Korean J Physiol Pharmacol.  2021 Jul;25(4):341-354. 10.4196/kjpp.2021.25.4.341.

Cardamonin exerts a protective effect against autophagy and apoptosis in the testicles of diabetic male rats through the expression of Nrf2 via p62-mediated Keap-1 degradation

• Samir SM
• Elalfy M
• Nashar EME
• Alghamdi MA
• Hamza E
• Serria MS
• Elhadidy MG

• KMID: 2516873
• DOI: http://doi.org/10.4196/kjpp.2021.25.4.341

Abstract

Cardamonin (CARD) is a chalconoid with anti-inflammatory and antioxidant properties, and it is present in several plants. We sought to explore whether CARD exerts any positive effects against hyperglycemia-induced testicular dysfunction caused by type 2 diabetes and aimed to identify its possible intracellular pathways. Adult male rats were subdivided into six groups: control, CARD, diabetic (DM), DM + glibenclamide (GLIB), DM + CARD and DM + GLIB + CARD. Type 2 DM induced a significant increase in blood glucose and insulin resistance, along with diminished serum insulin, testosterone and gonadotropins levels, which were associated with the impairment of key testicular androgenic enzymes and cellular redox balance. Administration of CARD at a dose of 80 mg/kg for 4 weeks effectively normalized all of these alterations, and the improvement was confirmed by epididymal sperm analysis. After treatment with CARD, the pathological changes in spermatogenic tubules were markedly improved. Significantly, CARD upregulated testicular glucose transporter-8 (GLUT-8) expression and had inhibitory effects on elevated autophagy markers and caspase-3 immunoreactive cells. Furthermore, our results revealed that CARD was able to attenuate damage via activation of Nrf2 through the p62-dependent degradation of testicular anti-Kelch-like ECH-associated protein-1 (Keap-1). In conclusion, this study suggests that CARD provides protection against diabetic stress-mediated testicular damage. The use of CARD with conventional anti-diabetic therapy was associated with improved efficacy compared with conventional therapy alone.

Keyword

Apoptosis; Autophagy; Gonadotropins; Insulin sensitivity; Reproductive capacity

Figure

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  Fig. 1 Time schedule for the experimental design. CARD, normal rats supplemented with cardamonin; DM, untreated diabetic group; DM + GLIB, diabetic rats treated with glibenclamide; DM + CARD, diabetic rats treated with cardamonin; DM + GLIB + CARD, diabetic rats treated with both glibenclamide and cardamonin. #Represents the administration of STZ.

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  Fig. 2 Effect of cardamonin on key testicular steroid enzymes in different experimental groups. Values are presented as mean ± SD. CARD, normal rats treated with cardamonin; DM, untreated diabetic group; DM + GLIB, diabetic rats treated with glibenclamide; DM + CARD, diabetic rats treated with cardamonin; DM + GLIB + CARD, diabetic rats treated with both glibenclamide and cardamonin. Tests used: One-way ANOVA followed by post-hoc Tukey’s test. p < 0.001. *Represents significance compared with the control group; #represents significance compared with the DM group; $represents significance compared with the DM + GLIB group; @represents significance compared with the DM + CARD group.

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  Fig. 3 Effect of cardamonin on testicular oxidative stress markers and antioxidant activities in different experimental groups. Values are presented as mean ± SD. CARD, normal rats treated with cardamonin; DM, untreated diabetic group; DM + GLIB, diabetic rats treated with glibenclamide; DM + CARD, diabetic rats treated with cardamonin; DM + GLIB + CARD, diabetic rats treated with both glibenclamide and cardamonin. Tests used: One-way ANOVA followed by post-hoc Tukey’s test. p < 0.001. *Represents significance compared with the control group; #represents significance compared with the DM group; $represents significance compared with the DM + GLIB group; @represents significance compared with the DM + CARD group.

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  Fig. 4 Effect of cardamonin on the testicular mRNA expression of Nrf2, Keap-1 and p62 in different experimental groups. Values are presented as mean ± SD. CARD, normal rats treated with cardamonin; DM, untreated diabetic group; DM + GLIB, diabetic rats treated with glibenclamide; DM + CARD, diabetic rats treated with cardamonin; DM + GLIB + CARD, diabetic rats treated with both glibenclamide and cardamonin. Tests used: One-way ANOVA followed by post-hoc Tukey’s test. p < 0.001. *Represents significance compared with the control group; #represents significance compared with the DM group; $represents significance compared with the DM + GLIB group; @represents significance compared with the DM + CARD group.

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  Fig. 5 Effect of cardamonin on the testicular mRNA expression of autophagy markers (LC3 and beclin-1) in different experimental groups. Values are presented as mean ± SD. CARD, normal rats treated with cardamonin; DM, untreated diabetic group; DM + GLIB, diabetic rats treated with glibenclamide; DM + CARD, diabetic rats treated with cardamonin; DM + GLIB + CARD, diabetic rats treated with both glibenclamide and cardamonin. Tests used: One-way ANOVA followed by post-hoc Tukey’s test. p < 0.001. *Represents significance compared with the control group; #represents significance compared with the DM group; $represents significance compared with the DM + GLIB group; @represents significance compared with the DM + CARD group.

