Salazinic acid attenuates male sexual dysfunction and testicular oxidative damage in streptozotocin-induced diabetic albino rats

Abstract

Male sexual dysfunctions such as infertility and impotence are recognized as the consequences of diabetes. Salazinic acid (Sa) is a depsidone found in lichen genera of Lobaria, Parmelia, and Usnea, which has prominent free radical and α-glucosidase inhibitory actions. The present study establishes the beneficial role of salazinic acid (Sa) to combat the deleterious effects of streptozotocin-induced diabetes on the male reproductive system of rats. In a dose-dependent manner, Sa significantly restored the reproductive organs weight, sperm characteristics, and testicular histoarchitecture in diabetic rats. Further, a significant recovery of insulin, follicle-stimulating hormone, luteinizing hormone and testosterone levels in serum was recorded in Sa-treated diabetic rats. The malondialdehyde levels were significantly lowered, and the activities of glutathione, superoxide dismutase, glutathione peroxidase and catalase, markedly elevated in the blood serum, as well as testicular tissue after Sa-supplementation. Sa also suppressed the protein expression levels of tumor necrosis factor-α in serum. The high dose of Sa showed significant improvement in glycemia and testicular protection, similar to sildenafil citrate. Moreover, the docking results showed that both Sa and sildenafil have a high affinity toward the target protein, PDE5 with binding affinity values found to be -9.5 and -9.2 kcal mol^-1, respectively. Molecularly, both Sa and sildenafil share similar hydrogen bonding patterns with PDE5. Hence, our study clearly showed the protective role of Sa against diabetic-induced spermatogenic dysfunction in rats, possibly by competing with cGMP to bind to the catalytic domain of PDE5 and thereby controlling the oxidative impairment of testes.

Fig. 1. In vitro (A) antioxidant and (B) antidiabetic activities of salazinic acid. Values are expressed as mean ± standard deviation (n = 3). ^aP < 0.05, ^bP < 0.001, and ^cP < 0.0001 statistically significant from the ascorbic acid. Statistical analyses were performed by one-way analysis of variance followed by Tukey's test at P ≤ 0.05.

Fig. 2. Effect of salazinic acid on oxidative stress parameters such as (A) malondialdehyde, (B) glutathione, (C) catalase, (D) superoxide dismutase, (E) glutathione peroxidase, and (F) reactive oxygen species (ROS), in the serum of streptozotocin-induced diabetic rats. Values are expressed as mean ± standard deviation (n = 6). ^aP < 0.05, ^bP < 0.001, and ^cP < 0.0001 statistically significant from the normal control group. ^xP < 0.05, ^yP < 0.001 and ^zP < 0.0001, statistically significant from the diabetic control group. Multiple group comparisons were performed by one-way analysis of variance followed by Tukey's multiple comparison post hoc test at P ≤ 0.05.

Fig. 3. Effect of salazinic acid on serum levels of testosterone, FSH, LH and TNF-α of streptozotocin-induced diabetic rats. Values are expressed as mean ± standard deviation (n = 6). ^aP < 0.05, ^bP < 0.001, and ^cP < 0.0001 statistically significant from the normal control group. ^xP < 0.05 and ^zP < 0.0001, statistically significant from the diabetic control group. Multiple group comparisons were performed by one-way analysis of variance followed by Tukey's multiple comparison post hoc test at P ≤ 0.05.

Fig. 4. Effect of salazinic acid on lipid parameters of streptozotocin-induced diabetic rats. Values are expressed as mean ± standard deviation (n = 6). ^aP < 0.05, ^bP < 0.001, and ^cP < 0.0001 statistically significant from the normal control group. ^zP < 0.0001, statistically significant from the diabetic control group. Multiple group comparisons were performed by one-way analysis of variance followed by Tukey's multiple comparison post hoc test at P ≤ 0.05.

Fig. 5. Effect of salazinic acid on liver biomarkers, namely (A) alanine transferase, (B) aspartate aminotransferase, (C) alkaline phosphatase, (D) creatinine, (E) urea, (F) total bilirubin and (G) total protein, of streptozotocin-induced diabetic rats. Values are expressed as mean ± standard deviation (n = 6). ^aP < 0.05, ^bP < 0.001, and ^cP < 0.0001 statistically significant from the normal control group. ^xP < 0.05 and ^zP < 0.0001, statistically significant from the diabetic control group. Multiple group comparisons were performed by one-way analysis of variance followed by Tukey's multiple comparison post hoc test at P ≤ 0.05.

Fig. 6. Effect of salazinic acid on antioxidant profile; SOD, GPx, CAT, GSH and MDA in the testicular homogenate of streptozotocin-induced diabetic rats. Values are expressed as mean ± standard deviation (n = 6). ^aP < 0.05, ^bP < 0.001, and ^cP < 0.0001 statistically significant from the normal control group. ^yP < 0.001 and ^zP < 0.0001, statistically significant from the diabetic control group. Multiple group comparisons were performed by one-way analysis of variance followed by Tukey's multiple comparison post hoc test at P ≤ 0.05.

Fig. 7. Histopathological examination (H&E x400) of the testicular tissues in (A) normal control group, (B) diabetic control group, (C) Sa-treated (5 mg kg⁻¹ b.w) group, (D) Sa-treated (10 mg kg⁻¹ b.w) group, and (E) sildenafil citrate-treated (5 mg kg⁻¹ b.w) group. (F) The bar chart represents testicular lesion score, where values are expressed as mean ± standard deviation. ^bP < 0.001, and ^cP < 0.0001 statistically significant from the normal control group. ^yP < 0.001 and ^zP < 0.0001, statistically significant from the diabetic control group. Multiple group comparisons were performed by one-way analysis of variance followed by Tukey's multiple comparison post hoc test at P ≤ 0.05.

Fig. 8. (A) Validation of docking protocol by superimposition of co-crystallized and re-docked poses of avanafil on PDE5; different non-bonding interactions between PDE5 and the native inhibitor avanafil, (B) Co-crystal pose, and (C) Re-docked pose.

Fig. 9. 2D interactions of PDE5 active site residues with (A) salazinic acid, and (B) sildenafil.

Fig. 10. The analysis of the molecular dynamics simulation trajectories (A) RMSD plot, (B) RMSF plot, (C) radius of gyration, and (D) hydrogen bonds of PDE5 with salazinic acid and sildenafil.

Fig. 11. The (A) enthalpic energy, (B) binding free energy and (C) per-residue energy contribution of active site residues of PDE5 with (I) salazinic acid and (II) sildenafil.