Modulatory effect of liraglutide on doxorubicin-induced testicular toxicity and behavioral abnormalities in rats: role of testicular-brain axis

Abstract

Doxorubicin (DOX) is a powerful chemotherapeutic agent used in many types of malignancies. However, its use results in testicular damage. DOX-induced testicular damage results in low level of serum testosterone which may affect cognitive function. The current study investigated the protective effect of liraglutide (50, 100 μg/kg/day) in testicular toxicity and the consequent cognitive impairment induced by DOX. DOX treatment reduced sperm count (62%) and sperm motility (53%) and increased sperm abnormalities (786%), as compared to control group. DOX also reduced serum testosterone level (85%) and the gene expression of testicular 3β-HSD (68%) and 17β-HSD (82%). Moreover, it increased testicular oxidative stress (MDA and GSH) by 103% and 59%, respectively, apoptotic (caspase-3 and P53) by 996% and 480%, respectively. In addition, DOX resulted in increasing autophagic markers including PAKT, mTOR, and LC3 by 48%, 56%, and 640%, respectively. Additionally, rats' behavior in Y-maze (60%) and passive avoidance task (85%) was disrupted. The histopathological results of testis and brain supported the biochemical findings. Treatment with liraglutide (100 μg/kg/day) significantly abrogated DOX-induced testicular damage by restoring testicular architecture, increasing sperm count (136%) and sperm motility (106%), and decreasing sperm abnormalities (84%) as compared to DOX group. Furthermore, liraglutide increased serum testosterone (500%) and steroidogenesis enzymes 3β-HSD (105%) and 17β-HSD (181%) along with suppressing oxidative stress (MDA and GSH) by 23% and 85%, respectively; apoptotic (caspase-3 and P53) by 59% and55%, respectively; and autophagic markers including PAKT, mTOR, and LC3 by 48%, 97%, and 60%, respectively. Moreover, it enhanced the memory functions in passive avoidance and Y-maze tests (132%). In conclusion, liraglutide is a putative agent for protection against DOX-induced testicular toxicity and cognitive impairment through its antioxidant, antiapoptotic, and antiautophagic effects.

Keywords: Autophagy; Cognitive impairment; DOX; Liraglutide; PI3 kinase/Akt/mTOR pathway; Testicular damage.

Fig. 1

A Dose range study timeline showing drug (liraglutide and DOX) administration schedule, sperm analysis, and histopathology of testes. B Mechanistic study timeline showing drug (liraglutide and DOX) administration schedule, the evaluated behavioral tests, and histopathology of brain

Fig. 2

Effect of different doses of liraglutide alone or in combination to DOX on the histopathological alterations in testes of rats administrated DOX. Representative photomicrographs of testis sections stained with hematoxylin–eosin stain (A, C, E, F, H, J, and K at 16 × magnification and B, D, G, and I at 40 × magnification). A and B Control group showing normal histological structure of the mature active seminiferous tubules with complete spermatogenic series. C DOX-treated group showing degeneration and lose of spermatogenic series with atrophy in some seminiferous tubules. D DOX-treated group showing plug formation in the tubule lumen. E DOX-treated group showing abnormal spermatids in central zone of the tubular lumen. F, G, H, and I Liraglutide (50, 100 µg) + DOX-treated groups showing normal histological structure. J and K Liraglutide (50, 100 µg)-alone-treated groups showing normal histological structure

Fig. 3

Effect of liraglutide (50, 100 µg) alone or in combination to DOX on A sperm motility, B sperm count, and C sperm abnormalities in rats. Data are expressed as mean ± SD using one-way ANOVA followed by Tukey multiple comparison test (n = 6). ***Significantly different from control group at p < 0.001. ^#Significantly different from DOX-treated group at p < 0.05. ^##Significantly different from DOX-treated group at p < 0.01. ^###Significantly different from DOX-treated group at p < 0.001. D Microphotographs illustrating morphologically of normal sperm and various sperm defects. D-I, D-IV, and D-V Control group, liraglutide (100 µg) + DOX, and liraglutide (100 µg) alone, respectively, showing normal sperm. D-II and D-III DOX-treated rats showed sperm defects. Coiled tail (red circle), zigzag-shape tail (red arrow). Liraglutide (100 µg) treatment counteracted sperm abnormalities induced by DOX

Fig. 4

Effect of liraglutide (50, 100 µg) alone or in combination to DOX on A serum testosterone concentration and B serum alkaline phosphatase (ALP) in rats. Data are expressed as mean ± SD using one-way ANOVA followed by Tukey multiple comparison test (n = 6). ***Significantly different from control group at p < 0.001. ^#Significantly different from DOX-treated group at p < 0.05. ^##Significantly different from DOX-treated group at p < 0.01. ^###Significantly different from DOX-treated group at p < 0.001

