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. 2019 Jan 15;39(1):BSR20180515.
doi: 10.1042/BSR20180515. Print 2019 Jan 31.

The molecular and biochemical insight view of grape seed proanthocyanidins in ameliorating cadmium-induced testes-toxicity in rat model: implication of PI3K/Akt/Nrf-2 signaling

Nazima Bashir  1 , Kalist Shagirtha  2 , Vaikundam Manoharan  1 , Selvaraj Miltonprabu  3
Affiliations

The molecular and biochemical insight view of grape seed proanthocyanidins in ameliorating cadmium-induced testes-toxicity in rat model: implication of PI3K/Akt/Nrf-2 signaling

Nazima Bashir et al. Biosci Rep. .

Expression of concern in

Abstract

The present study aims to evaluate the protective effect of grape seed proanthocyanidins (GSP) on cadmium (Cd)-induced testicular apoptosis, inflammation, and oxidative stress in rats. A total of 24 male Wistar rats were divided into four groups, namely control, GSP (100 mg/kg BW), Cd (5 mg/kg BW), and Cd+GSP. Cd-treated rat testes exhibited a significant increment in oxidative stress mediated inflammation and apoptosis. Pre-administration of GSP exhibit significant protection against the apoptotic and inflammatory damages elicited by Cd and uphold the intercellular antioxidant status in testes. Histological changes were studied and the immunohistochemical staining for caspase 3, HSP70, and eNOS protein expressions were also analyzed to justify the protective action of GSP. Furthermore, GSP prevented DNA damage, and enhanced the expression of antioxidant responsive elements Nrf2/HO-1 by PI3K/Akt-dependent pathway. Therefore, our results suggest that GSP acts as a multipotent antioxidant entity against Cd-induced oxidative testicular toxicity in rats.

Keywords: Nrf2/HO-1; antioxidant; cadmium; grape seed proanthocyanidins; oxidative stress; testes.

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Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Graphical Abstract
Graphical Abstract. Mechanism of GSP protection against Cd testes toxicity
Figure 1
Figure 1. Effect of GSP on Cd induced ROS concentration in experimental rats
Values are mean ± S.D. for six rats in each group. Values not sharing common superscript letters (a–d) differ significantly at P<0.05 (DMRT).
Figure 2
Figure 2. Effect of GSP on Cd-induced changes in steroidogenic enzyme activity in experimental rats
Values are mean ± S.D. for six rats in each group. Values not sharing common superscript letters (a–c) differ significantly at P<0.05 (DMRT).
Figure 3
Figure 3. Effcet of GSP and Cd on Sperm parameters
Effect of GSP and Cd on sperm parameters (A), sperm motility (B), dead sperm (C), and abnormal sperms (D) in experimental rats. Values are mean ± S.D. for six rats in each group. Values not sharing common superscript letters (a–d) differ significantly at P<0.05 (DMRT).
Figure 4
Figure 4. Effect of GSP on Cd-induced changes in ATPases of experimental rats in testis
Values are mean ± S.D. for six rats in each group. Values not sharing common superscript numbers (a, b, c) differ significantly at P<0.05 (DMRT).
Figure 5
Figure 5. Effect of GSP and Cd on apoptotic markers
Effect of GSP on Bcl-2, Bad, Bax, cytochrome c, and Caspase 3 protein expressions in the testes tissues of experimental rats by Western blot: effect of GSP on Lane 1. Control, Lane 2. Cd (5 mg/kg BW), Lane 3. GSP control (100 mg/kg bw) Lane 4. Cd (5 mg/kg BW) + GSP (100 mg/kg BW). Effect of GSP on Bcl-2, Bad, Bax, cytochrome c, and Caspase 3 protein band intensities scanned by densitometer. Values are mean ± S.D. for six rats in each group. Values not sharing common superscript letters (a–d) differ significantly at P<0.05 (DMRT).
Figure 6
Figure 6. Effect of GSP and Cd on testes antioxidative markers
Effect of GSP on Nrf2, Keap1, γ-GCS, NQO1, HO-1, anti-PI3k, p-PI3k, anti-Akt, and p-Akt protein expressions in the testes of experimental rats by Western blot: β-actin and Lamin B1 was used as an internal control. Lane 1. Control, Lane 2. Cd (5 mg/kg BW), Lane 3. GSP control (100 mg/kg bw) Lane 4. Cd (5 mg/kg BW) + GSP (100 mg/kg BW). Effect of GSP on Nrf2, Keap1, γ-GCS, NQO1, HO-1, anti-PI3k, p-PI3k, anti-Akt and p-Akt protein expressions in the testes scanned by densitometer: values are mean ± S.D. for six rats in each group. Values not sharing common superscript letters (a–d) differ significantly at P<0.05 (DMRT).
Figure 7
Figure 7. Effect of GSP on DNA damage of testicular cells induced by Cd intoxication
(A’) The testicular DNA damage was measured by comet assay. (A) Control rats show no DNA migration. (B) GSP alone administered rats show no DNA migration. (C) Cd-treated rats show extensive DNA migration. (D) GSP pre-administered Cd intoxicated rats show minimal DNA migration. (B’) Graphical representation of Comet assay(% Tail DNA).values are mean ± S.D. for six rats in each group. Values not sharing common superscript letters (a–c) differ significantly at P<0.05 (DMRT).
Figure 8
Figure 8. Effect of GSP and Cd on Immunohistochemical staining of iNOS, caspase-3, eNOS, HSP70 in rat testis
Immunohistochemical staining of iNOS, caspase-3, eNOS, HSP70 in rat testis: Testis of control rats shows no immunoreaction. Testis of GSP-treated rats seems normal as control group. Testis of Cd-treated rats, the intensity of iNOS, caspase-3, eNOS, HSP70 immunostaining is predominant on spermatogonia and seminiferous tubules of treated group. Testis of Cd+GSP rats shows moderate immunoreaction.
Figure 9
Figure 9. Effect of GSP and Cd on the histomrphology of rat testes
Photomicrographs of rat testes from control and experimental rats (H & E, ×200): the testes from control and GSP (A,B) rats showed normal histological architecture of seminiferous tubules, Sertoli cell with spermatogenesis. (C) Section of Cd-treated rat displayed marked disorder of the normal architecture of the testicular tissues, where the seminiferous tubules swell and enlarge with focal hemorrhage and necrosis of Leydig cells. (D) Testis section of GSP pre-treated Cd-intoxicated rats show significant improvement of histological architecture with normal seminiferous, spermatocytes, and interstitial cells.
Figure 10
Figure 10. Representative TEM photographs from the testes of control and experimental rats
(A) Normal histology of the control rat testis where seminiferous tubules, interstitial cells, and various stages of spermatogenesis are clearly visible (N-Spermatogonia Nuclei; M-Mitochondria). (B) The testis of GSP alone treated rat, show normal Sertoli cells, spermatogonia nuclei (N), basement membranes (Bm), and the inner most layer of sperm cells. (C) Image of Cd alone treated rat displaying irregular membrane of the spermatogonia nucleus with vacuolated cytoplasm where endoplasmic reticulum found to dilate and mitochondria is seeming to be swollen and vacuolated, (N-Spermatogonia nucleus). (D) Pre-administration of GSP to Cd-treated rats’ shows a significant recovery which is supported by the normal spermatid nucleus, endoplasmic reticulum, and mitochondria, (N-Spermatid Nuclei) (bars 2 µm) (×12000).
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