Wu-zhu-yu Decoction reduces early brain injury following subarachnoid hemorrhage in vivo and in vitro by activating the Nrf2 antioxidant system via SIRT6 targeting.

Early brain damage (EBI) following subarachnoid hemorrhage (SAH) is a long-lasting condition with a high occurrence. However, treatment options are restricted. Wu-zhu-yu Decoction (WZYD) can treat headaches and vomiting, which are similar to the early symptoms of subarachnoid hemorrhage (SAH). However, it is yet unknown if WZYD can reduce EBI following SAH and its underlying mechanisms. This study aimed to investigate whether WZYD protects against EBI following SAH by inhibiting oxidative stress through activating nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling via Sirtuin 6 (SIRT6)-mediated histone H3 lysine 56 (H3K56) deacetylation. In the current investigation, the principal components of WZYD were identified using high-performance liquid chromatography-diode array detection (HPLC-DAD). The SAH model in rats using the internal carotid artery plug puncture approach and the SAH model in primary neurons using hemoglobin incubation were developed. WZYD with different doses (137 mg kg−1, 274 mg kg−1, 548 mg kg−1) and the positive drug–Nimodipine (40 mg kg−1) were intragastrically administered in SAH model rats, respectively. The PC12 cells were cultured with corresponding medicated for 24h. In our investigation, neurological scores, brain water content, Evans blue leakage, Nissl staining, TUNEL staining, oxidative stress, expression of apoptosis-related proteins, and Nrf2/HO-1 signaling were evaluated. The interaction between SIRT6 and Nrf2 was detected by co-immunoprecipitation. SIRT6 knockdown was used to confirm its role in WZYD's neuroprotection. The WZYD treatment dramatically reduced cerebral hemorrhage and edema, and enhanced neurological results in EBI following SAH rats. WZYD administration inhibited neuronal apoptosis via reducing the expression levels of Cleaved cysteinyl aspartate specific proteinase-3(Cleaved Caspase-3), cysteinyl aspartate specific proteinase-3(caspase-3), and Bcl-2, Associated X Protein (Bax) and increasing the expression of B-cell lymphoma-2(Bal2). It also decreased reactive oxygen species and malondialdehyde levels and increased Nrf2 and HO-1 expression in the rat brain after SAH. In vitro, WZYD attenuated hemoglobin-induced cytotoxicity, oxidative stress and apoptosis in primary neurons. Mechanistically, WZYD enhanced SIRT6 expression and H3K56 deacetylation, activated Nrf2/HO-1 signaling, and promoted the interaction between SIRT6 and Nrf2. Knockdown of SIRT6 abolished WZYD-induced neuroprotection. WZYD attenuates EBI after SAH by activating Nrf2/HO-1 signaling through SIRT6-mediated H3K56 deacetylation, suggesting its therapeutic potential for SAH treatment. The graph created with BioRender (https://biorender.com). The caption for the abstract graphic: WZYD alleviated SAH symptoms by activating SIRT6/Nrf2/HO-1 antioxidant system and reducing pyroptosis in SAH rats. SIRT6 could directly bind to Nrf2, and SIRT6 could deacetylate the H3K56Ac site of the promoter of Nrf2 target genes, thus promoting the expression antioxidant of gene HO-1. [Display omitted] • Wu-zhu-yu Decoction (WZYD) ameliorated EBI after SAH in rats. • WZYD activated the Nrf2 antioxidant system and reduced oxidative stress and cell death. • WZYD activated the Nrf2 antioxidant system via targeting of SIRT6. • The knockdown of SIRT6 reversed the positive effects of activating the antioxidant system in vivo by WZYD. [ABSTRACT FROM AUTHOR]