Fig. 5

Effect of liraglutide (100 µg) treatment on DOX-induced behavioral changes. A Y-maze percent of spontaneous alternation (SAP). B Y-maze total arm entries (TAE). Data are presented as mean ± SD (n = 6). Statistical analysis was carried out by one-way ANOVA followed by Tukey multiple comparison test. Data are presented as mean ± SD. ***Significantly different from control group at p < 0.001. ^###Significantly different from DOX-treated group at p < 0.001. C Correlation analysis: analysis of the correlation coefficient between Y-maze % of alternation (SAP) and Y-maze total arm entries (TAE). Non-significant positive correlation was observed between SAP and TAE (r = 0.018, p > 0.05). D Step-through passive avoidance acquisition test. E Step-through passive avoidance retention test. Data are presented as medians (25th, 75th percentile) (n = 6). Statistical analysis was carried out using Kruskal–Wallis non-parametric test followed by Dunn’s test. **Significantly different from control group at p < 0.01. ^##Significantly different from DOX-treated group at p < 0.01. ^###Significantly different from DOX-treated group at p < 0.001

Fig. 6

Histopathological examination of the effects of DOX, liraglutide treatments, or their combination (40 ×). Control group shows normal histological structure of the neurons in the cerebral cortex (I), subiculum (II), fascia dentata and hilus (III), and striatum (VI). DOX-treated group showing diffuse nuclear pyknosis (p) and neuronal degeneration in the cerebral cortex (I), subiculum (II), fascia dentata and hilus (III), and striatum (VI). Also, hemorrhage (H) was observed in striatum (VI). Liraglutide 100 + DOX-treated group showing normal histological structures of cerebral cortex (I), subiculum (II), fascia dentata and hilus (III), and striatum (VI). Liraglutide (100)-alone-treated group showing normal histological structures of cerebral cortex (I), subiculum (II), fascia dentata and hilus (III), and striatum (VI)

Fig. 7

Effect of 100 µg liraglutide on A testicular MDA concentration, B testicular reduced glutathione (GSH) concentration, C gene expression of testicular 3β-hydroxysteroid dehydrogenase (3β-HSD), and D gene expression of testicular 17β-hydroxysteroid dehydrogenase (17β-HSD). Data are expressed as mean ± SD, using one-way ANOVA followed by Tukey multiple comparison test (n = 6). **Significantly different from control group at p < 0.01. ***Significantly different from control group at p < 0.001. ^#Significantly different from DOX-treated group at p < 0.05. ^##Significantly different from DOX-treated group at p < 0.01. ^###Significantly different from DOX-treated group at p < 0.001

Fig. 8

Effect of 100 µg liraglutide on A testicular MTOR concentration, B testicular pAKT concentration, and C gene expression of testicular microtubule-associated protein 1A/1B-light chain (LC3). Data are expressed as mean ± SD, using one-way ANOVA followed by Tukey multiple comparison test (n = 6 for MTOR and pAKT and n = 3 for LC3). **Significantly different from control group at p < 0.01. ***Significantly different from control group at p < 0.001. ^#Significantly different from DOX-treated group at p < 0.05. ^###Significantly different from DOX-treated group at p < 0.001

Fig. 9

Effect of 100 µg liraglutide on immunostaining caspase-3 in testicular tissue from DOX-administered rats. A Representative photomicrographs of caspase-3 immunostain sections of testes showing brown immunopositive staining in DOX-treated rats. B Bar chart representation of caspase-3 immunoexpression % in the different groups. C Gene expression of testicular p53. Data are expressed as mean ± SD, using one-way ANOVA followed by Tukey multiple comparison test (n = 6 for caspase-3 and n = 3 for p53). ***Significantly different from control group at p < 0.001. ^###Significantly different from DOX-treated group at p < 0.001

Fig. 10

The proposed modulatory effect of liraglutide in DOX-testicular toxicity via the involved PI3K/Akt/mTOR signaling pathway. DOX exerts its toxic effect by increasing ROS which inhibits PI3K that results in downregulation of phosphorylation of Akt (active form) with subsequent inhibition of mTOR leading to increase autophagy (high level of gene expression of LC3) and apoptosis (high level of caspase-3 and p53). All of these signals contribute to testicular damage. Furthermore, testicular damage is accompanied by low level of serum testosterone which can negatively affect cognition. On the other hand, liraglutide counteracts these events through its antioxidant, antiapoptotic, and antiautophagic effects besides elevating serum testosterone level and enhancing memory performance