Irisin Contributes to Neuroprotection by Promoting Mitochondrial Biogenesis After Experimental Subarachnoid Hemorrhage

Abstract

Subarachnoid hemorrhage (SAH) is a devastating form of stroke, which poses a series of intractable challenges to clinical practice. Imbalance of mitochondrial homeostasis has been thought to be the crucial pathomechanism in early brain injury (EBI) cascade after SAH. Irisin, a protein related to metabolism and mitochondrial homeostasis, has been reported to play pivotal roles in post-stroke neuroprotection. However, whether this myokine can exert neuroprotection effects after SAH remains unknown. In the present study, we explored the protective effects of irisin and the underlying mechanisms related to mitochondrial biogenesis in a SAH animal model. Endovascular perforation was used to induce SAH, and recombinant irisin was administered intracerebroventricularly. Neurobehavioral assessments, TdT-UTP nick end labeling (TUNEL) staining, dihydroethidium (DHE) staining, immunofluorescence, western blot, and transmission electron microscopy (TEM) were performed for post-SAH assessments. We demonstrated that irisin treatment improved neurobehavioral scores, reduced neuronal apoptosis, and alleviated oxidative stress in EBI after SAH. More importantly, the administration of exogenous irisin conserved the mitochondrial morphology and promoted mitochondrial biogenesis. The protective effects of irisin were partially reversed by the mitochondrial uncoupling protein-2 (UCP-2) inhibitor. Taken together, irisin may have neuroprotective effects against SAH via improving the mitochondrial biogenesis, at least in part, through UCP-2 related targets.

Keywords: FNDC5/irisin; mitochondrial homeostasis; neuronal apoptosis; oxidative stress; subarachnoid hemorrhage.

Figure 1

Animal usage and SAH grade. (A) The record of the animal used in this experiment. (B) Representative pictures of brains in the sham and SAH groups. (C) SAH grade scores in each group. Data were represented with mean ± SD, n = 6 per group; the one-way ANOVA was used followed by the Tukey's HSD post-hoc test and the Holm–Bonferroni correction method. ***P < 0.001 vs. sham group. Vehicle group, sterile 0.9% of NaCl; SAH, subarachnoid hemorrhage.

Figure 2

Time course of FNDC5/irisin expression after SAH. (A) Representative western blotting images and relative density analysis of FNDC5/irisin at 6, 12, 24, 48, and 72 h after SAH. Endogenous FNDC5/irisin protein decreased at 6 h while it increased at 12 h and decreased again from 24 to 72 h after SAH. Data were represented as mean ± SD, n = 6 per group; the one-way ANOVA was used followed by the Tukey's HSD post-hoc test and the Holm–Bonferroni correction method. *P < 0.05, **P < 0.01, ***P < 0.001 vs. sham group. (B) FNDC5/irisin could be co-localized with neurons, and the number of FNDC5/irisin-positive neurons was decreased at 24 h after SAH. Scale bar = 50 μm, n = 3 per group; FNDC5, fibronectin domain-containing protein 5; SAH, subarachnoid hemorrhage.

Figure 3

Beneficial effects of irisin on neuronal function 24 h after SAH. (A,B) Treatment with irisin significantly improved neurological deficits at 24 h after SAH and 300 μg/kg irisin was selected as the effective dose, n = 6 per group. The modified Garcia scores were represented as mean ± SD; the one-way ANOVA was used followed by the Tukey's HSD post-hoc test and the Holm–Bonferroni correction method. The beam balance test scores were represented as median 25–75th percentiles, and the Mann–Whitney U-test and the Kruskal–Wallis test followed by the Steel–Dwass test for multiple comparisons were used to analyze the difference between groups. **P < 0.01, ^##P < 0.01 vs. SAH + Vehicle group. Vehicle group, sterile 0.9% of NaCl; SAH, subarachnoid hemorrhage.

Figure 4

Anti-apoptosis effects of exogenous irisin treatment on neurons 24 h after SAH. (A,B) TUNEL-positive neurons were significantly decreased after exogenous irisin administration. Scale bar = 50 μm, n = 3 for each group. (C–F) Representative western blotting images and relative density analysis of Bax, Bcl-2, and cleaved caspase-3 at 24 h after SAH. Exogenous irisin treatment significantly improved the expressions of Bcl-2 (E) and result in a decreased level of apoptotic marker cleaved caspase-3 (D) and Bax (F), n = 6 for each group. Data were presented as mean ± SD. The one-way ANOVA was used followed by the Tukey's HSD post-hoc test and the Holm–Bonferroni correction method. ***P < 0.001 vs. Sham group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. SAH+Vehicle group. Vehicle group, sterile 0.9% of NaCl; TUNEL, TdT-UTP nick end labeling.

