MEL Ameliorates Post-SAH Cerebral Vasospasm by Affecting the Expression of eNOS and HIF1α via H19/miR-138/eNOS/NO and H19/miR-675/HIF1α

Abstract

Melatonin (MEL) has been demonstrated to exert a protective effect against subarachnoid hemorrhage (SAH), and nitric oxide (NO) has been shown to play an important role in the pathogenesis of vasospasm. This study aims to explore the underlying molecular mechanisms of MEL in the control of vasospasm following SAH. MEL administration attenuates SAH-induced vasospasm and neurobehavioral deficits. Expressions of H19, eNOS, and miR-675 are low in the SAH group, while expressions of miR-138 and HIF1α are high in the SAH group. Also, MEL treatment upon SAH rats completely restores the dysregulation of H19, eNOS, miR-675, miR-138, and HIF1α to their normal levels. Moreover, MEL dose dependently increases the luciferase activity of H19 promoter and hence the expression of H19. Additionally, H19 directly targets miR-675 and miR-138 to increase miR-675 expression and inhibit miR-138 expression. As virtual target genes of miR-675 and miR-138, respectively, HIF1α and eNOS are also regulated by the treatment with MEL. In particular, MEL treatment increases the expression of miR-675 and eNOS level while decreasing the expression of miR-138 and HIF1α in a dose dependent manner. Our study found that MEL ameliorates post-SAH vasospasm by regulating the expression of eNOS and HIF1α via the H19/miR-138/eNOS/NO and H19/miR-675/HIF1α signaling pathways.

Keywords: H19; HIF1α; eNOS; melatonin; miR-138; miR-675; post-SAH vasospasm.

Figure 1

MEL Administration Attenuated Neurobehavioral Deficits (A) Rats in the SAH group showed a reduced MCA diameter, but the administration of MEL in SAH rats significantly increased MCA diameter. (B) SAH rats exhibited obvious neurobehavioral deficits, while the administration of MEL in SAH rats obviously reduced the severity of neurobehavioral deficits.

Figure 2

H19, miR-675, and miR-138 Were Differentially Expressed among Various Groups (A) H19 expressions in SHAM and SHAM+MEL rats were similar. H19 expression in SAH rats was much lower, while the administration of MEL in SAH rats obviously increased H19 expression (*p < 0.05, versus SHAM group; #p < 0.05, versus SAH+VEH group). (B) miR-675 expressions in SHAM and SHAM+MEL rats were similar. miR-675 expression in SAH rats was much lower, while the administration of MEL in SAH rats obviously increased miR-675 expression (*p < 0.05, versus SHAM group; #p < 0.05, versus SAH+VEH group). (C) miR-138 expressions in SHAM and SHAM+MEL rats were similar. miR-138 expression in SAH rats was much higher, while the administration of MEL in SAH rats obviously decreased miR-138 expression (*p < 0.05, versus SHAM group; #p < 0.05, versus SAH+VEH group).

Figure 3

Expression of HIF1α and eNOS Was Compared among Various Groups Using Real-Time PCR and Western Blot Analysis (A) Expressions of HIF1α mRNA in the SHAM and SHAM+MEL groups were similar, while the expression of HIF1α mRNA in SAH rats was much higher, and the administration of MEL in SAH rats obviously decreased the expression of HIFIA mRNA (*p < 0.05, versus SHAM group; #p < 0.05, versus SAH+VEH group). (B) Expressions of eNOS mRNA in the SHAM and SHAM+MEL groups were similar, while the expression of eNOS mRNA in SAH rats was much lower, and the administration of MEL in SAH rats obviously increased the expression of eNOS mRNA (*p < 0.05, versus SHAM group; #p < 0.05, versus SAH+VEH group). (C) Expression of HIF1α protein was increased in SAH rats, while expression of eNOS protein was decreased in SAH rats compared with the SHAM and SHAM+MEL groups, and administration of MEL in SAH rats decreased the expression of HIF1α protein and increased the expression of eNOS protein. (D) Expressions of HIF1α protein in the SHAM and SHAM+MEL group were similar, while the administration of MEL in SAH rats decreased the upregulated expression of HIF1α protein in SAH rats (*p < 0.05, versus SHAM group; #p < 0.05, versus SAH+VEH group). (E) Expressions of eNOS protein in the SHAM and SHAM+MEL group were similar, while the administration of MEL in SAH rats increased the downregulated expression of eNOS protein in SAH rats (*p < 0.05, versus SHAM group; #p < 0.05, versus SAH+VEH group).

Figure 4

Expressions of HIF1α Was Determined among Various Groups Using IHC Expression of HIF1α was determined among various groups using IHC, and it was found that evident increase in HIF1α protein expression was observed in SAH group, while it was decreased by MEL treatment (magnification, 200×).

Figure 5

Expressions of eNOS Was Compared among Various Groups Using IHC Expression of eNOS was compared among various groups using IHC, and the results showed that the protein expression of eNOS was evidently decreased in the SAH group, while the administration of MEL in SAH rats obviously increased the protein expression of eNOS (magnification, 200×).

Figure 6

Effect of MEL on the Expression of H19, miR-675, miR-138, HIF1α, and eNOS in SH-SY5Y Cells (A) Co-transfection of the cells with H19 and miR-138 mimics reduced the luciferase activity of the cells (*p < 0.05, versus control group). (B) Transfection of the cells with wild-type 3′ UTR of eNOS significantly decreased the luciferase activity of miR-138 (*p < 0.05, versus control group). (C) Transfection of the cells with miR-675 mimics significantly decreased the luciferase activity of wild-type 3′ UTR of HIF1α (*p < 0.05, versus control group). (D) MEL dose dependently increased the luciferase activity of the H19 promoter (*p < 0.05, versus untreated cells). (E) MEL dose dependently increased the expression of H19 (*p < 0.05, versus untreated cells). (F) MEL dose dependently increased the expression of miR-675 (*p < 0.05, versus untreated cells). (G) MEL dose dependently decreased the expression of miR-138 (*p < 0.05, versus untreated cells). (H) MEL dose dependently downregulated the expression of HIF1α mRNA (*p < 0.05, versus untreated cells). (I) MEL dose dependently upregulated the expression of eNOS mRNA (*p < 0.05, versus untreated cells). (J) MEL dose dependently downregulated the expression of HIF1Α protein while upregulating the expression of eNOS protein.

Figure 7

Effect of MEL on the Expression of H19, miR-675, miR-138, HIF1Α, and eNOS in U251 Cells (A) Co-transfection of the cells with H19 and miR-138 mimics reduced the luciferase activity of the cells (*p < 0.05, versus control group). (B) Transfection of the cells with wild-type 3′ UTR of eNOS significantly decreased the luciferase activity of miR-138 (*p < 0.05, versus control group). (C) Transfection of the cells with miR-675 mimics significantly decreased the luciferase activity of wild-type 3′ UTR of HIF1α (*p < 0.05, versus control group). (D) MEL dose dependently increased the luciferase activity of H19 promoter (*p < 0.05, versus untreated cells). (E) MEL dose dependently increased the expression of H19 (*p < 0.05, versus untreated cells). (F) MEL dose dependently increased the expression of miR-675 (*p < 0.05, versus untreated cells). (G) MEL dose dependently decreased the expression of miR-138 (*p < 0.05, versus untreated cells). (H) MEL dose dependently downregulated the expression of HIF1α mRNA (*p < 0.05, versus untreated cells). (I) MEL dose dependently upregulated the expression of eNOS mRNA (*p < 0.05, versus untreated cells). (J) MEL dose dependently downregulated the expression of HIF1α protein while upregulating the expression of eNOS protein.