Melatonin affects hypoxia-inducible factor 1α and ameliorates delayed brain injury following subarachnoid hemorrhage via H19/miR-675/HIF1A/TLR4

This study aimed to investigate the molecular mechanism of how melatonin (MT) interferes with hypoxia-inducible factor 1α (HIF1A) and toll-like receptor 4 (TLR4) expression, which is implicated in the management of delayed brain injury (DBI) after subarachnoid hemorrhage (SAH). Luciferase assay, real-time PCR, Western-blot analysis and immunohistochemistry (IHC) assays were utilized to explore the interaction among H19, miR-675, HIF1A and TLR4, and to evaluate the effect of MT on the expression of above transcripts in different groups. MT enhanced H19 expression by promoting the transcription efficiency of H19 promoter, and HIF1A was identified as a target of miR-675. HIF1A enhanced TLR4 expression via promoting the transcription efficiency of TLR4 promoter. Furthermore, administration of MT up-regulated miR-675 but suppressed the expressions of HIF1A and TLR4. Treatment with MT alleviated neurobehavioral deficits and apoptosis induced by SAH. According to the result of IHC, HIF1A and TLR4 protein levels in the SAH group were much higher than those in the SAH+MT group. Therefore, the administration of MT increased the levels of H19 and miR-675 which have been inhibited by SAH. In a similar way, treatment with MT decreased the levels of HIF1A and TLR4 which have been enhanced by SAH. MT could down-regulate the expression of HIF1A and TLR4 via the H19/miR-675/HIF1A/TLR4 signaling pathway, while TLR4 is crucial to the release of pro-inflammatory cytokines. Therefore, the treatment with MT could ameliorate post-SAH DBI.Running title: Melatonin ameliorates post-SAH DBI via H19/miR-675/HIF1A/TLR4 signaling pathways