Molecular, Immunomodulatory, and Histopathological Role of Mesenchymal Stem Cells and Beetroot Extract on Cisplatin Induced Testicular Damage in Albino Rats

Abstract

Cisplatin (Cis) a drug commonly used as a chemotherapeutic agent to treat various types of cancer, inducing testicular damage. The present study aimed to investigate the inhibitory potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) and beetroot extract (BRE) in albino rats after testicular toxicity induced by cisplatin. Thirty adult male albino rats were grouped into: the control group, Cis group receiving a single dose of 7 mg/kg i.p. (intraperitoneal) to induce testicular toxicity, Cis plus BM-MSCs injected Cis followed by 2 × 10⁶ of BM-MSCs; Cis plus BRE group receiving Cis followed by 300 mg/kg body weight/day of BRE, and Cis plus BM-MSCs and BRE group. In the current study, Cis reduced sperm count, serum testosterone level, and testicular activity of alkaline phosphatase (AKP), besides a marked inhibition of succinate dehydrogenase (SDH) activity. In addition, it significantly increased malondialdehyde (MDA) and along with a marked decrease in testis reduced glutathione content and total antioxidant capacity (TAC). At the same time, Cis administration resulted in a marked elevation in interleukine-6 and the iNOS and caspase-3 genes; however, it decreased the expression of steroidogenic acute regulatory protein (StAR). Combined treatment with BM-MSCs and BRE resulted in great improvement of all previous parameters. These results were also confirmed by histopathological and immunohistochemical examination. In conclusion, both MSCs and BRE were found to have potent potentials to inhibit testicular damage induced by cisplatin.

Keywords: StAR; beetroot extract; caspase-3; cisplatin; iNOS; mesenchymal stem cells; testicular damage.

Figure 1

Box and whisker plot for sperm counts in the treated groups. The groups are: the control group (NG); the Cisplatin-treated group (CG); the Cisplatin+ beetroot extract-treated group (CBG); the Cisplatin+ mesenchymal stem cells-treated group (CMG); and the Cisplatin+ mesenchymal stem cells + beetroot extract-treated group (CMBG). The interrelations between all the presented groups are statistically significant (p ≤ 0.05). Group mean is presented by x symbols in the boxplots.

Figure 2

Box and whisker plot for serum testosterone concentration (ng/mL) in the treatment groups. The groups are: the control group (NG); the Cisplatin-treated group (CG); the Cisplatin+ beetroot extract-treated group (CBG); the Cisplatin+ mesenchymal stem cells treated group (CMG); and the Cisplatin+ mesenchymal stem cells + beetroot extract-treated group (CMBG). The interrelations between all the presented groups are statistically significant (p ≤ 0.05). Group mean is presented by x symbols in the boxplots.

Figure 3

Histogram for succinate dehydrogenase (SDH) level (U/mg total proteins) in the treatment groups. The groups are: the control group (NG); the Cisplatin-treated group (CG); the Cisplatin+ beetroot extract treated group (CBG); the Cisplatin+ mesenchymal stem cells treated group (CMG); and the Cisplatin+ mesenchymal stem cells + beetroot extract treated group (CMBG). The interrelations between all the presented groups are statistically significant (p ≤ 0.05), except for the annotated groups (groups with the same letter have a statistically non-significant relationship).

Figure 4

Histogram for alkaline phosphatase (AKP) level (U/mg total proteins) in the treatment groups. The interrelations between all the presented groups are statistically significant (p ≤ 0.05), except for the annotated groups (groups with the same letter have a statistically non-significant relationship). The groups are: the control group (NG); the Cisplatin-treated group (CG); the Cisplatin+ beetroot extract-treated group (CBG); the Cisplatin+ mesenchymal stem cells-treated group (CMG); and the Cisplatin+ mesenchymal stem cells + beetroot extract-treated group (CMBG).

Figure 5

Representation for Interleukin-6 (IL-6) concentration (ng/mL) in the treatment groups. The groups are: the control group (NG); the Cisplatin treated-group (CG); the Cisplatin+ beetroot extract-treated group (CBG); the Cisplatin+ mesenchymal stem cells-treated group (CMG); and the Cisplatin+ mesenchymal stem cells + beetroot extract-treated group (CMBG). The interrelations between all the presented groups are statistically significant (p ≤ 0.05).

Figure 6

Histogram for testicular malondialdehyde (MDA) concentration (m mol/L) in the treated groups. The interrelations between all the presented groups are statistically significant (p ≤ 0.05). The groups are: the control group (NG); the Cisplatin-treated group (CG); the Cisplatin+ beetroot extract-treated group (CBG); the Cisplatin+ mesenchymal stem cells-treated group (CMG); and the Cisplatin+ mesenchymal stem cells + beetroot extract-treated group (CMBG).