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  Fig. 6 Effect of cardamonin on the testicular protein expression of Nrf2, Keap-1, p62 and autophagy markers in different experimental groups. The testicular expression of Nrf2, Keap-1, p62 and autophagy markers (LC3, beclin-1) proteins was detected by western blotting assay. Values are presented as mean ± SD. CARD, normal rats treated with cardamonin; DM, untreated diabetic group; DM + GLIB, diabetic rats treated with glibenclamide; DM + CARD, diabetic rats treated with cardamonin; DM + GLIB + CARD, diabetic rats treated with both glibenclamide and cardamonin. Tests used: One-way ANOVA followed by post-hoc Tukey’s test. p < 0.001. *Represents significance compared with the control group; #represents significance compared with the DM group; $represents significance compared with the DM + GLIB group; @represents significance compared with the DM + CARD group.

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  Fig. 7 Effect of cardamonin on testicular glucose transporter-8 (GLUT-8) mRNA expression in different experimental groups. Values are presented as mean ± SD. CARD, normal rats treated with cardamonin; DM, untreated diabetic group; DM + GLIB, diabetic rats treated with glibenclamide; DM + CARD, diabetic rats treated with cardamonin; DM + GLIB + CARD, diabetic rats treated with both glibenclamide and cardamonin. Tests used: One-way ANOVA followed by post-hoc Tukey’s test. p < 0.001. *Represents significance compared with the control group; #represents significance compared with the DM group; $represents significance compared with the DM + GLIB group; @represents significance compared with the DM + CARD group.

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  Fig. 8 Effect of cardamonin on histopathological testicular changes in different experimental groups. Testis of the control group showing normal seminiferous tubules lined with normal spermatogenic cells with the presence of numerous free spermatids in the lumen (arrow). (B) Testis of the CARD group showing normal seminiferous tubules lined with normal spermatogenic cells with the presence of numerous free spermatids in the lumen (arrow). (C) Testis of untreated DM rats showing a marked loss of spermatogenic epithelial layers and damaged appearance of the basal layer with the complete loss of spermatids in the lumen of seminiferous tubules (arrow). (D) Testis of the DM + GLIB group showing a decrease in the degenerative changes within the spermatogenic epithelial layers (arrow). (E) Testis of the DM + CARD group showing moderate restoration of the spermatogenic epithelial layers with mild appearance of spermatids (arrows). (F) Testis of the DM + GLIB + CARD group showing marked improvement in the spermatogenic epithelial layers with numerous spermatids (arrows). H&E, 200×, bar = 50 µm. CARD, normal rats treated with cardamonin; DM, untreated diabetic group; DM + GLIB, diabetic rats treated with glibenclamide; DM + CARD, diabetic rats treated with cardamonin; DM + GLIB + CARD, diabetic rats treated with both glibenclamide and cardamonin.

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  Fig. 9 Effect of cardamonin on the immunohistochemical testicular expression of an apoptotic marker (caspase-3) in different experimental groups. Immunohistochemical stain, 200×, bar = 50 μm. (A) Testis of the control group showing mild expression of caspase-3 antibody (arrows). (B) Testis of the CARD group showing little expression of caspase-3 antibody (arrows). (C) Testis of the untreated DM group showing marked expression of caspase-3 antibody in the spermatogenic cells of seminiferous tubules (arrows). (D) Testis of the DM + GLIB group showing a mild decrease in the expression of caspase-3 antibody in spermatogenic cells (arrows). (E) Testis of the DM + CARD group showing a moderate decrease in the expression of caspase-3 antibody in spermatogenic cells (arrows). (F) Testis of the DM + GLIB + CARD group showing a marked decrease in the expression of caspase-3 antibody in spermatogenic cells (arrows). (G) Results showing a significant increase in the caspase-3 labelling index in the untreated diabetic testicular tissue in comparison with the control. Treatment of diabetic rats with CARD produced a marked decrease in the labelling index of the caspase-3-stained area as compared with the untreated diabetic group. The maximum decrease in the immunoreactive stain was found in the DM + GLIB + CARD group when compared with the other treated diabetic groups. CARD, normal rats treated with cardamonin; DM, untreated diabetic group; DM + GLIB, diabetic rats treated with glibenclamide; DM + CARD, diabetic rats treated with cardamonin; DM + GLIB + CARD, diabetic rats treated with both glibenclamide and cardamonin. *Represents significance compared with the control group; #represents significance compared with the DM group; $represents significance compared with the DM + GLIB group; @repre-sents significance compared with the DM + CARD group.

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