The effects of MOTILIPERM on cisplatin induced testicular toxicity in Sprague-Dawley rats

Abstract

Background: Cisplatin causes male infertility but the exact mechanism have not been clarified, yet. MOTILIPERM has been implicated in alleviation of infertility in Sprague-Dawley rats caused by cisplatin. We evaluated recovery effect of MOTILIPERM on cisplatin (CIS)-induced testicular toxicity in Sprague-Dawley rats.

Methods: Five groups were included. The groups are control (CTR), CTR + MOTILIPERM 200 mg/kg/day per oral, CIS 10 mg/kg i.v., CIS 10 mg/kg + MOTILIPERM 100 mg/kg/day, CIS 10 mg/kg + MOTILIPERM 200 mg/kg/day. CIS 10 mg/kg i.v. single dose was given before 100 mg/kg, or 200 mg/kg MOTILIPERM per oral daily for 28 days. Body and genital organs weight, epididymis sperm count, sperm motility, sperm apoptosis, testosterone level, MDA of testis tissue, spermatogenic cell density, and Johnsen's score were evaluated. Steroidogenic acute regulatory (StAR) protein, and Glucose-regulated protein-78 (GRP-78), phosphorylated Inositol-Requiring Transmembrane Kinase/Endoribonuclease 1 (IRE1) and phosphorylated c-jun-N-terminal kinase (p-JNK) were quantitated by western blot to show its signaling pathway.

Results: The body weight was decreased significantly in CIS 10 mg/kg, CIS 10 mg/kg + MOTILIPERM 100 mg/kg/day, CIS 10 mg/kg + MOTILIPERM 200 mg/kg/day compared with CTR (p < 0.001) however, it was increased in CIS 10 mg/kg + MOTILIPERM 100 mg/kg/day, CIS 10 mg/kg + MOTILIPERM 200 mg/kg/day compared with CIS 10 mg/kg. The decreased weight of epididymis and prostate were increased significantly in CIS 10 mg/kg + MOTILIPERM 100 mg/kg/day compared with CIS 10 mg/kg. Sperm count, sperm motility, sperm apoptosis, MDA of testis tissue, spermatogenic cell density, Johnsen's score, and total testosterone were also significantly improved by MOTILIPERM treatment. The levels of decreased StAR protein was significantly improved by MOTILIPERM administration, increased GRP-78 protein p-IRE1and p-JNK was also significantly decreased with MOTILIPREM treatment.

Conclusion: The MOTILIPERM could be an effective medicine to reduce the toxic effect caused ER stress by CIS in the testis.

Keywords: Cispaltin (CIS); Glucose-regulated protein-78 (GRP-78); MOTILIPERM; Phosphorylated c-jun-N-terminal kinase (p-JNK); Phosphorylated inositol-requiring transmembrane kinase/endoribonuclease 1 (IRE1); Spermatogenic cell denity; Steroidogenic acute regulatory (StAR) protein.

Fig. 1

HPLC chromatogram of MOTILIPERM and ultraviolet spectra of major marker components from each herbal ingredients. Monotropein (1) and deacetyl asperulosidic acid (2) from the Morinda officinalis; Chlorogenic acid (3) and 3,5-dicaffeoylquinic acid (4) from the Cuscuta japonica; quercetin 4′-glcoside (5) and quercetin (6) from the Allium cepa. Each peak of MOTILIPERM in the HPLC chromatogram was identified by comparison with the retention times and UV spectra of standard compounds

Fig. 2

Evaluation of MDA for each group. Data are presented in mean ± SD. ^† p < 0.05 vs CTR + M 200 group, ^# p < 0.05 vs CIS group, ^** p < 0.001 vs CTR group, ^†† p < 0.001 vs CTR + M 200 group