Figure 7

Histogram for testicular total antioxidant capacity (TAC) concentration (m mol/L) in the treated groups. The interrelations between all the presented groups are statistically significant (p ≤ 0.05). The groups are: the control group (NG); the Cisplatin-treated group (CG); the Cisplatin+ beetroot extract-treated group (CBG); the Cisplatin+ mesenchymal stem cells-treated group (CMG); and the Cisplatin+ mesenchymal stem cells + beetroot extract-treated group (CMBG).

Figure 8

Histogram for glutathione concentration (m mol/L) in the treated groups. The interrelations between all the presented groups are statistically significant (p ≤ 0.05), except for the annotated groups (groups with same letter have insignificant relation). The groups are: the control group (NG); the Cisplatin-treated group (CG); the Cisplatin+ beetroot extract-treated group (CBG); the Cisplatin+ mesenchymal stem cells-treated group (CMG); and the Cisplatin+ mesenchymal stem cells + beetroot extract-treated group (CMBG).

Figure 9

Panel of histological (H&E ×400) pictures showing: (A) control group (NG) seminiferous tubules (ST) with average BM (basement membrane), spermatogonia, primary spermatocyte, and many spermatozoa (red arrows); (B,C) Cisplatin-treated group (CG) with tubules have detached and thick BM (black arrow), marked reduction of germinal lining with few sperms, and interstitial blood vessels congested (yellow arrow). (D) Cisplatin+ mesenchymal stem cells treated group (CMG) tubules with mildly thick BM, mild reduction of germinal lining with moderate number of sperms. (E) Cisplatin+ mesenchymal stem cells+ beetroot extract treated group (CBG) tubules with average BM, average germinal lining up to full spermatogenesis, and average interstitium with average Leydig cells. (F) Cisplatin+ mesenchymal stem cells+ beetroot extract treated group (CMBG) tubules with mildly thick BM, average germinal lining up to full spermatogenesis, and average interstitium with average Leydig cells.

Figure 10

Panel of proliferating cell nuclear antigen immuno-histochemical (IHC) pictures (×400). Degree of PCNA staining were referred to as mild staining (+), moderate staining (++), and strong staining (+++). (A) control group (NG) testicular tissue with mild nuclear reactivity (++) for PCNA in basal tubular lining, spermatogonia (red arrow) and primary spermatocytes; (B) Cisplatin-treated group (CG) has marked low nuclear reactivity (+) for PCNA in tubular lining from spermatogonia (red arrow) up to spermatids, and in interstitial cells; (C) Cisplatin+ beetroot extract treated group (CBG) with marked nuclear reactivity (++) for PCNA in tubular lining from spermatogonia (red arrow) up to spermatids, and in interstitial cells; (D) Cisplatin+ mesenchymal stem cells treated group (CMG) has moderate nuclear reactivity (++) for PCNA in tubular lining from spermatogonia (red arrows) up to spermatids; (E) Cisplatin+ mesenchymal stem cells+ beetroot extract treated group (CMBG) has marked more nuclear reactivity (+++) for PCNA in tubular lining from spermatogonia (red arrow) up to spermatids, and in interstitial cells.

Figure 11

Panel of Caspase-3 immune-histochemical (IHC) pictures (×400). Degree of caspase staining were referred to as mild staining (+), moderate staining (++), and strong staining (+++). (A) control group (NG) testicular tissue showing mild cytoplasmic reactivity (+) for Caspase-3 in basal tubular lining, spermatogonia (red arrow), and primary spermatocytes; (B) Cisplatin-treated group (CG) with more cytoplasmic reactivity (+++) for Caspase-3 in tubular lining from spermatogonia (red arrow) up to spermatids; (C) Cisplatin+ beetroot extract treated group (CBG) with mild cytoplasmic reactivity (++) for Caspase-3 in tubular lining, from spermatogonia (red arrows) up to spermatids, and in interstitial cells; (D) Cisplatin+ mesenchymal stem cells treated group (CMG) showing mild cytoplasmic reactivity (++) for Caspase-3 in tubular lining from spermatogonia (red arrow) up to spermatids, and in interstitial cells; (E) Cisplatin+ mesenchymal stem cells+ beetroot extract treated group (CMBG) figuring low nuclear reactivity (+) for Caspase-3 in tubular lining from spermatogonia (red arrow) up to spermatids, and in interstitial cells.

Figure 12

Histogram for gene expression in response to the treatments. The genes were caspase-3; steroidogenic acute regulatory protein (StAR); and inducible nitric oxide synthase (iNOS). The groups are: the control group (NG); the Cisplatin treated group (CG); the Cisplatin+ beetroot extract treated group (CBG); the Cisplatin+ mesenchymal stem cells treated group (CMG); and the Cisplatin+ mesenchymal stem cells + beetroot extract treated group (CMBG). The interrelations between all the presented groups are statistically significant (p ≤ 0.05), except for the annotated groups (groups with the same letter have insignificant relationship).