Figure 5

Effect of exogenous irisin treatment on SAH-induced oxidative stress 24 h after SAH. (A,B) A lower red fluorescence intensity (DHE) was observed in the irisin-intervened group, Scale bar = 100 μm, n = 3 for each group. (C) Levels of oxidative markers were analyzed, MDA levels were significantly decreased after irisin treatment. (D,E) SOD and GSH-PX levels were increased with the irisin intervention, n = 6 for each group. Data were presented as mean ± SD. The one-way ANOVA was used followed by the Tukey's HSD post-hoc test and the Holm–Bonferroni correction method. ***P < 0.001 vs. Sham group; ^#P < 0.05, ^##P < 0.01 vs. SAH + Vehicle group. Vehicle, sterile 0.9% of NaCl; DHE, dihydroethidium; SOD, Superoxide Dismutase; GSH-PX, Glutathione Peroxidase Activity.

Figure 6

Effect of exogenous irisin treatment on mitochondrial morphology and mitochondrial biogenesis in mice brain 24 h after SAH. (A) Less mitochondrial swelling and vacuolization were observed under the TEM after irisin treatment, Scale bar = 2 μm, 500 nm, n = 3 for each group. (B–E) Representative western blotting images and relative density analysis of TFAM, PGC-1α, and UCP-2 24 h after SAH. Exogenous irisin treatment significantly improved the expressions of TFAM and PGC-1α. The UCP-2 expression increased after SAH and a higher level was observed after irisin treatment, n = 6 for each group. All data were presented as mean ± SD. The one-way ANOVA was used followed by the Tukey's HSD post-hoc test and the Holm–Bonferroni correction method. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Sham group; ^##P < 0.01, ^###P < 0.001 vs. SAH + Vehicle group. Vehicle group, sterile 0.9% of NaCl; UCP-2, Uncoupling Protein-2; SAH, subarachnoid hemorrhage.

Figure 7

Inhibition of UCP-2 pathway blunted the beneficial effect of irisin treatment on short-term neurologic function after SAH. (A,B) Genipin significantly blunted the protective effect of irisin treatment on neurological deficits at 24 h after SAH, n = 6 for each group. The modified Garcia test scores were represented as mean ± SD; the one-way ANOVA was used followed by the Tukey's HSD post-hoc test and the Holm–Bonferroni correction method. The beam balance test scores were represented as median 25th−75th percentiles, and the Mann–Whitney U-test and the Kruskal–Wallis test followed by the Steel–Dwass method of multiple comparisons were used to analyze the difference between groups. **P < 0.01, ***P < 0.001 vs. Sham group; ^##P < 0.01, ^###P < 0.001 vs. SAH + Vehicle group; ^&P < 0.05, ^&&&P < 0.001 vs. SAH + irisin + Vehicle group. Vehicle group, sterile 0.9% of NaCl; SAH, subarachnoid hemorrhage.

Figure 8

Inhibition of UCP-2 pathway reversed the beneficial effects of irisin on apoptosis, mitochondrial function, and oxidative stress after SAH. (A,B) TUNEL-positive neurons were significantly increased after genipin administration in the irisin treatment group. Scale bar = 50 μm, n = 3 for each group (The area of observation was circled in red). (C–I) Representative western blotting images and relative density analysis of proteins related to mitochondrial biogenesis (TFAM, PGC-1α), oxidative stress (SOD2), and apoptotic markers (Cleaved caspase-3, Bax, Bcl-2) at 24 h after SAH. As an exogenous UCP-2 inhibitor, genipin significantly blunted the expressions of PGC-1α, TFAM, SOD2, and Bcl-2 (D,E,F,I) and resulted in an increased level of cleaved caspase-3 and Bax (G,H), n = 6 for each group. All data were presented as mean ± SD. The one-way ANOVA was used followed by the Tukey's HSD post-hoc test and the Holm–Bonferroni correction method. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Sham group; ^##P < 0.01, ^###P < 0.001 vs. SAH + Vehicle group. ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001 vs. SAH + irisin + Vehicle group. Vehicle group, sterile 0.9% of NaCl; TUNEL, TdT-UTP nick end labeling; SOD, Superoxide Dismutase; UCP-2, Uncoupling Protein-2.

Figure 9

The schematic of irisin on mitochondrial biogenesis following SAH. SAH, subarachnoid hemorrhage.