Kaempferol mitigates reproductive dysfunctions induced by Naja nigricollis venom through antioxidant system and anti-inflammatory response in male rats

Abstract

Naja nigricollis Venom (NnV) contains complex toxins that affects various vital systems functions after envenoming. The venom toxins have been reported to induce male reproductive disorders in envenomed rats. This present study explored the ameliorative potential of kaempferol on NnV-induced male reproductive toxicity. Fifty male wistar rats were sorted randomly into five groups (n = 10) for this study. Group 1 were noted as the control, while rats in groups 2 to 5 were injected with LD₅₀ of NnV (1.0 mg/kg bw; i.p.). Group 2 was left untreated post envenomation while group 3 was treated with 0.2 ml of polyvalent antivenom. Groups 4 and 5 were treated with 4 and 8 mg/kg of kaempferol, respectively. NnV caused substantial reduction in concentrations of follicle stimulating hormone, testosterone and luteinizing hormone, while sperm motility, volume and counts significantly (p < 0.05) decreased in envenomed untreated rats. The venom enhanced malondialdehyde levels and substantially decreased glutathione levels, superoxide dismutase and glutathione peroxidase activities in the testes and epididymis of envenomed untreated rats. Additionally, epididymal and testicular myeloperoxidase activity and nitric oxide levels were elevated which substantiated severe morphological defects noticed in the reproductive organs. However, treatment of envenomed rats with kaempferol normalized the reproductive hormones with significant improvement on sperm functional parameters. Elevated inflammatory and oxidative stress biomarkers in testis and epididymis were suppressed post kaempferol treatment. Severe histopathological lesions in the epididymal and testicular tissues were ameliorated in the envenomed treated groups. Results highlights the significance of kaempferol in mitigating reproductive toxicity induced after snakebite envenoming.

Figure 1

Concentrations of serum male reproductive hormones in envenomed rats treated with kaempferol. Data are represented as mean ± SE (n = 10). Bar with different lower-case letter represent significant difference among the groups at p < 0.05 using DMRT. FSH follicle stimulating hormone, TEST testosterone hormone, LH luteinizing hormone. Group 1: Injected with saline (Control), Group 2: Envenomed not treated (Venom control), Group 3: Envenomed and treated with 0.2 ml of antivenom, Group 4: Envenomed and treated with 4 mg/kg⁻¹ of kaempferol, Group 5: Envenomed and treated with 8 mg/kg⁻¹ of kaempferol.

Figure 2

Stress profiles of the testis and epididymis of envenomed rats treated with kaempferol. Data are represented as mean ± SE (n = 5). Bar with different lower-case letter represent significant difference in stress profile of testis among the groups at p < 0.05 using DMRT. Bar with different upper-case letter represent significant difference in stress profile of epididymis among the groups at p < 0.05 using DMRT. MDA malondialdehyde, GSH glutathione, SOD superoxide dismutase, GPX glutathione peroxidase. Group 1: Injected with saline (Control), Group 2: Envenomed not treated (Venom control), Group 3: Envenomed and treated with 0.2 ml of antivenom, Group 4: Envenomed and treated with 4 mg/kg⁻¹ of kaempferol, Group 5: Envenomed and treated with 8 mg/kg⁻¹ of kaempferol.

Figure 3

Inflammatory profiles of the testis and epididymis of envenomed rats treated with kaempferol. Data are represented as mean ± SE (n = 5). Bar with different lower-case letter represent significant difference in inflammatory markers of the testis among the groups at p < 0.05 using DMRT. Bar with different upper-case letter represent significant difference in inflammatory markers of epididymis among the groups at p < 0.05. NO nitric oxide, MPO myeloperoxidase. Group 1: Injected with saline (Control), Group 2: Envenomed not treated (Venom control), Group 3: Envenomed and treated with 0.2 ml of antivenom, Group 4: Envenomed and treated with 4 mg/kg⁻¹ of kaempferol, Group 5: Envenomed and treated with 8 mg/kg⁻¹ of kaempferol.

Figure 4

Photomicrographs of the testicular histomorphology of envenomed treated rats. BM The seminiferous epithelium and basement membrane, L The lumen, IS interstitial space containing interstitial cells, SG spermatogonium (1⁰,2⁰,3⁰). Group 1 (control): showed normal seminiferous tubules lined with spermatogenic cell layers with their lumen (L) with no observable lesion. Group 2 (venom control): revealed severe testicular degeneration within the spermatogenic cell layers, hyperplasia of Leydig cells within edematous and atypia within some spermatogenic cells (red arrows). Groups 3 (venom/antivenom): showed mild testicular degeneration within the spermatogenic cell layers (red arrow) with a core of desquamative spermatogenic cells. Groups 4 (venom/4 mg/kg kaempferol): showed mild testicular degeneration within the spermatogenic cell layers (arrows) with a core of desquamative spermatogenic cells, Groups 5 (venom/4 mg/kg kaempferol): No observable pathomorphology was seen.

Figure 5

Photomicrographs of the epididymal histomorphology of envenomed treated rats. ET epididymal tubules, PE pseudostratified columnar epithelium, and arrows: hemorrhage in the lumen. Group 1 (control): showed regular and circular tubules with a pseudostratified columnar epithelium that exhibits stereocilia with no alteration in epididymal structural integrity, Group 2 (venom control): revealed extra tubular space contains blood vessels in the interstitial connective tissue and evidence of hemorrhage (red arrows). Groups 3 (venom/antivenom): The epithelium was separated from the connective tissue with extra tubular space containing blood vessels in the interstitial connective tissue (red arrows), Groups 4 (venom/4 mg/kg kaempferol): There is a vacuole formation within pseudostratified epithelium with no marked histological alteration, Groups 4 (venom/4 mg/kg kaempferol): There is a vacuole formation within pseudostratified epithelium with no observable marked histological alteration.