PLUMBAGIN-INDUCED TESTICULAR MITOCHONDRIAL-DEPENDENT CELL DEATH AND INFERTILITY IN MALE WISTAR RATS

Abstract

The opening of the mitochondrial Permeability Transition (mPT) pore is an important event in mitochondrial-mediated cell death. Plumbagin, the active principle in Plumbago zeylanica, induces cell death in rapidly dividing cells and conditions such as prostate tumor but its effects on testicular cell death and fertility are not well understood. This study was designed to investigate the effects of plumbagin on testicular cell death and fertility in male Wistar rats. This study was in two phases. In phase 1, twenty male Wistar rats (80-100g) were grouped into four (n= 5) and were treated once daily as follows: Group I (Control) received 10ml/kg distilled water, groups II, III and IV were orally treated with 2.5, 5.0 and 10.0mg/kg of plumbagin for 14 days. In study 2, fifteen male Wistar rats (100-120g) were grouped into three (n=5) and were orally treated once daily with 10 ml/kg distilled water (control), 30 and 100mg/kg of plumbagin for 72 hours. In study 1, testes mitochondria were isolated using differential centrifugation. The mPT pore opening, mitochondrial Lipid Peroxidation (mLPO) and mitochondrial ATPase (mATPase) were assessed using spectrophotometry. Caspases 3 (C3) and 9 (C9) activities were assessed using ELISA. Sperm count, motility and morphology were determined using microscopy. Expressions of p53, Bax, Cytochrome C Release (CCR) and Bcl-2 were determined using immunohistochemistry. In study 2, expressions of Follicle Stimulating Hormone (FSH), Progesterone Receptors (PR), Testis Specific Protein Kinase-1 (TESK-1) and aromatase were determined using polymerase chain reaction. Interactions between plumbagin, Mouse Double Minute Homolog 2 (MDM2) and Bcl-2 were assessed using docking method. Statistical analysis was done using descriptive statistics and ANOVA at α 0.05 Plumbagin at 2.5, 5.0 and 10.0mg/kg induced mPT pore opening in testis by 2.3, 4.6 and 8.0 folds relative to control. Plumbagin also increased mLPO (2.3, 5.8, 8.0µmoleMDA/mg protein), mATPase (10.1, 12.7, 13.6mmolePi/mg protein/min), C3 (5.1, 7.3, 12.2ng/mL) and C9 (6.7, 8.4, 11.8ng/mL) activities respectively compared to control (1.1µmoleMDA/mg protein, 8.2mmolePi/mg protein/min, 2.6 and 1.8ng/mL), respectively. Sperm analysis revealed decrease in sperm count (63, 61, 62.5 million/mL), motility (80.1,78.3,77.0%) and increased sperm abnormality (7.2+1.41, 9.5+ 0.71, 11.5+0.71million/mL) at 2.5, 5.0 and 10.0mg/kg, compared to control (120million/mL, 98% and 4.3+0.21million/mL) respectively. Furthermore, plumbagin increased the expressions of p53 (6.3, 7.5 and 12.3%), Bax (6.1, 7.5 and 9.5%), CCR (7.2, 8.4 and 12.3%) and decreased Bcl-2 (25.0, 18.0 and 15.5%) at the same dose compared to control (5.0, 3.4, 6.0 and 27.0%,). Expressions of FSH (0.9, 0.7), PR (0.8, 0.3), TESK-1 (0.7, 0.3) and aromatase (0.8, 0.3) folds decreased relative to control. Plumbagin interacted with MDM2 and Bcl-2 (∆G = -6.0 and -5.9kcal/mol) respectively compared to 7-hydroxy-4-methylcoumarin (∆G = -10 kcal/mol) used as control in docking studies. Plumbagin decreased spermatogenesis in male Wistar rats by decreasing the expressions of Follicle Stimulating Hormone and masking the Progesterone Receptor. It also decreased spermatogenesis by down-regulating the activities of Testis Specific Protein Kinase-1 and Aromatase. It caused testicular damage via mitochondrial-dependent cell death with increased sperm abnormality.