Fig. 3

Effects of cisplatin (CIS) and MOTILIPERM on total testosterone and StAR protein by Western blot. a Total testosterone level. b StAR protein. c Western blotting of StAR protein for each group. Data are presented in mean ± SD. CTR control; CTR + M 200 CTR + MOTILIPERM 200 mg/kg/day; CIS cisplatin 10 mg/kg i intravenous (i.v.); CIS + M 100 CIS 10 mg/kg, i.v. + MOTILIPERM 100 mg/kg/day; CIS + M 200 CIS 10 mg/kg, i.v. + MOTILIPERM 200 mg/kg/day p.o. M MOTILIPERM. ^* p < 0.05 vs CTR group, ^† p < 0.05 vs CTR + M 200 group, ^# p < 0.05 vs CIS group

Fig. 4

Light microscope evaluations of the testis. a Hematoxylin & Eosin (H&E) staining of the testis. b Spermatogenic cell density. c Johnsen’s score in seminiferous tubules. Data are presented in mean ± SD. CTR control; CTR + M 200 CTR + MOTILIPERM 200 mg/kg/day; CIS cisplatin 10 mg/kg i intravenous (i.v.); CIS + M 100 CIS 10 mg/kg, i.v. + MOTILIPERM 100 mg/kg/day; CIS + M 200 CIS 10 mg/kg, i.v. + MOTILIPERM 200 mg/kg/day p.o. M MOTILIPERM. ^* p < 0.05 vs CTR group, ^† p < 0.05 vs CTR + M 200 group, ^# p < 0.05 vs CIS group, ^** p < 0.001 vs CTR group, ^†† p < 0.001 vs CTR + M 200 group

Fig. 5

Evaluation of GRP-78. a Western blot of testis. b Levels of GRP-78 for each group. Data are presented in mean ± SD. CTR control; CTR + M 200 CTR + MOTILIPERM 200 mg/kg/day; CIS cisplatin 10 mg/kg i intravenous (i.v.); CIS + M 100 CIS 10 mg/kg, i.v. + MOTILIPERM 100 mg/kg/day; CIS + M 200 CIS 10 mg/kg, i.v. + MOTILIPERM 200 mg/kg/day p.o. M MOTILIPERM. ^† p < 0.05 vs CTR + M 200 group, ^# p < 0.05 vs CIS group

Fig. 6

Evaluation of p-IRE1, IRE1, a Western blot of testis. b Levels of pIRE1 and total IRE1 for each group. Data are presented in mean ± SD. CTR control; CTR + M 200 CTR + MOTILIPERM 200 mg/kg/day; CIS cisplatin 10 mg/kg i intravenous (i.v.); CIS + M 100 CIS 10 mg/kg, i.v. + MOTILIPERM 100 mg/kg/day; CIS + M 200 CIS 10 mg/kg, i.v. + MOTILIPERM 200 mg/kg/day p.o. M MOTILIPERM. ^* p < 0.005 vs CTR group, ^† p < 0.05 vs CTR + M 200 group

Fig. 7

Evaluation of pJNK and JNK a Western blot of testis. b Levels of pJNK and total JNK for each group. Data are presented in mean ± SD. CTR control; CTR + M 200 CTR + MOTILIPERM 200 mg/kg/day; CIS cisplatin 10 mg/kg i intravenous (i.v.); CIS + M 100 CIS 10 mg/kg, i.v. + MOTILIPERM 100 mg/kg/day; CIS + M 200 CIS 10 mg/kg, i.v. + MOTILIPERM 200 mg/kg/day p.o. M MOTILIPERM.^{. *} p < 0.05 vs CTR group, ^† p < 0.05 vs CTR + M 200 group, ^** p < 0.001 vs CTR group

Fig. 8

Schematic view of endoplasmic reticulum (ER) stress signaling pathway explained. GRP78 Glucose-regulated protein-78 (GRP-78), pIRE1 phosphorylated inositol-requiring transmembrane kinase/endoribonuclease1; IRE1 inositol-requiring transmembrane kinase/endoribonuclease1; ATF6 activating transcription factor-6; TRAF2 tumor necrosis factor receptor associated factor2; PERK protein kinase RNA-like endoplasmic reticulum kinase; ASK1 apoptosis signal-regulating kinase; pJNK phosphorylated c-jun-N-terminal kinase; JNK c-jun-N-terminal kinase