-inflammatory, and antiapoptotic effects of agmatine were explained by increased expression of sirtuin-1. Conclusions: agmatine effectively attenuated testicular injuries induced by tacrolimus and enhanced spermatogenesis. This protective effect of agmatine might be mediated via the upregulation of sirtuin-1 expression that in turn restores oxidative status and regulates nuclear factor erythroid 2-related factor2/nuclear factor kappa B/Bcl-2 signaling.

Keywords: agmatine; oxidative stress; tacrolimus; testicular toxicity.

Figure 1

Impact of AGM₁₀ and AGM₄₀ on TAC-induced changes in sperm count, serum TT, and LH concentration. TAC was administered along with AGM for 14 days. (A) Sperm count, (B) serum TT, and (C) LH concentration. Data are expressed as means ± SD (n = 6 rats/group). Mean values were compared using one-way ANOVA followed by post hoc Tukey’s multiple comparison test. * indicates significance at p value of <0.05 vs. control group; ^# indicates significance at p value of <0.05 vs. TAC group; and ^$ indicates significance at p value of <0.05 vs. TAC + AGM₁₀ group. AGM: agmatine; CTR: control; LH: luteinizing hormone; TAC: tacrolimus; TT: total testosterone.

Figure 2

Impact of AGM₁₀ and AGM₄₀ on TAC-induced histopathological changes. Microscopic images of H&E-stained testicular sections. Upper panel: magnification 100x (scale bar 100 µm), and lower panel: magnification 200× (scale bar 50 µm). Black arrow: spermatogonia; red arrow: spermatocytes; blue arrow: rounded and longitudinal spermatids; yellow arrow: Leydig cell; black arrowhead: irregularly shaped seminiferous tubule; red arrowhead: sloughing of germinal epithelium; and yellow arrowhead: vacuolation with pyknotic or necrosis of Leydig cell. AGM: agmatine; CTR: control; TAC: tacrolimus.

Figure 3

Impact of AGM₁₀ and AGM₄₀ on TAC-induced changes in oxidative status. (A) MDA content, (B) GSH level, and (C) iNOS level. TAC was administered along with AGM₁₀ or AGM₄₀ for 14 days. Data are expressed as means ± SD (n = 6 rats per group). Mean values were compared via one-way ANOVA followed by post hoc Tukey’s multiple comparison test. * indicates significance at p value of <0.05 vs. CTR group. ^# indicates significance at p value of <0.05 vs. TAC group and ^$ indicates significance at p value of <0.05 vs. TAC + AGM₁₀ group. AGM: agmatine; CTR: control; GSH: reduced glutathione; iNOS: inducible nitric oxide; MDA: malondialdehyde; synthase; TAC: tacrolimus.

Figure 4

Impact of AGM₁₀ and AGM₄₀ on TAC-induced changes in Nrf2 expression and HO-1 and SIRT1 levels. TAC was administered along with AGM₁₀ or AGM₄₀ for 14 days. (A) Photomicrograph of immunostained testicular sections against Nrf2 (magnification 200× and scale bar 50 µm), (B) semiquantification of Nrf2, (C) HO-1 level, and (D) SIRT1 level. Data are expressed as means ± SD (n = 6 rats per group). Mean values were compared via one-way ANOVA followed by post hoc Tukey’s multiple comparison test. * indicates significance at p value of <0.05 vs. CTR group, ^# indicates significance at p value of <0.05 vs. TAC group, and ^$ significance at p value of <0.05 vs. TAC + AGM₁₀ group. AGM: agmatine; CTR: control; HO-1: Heme Oxygenase-1; Nrf2: nuclear factor erythroid 2; SIRT1: Nad-dependent protein deacetylase sirtuin 1; TAC: tacrolimus.

Figure 5

Impact of AGM₁₀ and AGM₄₀ on TAC-induced changes in NF-κB and IL-17 levels. TAC was administered along with AGM₁₀ or AGM₄₀ for 14 days. Testes were collected to assess the following: (A) photomicrograph of immunostained testicular sections against NF-κB (magnification 200× and scale bar 50 µm), (B) NF-κB expression, and (C) IL-17 levels. Data are expressed as means ± SD (n = 6 rats/group). Mean values were compared via one-way ANOVA followed by post hoc Tukey’s multiple comparison test. * indicates significance at p value of <0.05 vs. CTR group. ^# indicates significance at p value of <0.05 vs. TAC group, and ^$ indicates significance at p value of <0.05 vs. TAC + AGM₁₀ group. AGM: agmatine; CTR: control; IL-17: interleukin 17; NF-κB: nuclear factor kappa B; TAC: tacrolimus.

Figure 6

Impact of AGM₁₀ and AGM₄₀ on TAC-induced changes in caspase 3 and Bcl-2 expression. TAC was administered along with AGM₁₀ or AGM₄₀ for 14 days. (A) Photomicrograph of immunostained testicular sections against caspase-3 (magnification 200× and scale bar 50 µm), (B) Caspase-3 expression, and (C) Bcl-2 levels. Data are expressed as means ± SD (n = 6 rats/group). Mean values were compared via one-way ANOVA followed by post hoc Tukey’s multiple comparison test. * indicates significance at p value of <0.05 vs. CTR group. ^# indicates significance at p value of <0.05 vs. TAC group, and ^$ indicates significance at p value of <0.05 vs. TAC + AGM₁₀ group. AGM: agmatine; CTR: control; TAC: tacrolimus.

Figure 7

The proposed protective mechanism of AGM on TAC-induced testicular toxicity. GSH: reduced glutathione; iNOS: inducible nitric oxide synthase; IL-17: interleukin 17; HO-1: heme oxygenase-1; NF-κB: nuclear factor kappa B; Nrf2: nuclear factor erythroid 2.