SIRT3 protects against early brain injury following subarachnoid hemorrhage via promoting mitochondrial fusion in an AMPK dependent manner

Abstract

Background: Subarachnoid hemorrhage (SAH), an acute cerebrovascular accident, features with its high death and disability rate. Sirtuin3 (SIRT3) is a NAD+ dependent deacetylase which mainly located in mitochondria. Reduced SIRT3 function was indicated to involve in many disorders of central nervous system. Herein, we aimed to explore the neuroprotective effects of SIRT3 on SAH and to furtherly explore the underlying mechanisms.

Methods: Adult C57BL/6 J male mice (8-10 weeks) were used to establish SAH models. The pharmacological agonist of SIRT3, Honokiol (HKL), was injected in an intraperitoneal manner (10 mg/kg) immediately after the operation. Brain edema and neurobehavioral score were assessed. Nissl staining and FJC staining were used to evaluate the extent of neuronal damage. The changes of mitochondria morphology were observed with transmission electron microscopy. Western blot was used for analyzing the protein level of SIRT3 and the downstream signaling molecules.

Result: SIRT3 was downregulated after SAH, and additional treatment of SIRT3 agonist HKL alleviated brain edema and neurobehavioral deficits after SAH. Additionally, electron microscopy showed that HKL significantly alleviated the morphological damage of mitochondria induced by SAH. Further studies showed that HKL could increase the level of mitochondrial fusion protein Mfn1 and Mfn2, thus maintaining (mitochondrial morphology), protecting mitochondrial function and promoting neural survival. While, additional Compound C (CC) treatment, a selective AMPK inhibitor, abolished these protective effects.

Conclusions: Activation of SIRT3 protects against SAH injury through improving mitochondrial fusion in an AMPK dependent manner.

Keywords: AMPK; Honokiol; Mfn1; Mfn2; Mitochondrial fusion; SIRT3; Subarachnoid hemorrhage.

Fig. 1

SIRT3 expression after SAH in mice. a Western blot analysis of the protein level of SIRT3 after SAH. b The bar graph shows the statistical results of SIRT3 protein level. c Western blot analysis of the acetylation level of mitochondrial proteins. Values were represented as mean ± SD, n = 8 for each group. *P < 0.05 and **P < 0.01 vs. sham group

Fig. 2

SIRT3 agonist HKL alleviated neurological deficits and brain edema after SAH in mice. a, b HKL treatment significantly reversed the low expression of SIRT3 induced by SAH injury. c The modified neurological severity scores in each group. d Brain water content in each group. Values are represented as mean ± SD. n = 8 for each group. *P < 0.05 and **P < 0.01 vs. sham group, ^#P < 0.05 and ^##P < 0.01 vs. SAH group

Fig. 3

HKL ameliorated neuronal damage after SAH in mice. a Representative images of Nissl staining and c quantitative analyses of Nissl positive neurons in each group 24 h after the operation are shown. Scale bar 50 μm. b Representative images of FJC staining and d Quantitative analyses of FJC-positive cells in each group 24 h after the operation are shown. Scale bar 50 μm. Values were represented as mean ± SD, n = 8 for each group. *P < 0.05 and **P < 0.01 vs. sham group, ^#P < 0.05 and ^##P < 0.01 vs. SAH group

Fig. 4

Effects of HKL on the ultrastructure of neurons 24 h after SAH, as detected using electron microscopy. a–c Representative ultrastructure of neurons is shown in each group. a1–c1 Representative ultrastructure of neurons are enlargements of a–c respectively. Scale bar = 2 μm

Fig. 5

SIRT3 protected mitochondrial fusion proteins Mfn1 and Mfn2 after SAH in an AMPK-dependent manner. (a selective inhibitor of AMPK). a Western blotting analysis of SIRT3; p-AMPK; AMPK; Mfn1, Mfn2; Bcl2; Bax in each group. b The bar graph shows the statistical results of protein level. CC: Compound C. Values were represented as mean ± SD, n = 8 for each group. *P < 0.05 and **P < 0.01 vs. sham group, ^#P < 0.05 and ^##P < 0.01 vs. SAH group. ^&P < 0.05 vs. SAH + HKL group