The Possible Contribution of P-Glycoprotein in the Protective Effect of Paeonol against Methotrexate-Induced Testicular Injury in Rats

Abstract

Paeonol, a phenolic ingredient in the genus Paeonia, possesses antioxidant and anti-inflammatory effects. Methotrexate (MTX) is a commonly used chemotherapeutic agent; however, its germ cell damage is a critical problem. P-glycoprotein (P-gp), an efflux transporter, is a member of the blood-testis barrier. The present study evaluated the protective effect of paeonol on MTX-induced testicular injury in rats with the exploration of its mechanism and the possible contribution of P-gp in such protection. Testicular weight, serum testosterone, and testicular P-gp levels were measured. Testicular oxidant/antioxidant status was evaluated via determining the levels of malondialdehyde, total nitrite, reduced glutathione, and superoxide dismutase activity. The inflammatory cytokine tumor necrosis factor-alpha (TNF-α) and the apoptotic marker caspase 3 were estimated immunohistochemically. Testicular histopathology and spermatogenesis scores were also examined. MTX caused histopathologically evident testicular damage with decreased testicular weight, testosterone level, and spermatogenesis score, as well as significant increases in oxidative, inflammatory, and apoptotic responses. Paeonol significantly restored testicular weight, testosterone level, spermatogenesis score, and oxidant/antioxidant balance. Moreover, paeonol increased the testicular P-gp level and significantly decreased TNF-α and caspase 3 immunostaining. In conclusion, paeonol offered a protective effect against MTX-induced testicular injury through its antioxidant, anti-inflammatory, and antiapoptotic effects, as well as by increasing testicular P-gp level.

Keywords: P-glycoprotein; caspase 3; methotrexate; oxidative stress; paeonol; testicular injury; tumor necrosis factor-α.

Figure 1

Photomicrographs of the testis (hematoxylin and eosin ×100). (A,B) Control and paeonol groups respectively, showing the same histological picture. The seminiferous tubules (stars) are seen lined with germinal epithelium. The interstitial cells of Leydig (circles) are observed between these tubules. Notice the sperms occupying the lumen of seminiferous tubules. The tubules are seen laying on the basement membrane (arrows). (C) MTX group showing several distorted seminiferous tubules (S) lined by darkly stained acidophilic cells (rectangle) with dense nuclei. Notice the focal reduction of the germinal cells (circle). A widened interstitium (star) is also noticed. The insert shows some tubules (T) that have nearly lost their germinal epithelium lining. The separated basement membrane (short arrow) can also be seen. Dilated congested blood vessels (curved arrow) occupying the interstitium are also noticed. (D) Paeonol + MTX group showing apparent normalization of the seminiferous tubules, but still few tubules appear with some distortion (S). Less widening of blood vessels (notched arrow) are seen.

Figure 2

Immunohistochemical expression of testicular tumor necrosis factor-alpha (TNF-α) (×400). (A,B) Control and paeonol groups respectively, showing negative TNF-α immunoreactivity (stars) in the germinal cells. (C) MTX group showing cytoplasmic TNF-α immunoreactivity in the germinal cells and in the interstitial cells (dashed arrows). (D) Paeonol + MTX group showing faint cytoplasmic TNF-α expression in the germinal and interstitial cells (dashed arrows). (E) Mean area fraction of TNF-α immunostaining. Results are mean ± SEM (n = 8). *^,# Significantly different (p < 0.05) from control and MTX groups, respectively. MTX: methotrexate.

Figure 3

Immunohistochemical expression of testicular caspase 3 (×400). (A,B) Control and paeonol groups respectively, showing negative caspase 3 immunoreactivity (stars) in the germinal cells. (C) MTX group showing positive diffuse intense cytoplasmic caspase 3 immunoreactivity (arrows) in the germinal cells. (D) Paeonol + MTX group showing less cytoplasmic caspase 3 immunoreactivity (arrows) in germinal cells. (E) Mean area fraction of caspase 3 immunostaining. Results are mean ± SEM (n = 8). *^,# Significantly different (p < 0.05) from control and MTX groups, respectively. MTX: methotrexate.