HO-1 attenuates testicular ischemia/reperfusion injury through activating the phosphorylated c-Jun-miR-221/222-TOX pathway [miRNA]

Heme oxygenase(HO-1) has been shown to protect against I/R injury, but its effects on testicular I/R is very limited. Therefore, this study aims to investigate the effects of HO-1 on testicular I/R and the underlying mechanism.Knockout of HO-1 rats by TALEN technique.Immunofluorescence and immunohistochemistry was used to detect HO-1 nuclear translocation. Flow cytometry was used to detect cell apoptosis and cell cycle.High-resolution miRNA,mRNA sequencing, real-time PCR and western blot were performed to select testicular I/R injury related genes with strong conservation in HO-1 knockout rats.Double luciferase reporter assay was used to verify the relationship between c-Jun and miR-221/222.We found that HO-1 improved the pathological damage caused by testicular I/R in vivo. We verified the nuclear translocation of HO-1 and its protective effect on hypoxia/reoxygenation(H/R) damage in GC-1 cells.And our results indicated that HO-1 protein itself rather than heme breakdown metabolites might play a key role in testicular I/R.Gene sequencing was performed to screen out MiR221/222 and its downstream gene-thymocyte selection associated high mobility group box(TOX). In addition, we found that HO-1 increased phosphorylation of c-Jun in H/R group.We knocked down c-Jun in GC-1 cells and observed a decrease in the expression of miR-221/222.And we found by inhibiting HO-1,we observed a decrease in the expression of c-jun and miR-221/222,which could be rescued by adding c-jun. The dual luciferase reporter assay confirmed the interaction between c-Jun and miR-221/222. Collectively, HO-1 possesses a protective effect on testicular I/R via phosphorylated c-Jun-miR-221/222-TOX pathway. After anesthetizing the HO-1 wild-type (WT) rats or HO-1 knockout (HO-1-/-) rats, the left testicle was twisted clockwise 720°and fixed to the scrotal skin with silk thread. After 2 h of torsion, the testes were allowed to return to their natural position for 3 days,then the animals were sacrificed and testicular tissue was harvested.