Carnosic Acid Mitigates Early Brain Injury After Subarachnoid Hemorrhage: Possible Involvement of the SIRT1/p66shc Signaling Pathway

Abstract

Carnosic acid (CA) has been reported to exhibit a variety of bioactivities including antioxidation, neuroprotection, and anti-inflammation; however, the impact of CA on subarachnoid hemorrhage (SAH) has never been elucidated. The current study was undertaken to explore the role of CA in early brain injury (EBI) secondary to SAH and the underlying mechanisms. Adult male Sprague-Dawley rats were perforated to mimic a clinical aneurysm with SAH. CA or vehicle was administered intravenously immediately after the SAH occurred. Mortality, SAH grade, neurologic function scores, brain water content, Evans blue extravasation, and the levels of reactive oxygen species (ROS) levels in the ipsilateral cortex were determined 24 h after the SAH occurred. Western blot, immunofluorescence, Fluoro-Jade C (FJC) and TUNEL staining were also performed. Our results showed that CA decreased ROS levels, alleviated brain edema and blood-brain barrier permeability, reduced neuronal cell death, and promoted neurologic function improvement. To probe into the potential mechanisms. We showed that CA increased SIRT1, MnSOD, and Bcl-2 expression, as well as decreased p66shc, Bax, and cleaved caspase-3 expression. Interestingly, sirtinol, a selective inhibitor of SIRT1, abolished the anti-apoptotic effects of CA. Taken together, these data revealed that CA has a neuroprotective role in EBI secondary to SAH. The potential mechanism may involve suppression of neuronal apoptosis through the SIRT1/p66shc signaling pathway. CA may provide a promising therapeutic regimen for management of SAH.

Keywords: apoptosis; carnosic acid; early brain injury; p66shc; subarachnoid hemorrhage.

FIGURE 1

(A) Representative Western blot bands of p66shc at different time course in ipsilateral basal cortex after subarachnoid hemorrhage (SAH) induction. (B) Quantitative analysis of p66shc expression. n = 6 for each group. The bars represent the mean ± SD. ^∗P < 0.05 versus sham, ^#P < 0.05 versus every other group. The densities of the protein bands were analyzed in relation to β-actin and normalized to the sham group. (C) Representative microphotographs of immunofluorescence staining showing localization of p66shc (green) with NeuN (red) in ipsilateral basal cortex of SAH 24 h group (scale bar = 50 μm). The rightmost image was magnified by digital zoom.

FIGURE 2

Typical representation of brains from each group and SAH grade, neurological scores, brain water content, and Evans blue dye extravasation at 24 h after SAH. (A) Representative brains from the sham, SAH, SAH + vehicle, SAH + CA group. (B) Quantitative analyses of neurological scores. The bars represent the mean ± SD. n = 30. (C) Quantitative analyses of SAH severity. The bars represent the mean ± SD. n = 30. (D) Quantitative analyses of brain water content. The bars represent the mean ± SD. n = 6. (E) Quantitative analyses of Evans blue dye extraversion. The bars represent the mean ± SD. n = 6. ^∗#P < 0.05 vs. Sham, ^∗P < 0.05 vs. SAH.

FIGURE 3

Carnosic acid attenuated apoptosis 24 h after subarachnoid hemorrhage (SAH) via activating SIRT1/p66shc signaling pathway. (A) Representative Western blot bands of SIRT1, p66shc. (B,C) Quantitative analyses of SIRT1 (B), p66shc (C). (D) Activity of SIRT1 in the brain. (E) The effect of CA on SIRT1 expression. (F) Representative Western blot bands of MnSOD, Bax, Bcl-2, and cleaved caspase-3. (G–J) Quantitative analyses of, MnSOD (G), Bax (H), Bcl-2 (I), cleaved caspase-3 (J). The densities of the protein bands were analyzed in relation to β-actin and normalized to the sham group. (K) Quantitation of ROS. The histograms represent the median with interquartile range, n = 6. The statistical differences between two groups were analyzed by Kruskal–Wallis test. ^∗#P < 0.05 vs. Sham, ^∗P < 0.05 vs. SAH.

FIGURE 4

Carnosic acid reduced degenerating neurons and neuronal cells death in the ipsilateral cortex at 24 h after SAH. (A) Representative co-labeling TUNEL/NeuN photomicrographs of the ipsilateral cortex in the different groups (scale bar = 100 μm). (B) Representative FJC photomicrographs in the different groups (scale bar = 100 μm). (C) Quantification of TUNEL-positive neurons, expressed as percentage of total (NeuN⁺) cells. (D) Quantification of FJC-positive degenerating neurons. The bars represent the mean ± SD. n = 6. ^∗#P < 0.05 vs. Sham, ^∗P < 0.05 vs. SAH.

FIGURE 5

SIRT1 inhibitor sirtinol abolished the beneficial effect of carnosic acid on apoptosis. (A) Quantitative analyses of neurological scores. The bars represent the mean ± SD. n = 18. (B) Quantitative analyses of ROS. The histograms represent the median with interquartile range, n = 6. (C–E) Western blot assay and quantitative analyses of SIRT1 (D), p66shc (E) in PC12 cells after treatment with CA alone or together with sirtinol. ^∗P < 0.05 vs. Vehicle, ^#P < 0.05 vs. CA. (F–L) Western blot assay (F) and quantitative analyses of SIRT1 (G), p66shc (H), MnSOD (I), Bcl-2 (J), Bax (K), cleaved caspase-3 (L) expressions in ipsilateral cortex. The densities of the protein bands were analyzed in relation to β-actin and normalized to the sham group. The bars represent the mean ± SD. n = 6. ^∗P < 0.05 vs. SAH, ^#P < 0.05 vs. SAH + CA.

FIGURE 6

SIRT1 inhibitor sirtinol abolished Carnosic acid-reduced degenerating neurons and neuronal cells death in the ipsilateral cortex at 24 h after SAH. (A) Representative co-labeling TUNEL/NeuN photomicrographs of the ipsilateral cortex in the different groups (scale bar = 100 μm). (B) Representative FJC photomicrographs in the different groups (scale bar = 100 μm). (C) Quantification of TUNEL-positive neurons, expressed as percentage of total (NeuN⁺) cells. (D) Quantification of FJC-positive degenerating neurons. The bars represent the mean ± SD. n = 6. ^∗P < 0.05 vs. SAH, ^#P < 0.05 vs. SAH + CA.