Resveratrol Attenuates Early Brain Injury after Experimental Subarachnoid Hemorrhage via Inhibition of NLRP3 Inflammasome Activation

Abstract

Previous studies have demonstrated resveratrol (RSV) has beneficial effects in early brain injury (EBI) after subarachnoid hemorrhage (SAH). However, the beneficial effects of RSV and the underlying mechanisms have not been clearly identified. The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation plays a crucial role in the EBI pathogenesis. The aim of this study was to investigate the role of RSV on the NLRP3 inflammasome signaling pathway and EBI in rats after SAH. A prechiasmatic cistern injection model was established in rats, and the primary cultured cortical neurons were stimulated with oxyhemoglobin (oxyHb) to induce SAH in vitro. It showed that the NLRP3 inflammasome components, including NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, mature interleukin-1β (IL-1β), and interleukin-18 (IL-18) were upregulated after SAH, and the enhanced NLRP3 after SAH was mainly located in microglia. Treatment with 60 or 90 mg/kg RSV after SAH dramatically inhibited the expression of NLRP3, but there was no significant difference in the expression of NLRP3 between the SAH + 60 mg/kg RSV and SAH + 90 mg/kg RSV groups. In addition, treatment with 30 mg/kg RSV did not significantly reduced the expression of NLRP3. We next evaluated the neuroprotective effects of RSV against SAH. We determined that SAH-induced NLRP3 inflammasome activation was significantly inhibited in the SAH + 60 mg/kg RSV group. Meanwhile, 60 mg/kg RSV administration could markedly inhibit microglia activation and neutrophils infiltration after SAH. Concomitant with the decreased cerebral inflammation, RSV evidently reduced cortical apoptosis, brain edema, and neurobehavioral impairment after SAH. In vitro experiments, RSV treatment also clearly protected primary cortical neurons against oxyHb insults, including reduced the proportion of neuronal apoptosis, alleviated neuronal degeneration, and improved cell viabilities. These in vitro data further confirm that RSV has an efficient neuroprotection against SAH. Taken together, these in vivo and in vitro findings suggested RSV could protect against EBI after SAH, at least partially via inhibiting NLRP3 inflammasome signaling pathway.

Keywords: NLRP3; early brain injury; inflammation; resveratrol; subarachnoid hemorrhage.

Figure 1

Expression and cellular distribution of the NLRP3 inflammasome components in the basal cortex at 24 h post-subarachnoid hemorrhage (SAH). (A) Western blot assay for the expression of NLRP3, ASC, caspase-1 P20 subunit, IL-1β and IL-18; (B) Quantification of NLRP3, ASC, caspase-1 P20 subunit, IL-1β and IL-18 expression is shown; (C) Representative photomicrographs of NLRP3 immunohistochemistry in the sham and 24 h post-SAH groups; (D) Quantification of the NLRP3 immunohistochemistry; (E) Representative photographs of NLRP3 immunofluorescence staining in microglia in the basal cortex at 24 h following SAH; (F) Schematic representation of the areas taken for assay. Bars represent the mean ± SEM. ^**P < 0.01, ^*P < 0.05, and ns means non-significant. Scale Bars = 50 μm.

Figure 2

Effects of different doses of RSV on the expression of NLRP3 at 24 h post-SAH. (A) Western blot analysis for NLRP3 expression in all different groups; (B) Representative photographs of NLRP3 immunofluorescence staining in microglia in all groups; (C) Quantification of the NLRP3 immunofluorescence staining in different groups. Bars represent the mean ± SEM. ^**P < 0.01, ^*P < 0.05, and ns means non-significant. Scale Bars = 50 μm.

Figure 3

Effects of RSV on the activation of NLRP3 inflammasome activation-related pathway at 24 h post-SAH. (A) Representative images of Western blot for NLRP3, ASC, caspase-1 P20 subunit, IL-1β, IL-18, and TNF-α; (B–G) Quantitative analyses of NLRP3, ASC, caspase-1 P20 subunit, IL-1β, IL-18, and TNF-α among all experimental groups, respectively. Bars represent the mean ± SEM. ^**P < 0.01, ^*P < 0.05, and ns means non-significant.

Figure 4

Effects of RSV on the NLRP3 inflammasome components distribution at 24 h post-SAH. (A) Representative images of NLRP3, ASC, and caspase-1 P20 subunit immunohistochemistry in all groups; (B) Quantification of NLRP3, ASC, and caspase-1 P20 subunit immunohistochemistry in all experimental groups. Bars represent the mean ± SEM. ^**P < 0.01, ^*P < 0.05, and ns means non-significant. Scale Bars = 50 μm.

Figure 5

Effects of RSV treatment on microglia activation and neutrophils infiltration at 24 h post-SAH. (A) Western blot assay for the Iba-1 and MPO levels in the brain samples at 24 h after SAH; (B,C) Quantitative analyses of Iba-1 and MPO among all experimental groups; (D,F) Representative photomicrographs of Iba-1 and MPO immunofluorescence staining in different groups at 24 h following SAH; (E,G) Quantification the number of Iba-1- and MPO- positive cells in all groups. Bars represent the mean ± SEM. ^**P < 0.01, ^*P < 0.05, and ns means non-significant. Scale Bars = 50 μm.

Figure 6

Effects of RSV on neuronal cell apoptosis, brain edema, and neurological function at 24 h following SAH. (A) Western blot assay for the expression of Bcl2, Bax and caspase-3 in the groups; (B) Quantification of the protein levels of Bcl2, Bax and caspase-3; (C) Quantification of TUNEL-positive neurons in all groups; (D) Representative photomicrographs of TUNEL staining in different groups; (E,F) Quantification of brain water content and neurological functions in different groups. Bars represent the mean ± SEM. ^***P < 0.001, ^**P < 0.01, ^*P < 0.05, and ns means non-significant. Scale Bars = 50 μm.

Figure 7

Effects of RSV on neuronal damage and cell viabilities in primary cortical neurons exposed to oxyHb. (A) Representative images of light microscopic for primary cortical neurons in all different groups; (B) Western blot assay for the apoptotic pathways; (C) Quantification of the protein levels of Bcl2, Bax and caspase-3 in all groups; (D) Representative photomicrographs of TUNEL staining in different groups; (E) Quantification of TUNEL-positive neurons in all groups; (F) Quantitative analyses of LDH activity in all groups. Bars represent the mean ± SEM. ^***P < 0.001, ^**P < 0.01, ^*P < 0.05, and ns means non-significant.