Abstract
Background Di-N-butylphthalate (DBP) is a kind of unique endocrine toxicity linked to hormonal disruptions that affects the male reproductive system and has given rise to more and more attention. However, the mechanism of DBP-induced testicular injury remains unclear. Here, the objective of this study was to investigate the potential molecular mechanism of miR-506-3p in DBP-induced rat testicular oxidative stress injury via ANXA5 (Annexin A5)/Nrf2/HO-1 signaling pathway. Methods In vivo, a total of 40 adolescent male rats were treated from 2 weeks with 800 mg/kg/day of DBP in 1 mL/kg corn oil administered daily by oral gavage. Among them, some rats were also injected subcutaneously with 2 nmol agomir-506-3p and/or 10 nmol recombinant rat ANXA5. The pathomorphological changes of testicular tissue were assessed by histological examination, and the antioxidant factors were evaluated. Subsequently, ANXA5, Nrf2, and its dependent antioxidant enzymes, such as HO-1, NQO1, and GST, were detected by Western blotting or immunohistochemical staining. In vitro, TM3 cells (Leydig cells) were used to detect the cell activity by CCK-8 and the transfection in the DBP-treated group. Results Differentially expressed miRNAs between the DBP-treated and normal rats were analyzed, and qRT-PCR showed miR-506-3p was highly expressed in testicular tissues of the DBP-treated rats. DBP-treated rats presented severe inflammatory infiltration, increased abnormal germ cells, and missed cell layers frequently existed in seminiferous tubules, resulted in oxidative stress and decreased testicular function. Meanwhile, upregulation of miR-506-3p aggravated the above changes. In addition, miR-506-3p directly bound to ANXA5, and overexpression of miR-506-3p could reduce the ANXA5 expression and also decrease the protein levels of Nrf2/HO-1 signaling pathway. Additionally, we found that recombinant rat ANXA5 reversed the DBP-treated testicular oxidative stress promoting injury of miR-506-3p in rats. In vivo results were reproduced in in vitro experiments. Conclusions This study provided evidence that miR-506-3p could aggravate the DBP-treated testicular oxidative stress injury in vivo and in vitro by inhibiting ANXA5 expression and downregulating Nrf2/HO-1 signaling pathway, which might provide novel understanding in DBP-induced testicular injury therapy.
Highlights
As one kind of phthalic acid esters (PAEs), Di-Nbutylphthalate (DBP) is worldwide used in industrial products [1, 2]
Testicular cell dysplasia, abnormal cholesterol transport and steroidogenesis, and low testosterone production accompanied by demasculinization have been reported in rodents prenatally exposed to DBP [8,9,10,11,12]
Our findings indicated that miR-506-3p downregulated the expression of Annexin A5 (ANXA5) via Nrf2/heme oxygenase 1 (HO-1) signaling pathway in rats and provided a novel potential therapeutic target for DBP-induced testicular injury
Summary
As one kind of phthalic acid esters (PAEs), Di-Nbutylphthalate (DBP) is worldwide used in industrial products [1, 2]. The mechanism of DBP-induced testicular injury remains unclear. The objective of this study was to investigate the potential molecular mechanism of miR-506-3p in DBPinduced rat testicular oxidative stress injury via ANXA5 (Annexin A5)/Nrf2/HO-1 signaling pathway. DBP-treated rats presented severe inflammatory infiltration, increased abnormal germ cells, and missed cell layers frequently existed in seminiferous tubules, resulted in oxidative stress and decreased testicular function. We found that recombinant rat ANXA5 reversed the DBP-treated testicular oxidative stress promoting injury of miR-506-3p in rats. This study provided evidence that miR-506-3p could aggravate the DBP-treated testicular oxidative stress injury in vivo and in vitro by inhibiting ANXA5 expression and downregulating Nrf2/HO-1 signaling pathway, which might provide novel understanding in DBP-induced testicular injury therapy
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