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.
