Corrigendum: Resveratrol improves mitochondrial biogenesisfunction and activates PGC-1α pathway in a preclinical model of early brain injury following subarachnoid hemorrhage

Jian Zhou ^* Zaijia Yang Ruiming Shen Huiduan Zhen Wangwang Zhong Zhenggang Chen Jianjian Tang Juan Zhu

• Hainan Medical University, Haikou, China

Introduction Subarachnoid hemorrhage (SAH) is a fatal cerebrovascular disease with high morbidity and mortality rates. Accumulating evidence indicates that early brain injury (EBI) is the major cause of the deterioration of patients with SAH. Therefore, the treatment of EBI has been believed to be the principal target for patients with the disease. Studies have indicated that numerous pathophysiological processes are involved in the pathogenesis of EBI, such as mitochondrial dysfunction, oxidative stress, apoptosis, inflammation, autophagy, and brain edema [1]. Among them, mitochondrial dysfunction plays a pivotal role in EBI. Hence, improving mitochondria dysfunction may be a promising therapeutic strategy for the treatment of EBI following SAH. Mitochondria play a fundamental role in cellular homeostasis. A recent analysis indicated that mitochondrial dysfunction increases oxidative stress. Excessive reactive oxygen species (ROS) production contributes to oxidative damage causing persistent injury to the brain tissue after SAH. However, superfluous ROS generation induced by SAH can lead to exhaustion of the innate antioxidation and disturbance of the redox system, resulting in severe complications. Brain tissue is more susceptible to free radical attacks, leading to mitochondrial dysfunction, ultimately triggering the cascade of cell apoptosis involving P53, Bcl-2 family, cytochrome c, and caspase3, which induces lipid peroxidation, nucleic acid oxidation, and DNA breakdown [2]. Therefore, reversing mitochondrial dysfunction and promoting mitochondrial biogenesis after SAH can improve mitochondria function and maintain cell homeostasis. However, the exact mechanisms underlying these processes have not been investigated in SAH. Mitochondria, especially mitochondrial DNA (mtDNA), are affected by ROS, and this may be modulated by the peroxisome proliferator-activated receptor (PPAR) γ coactivator-1 (PGC-1) family of transcriptional coactivators and is susceptible to oxidative impairment. PGC-1α, a transcriptional coactivator of mitochondrial biogenesis, which directly links external physiological stress to the regulation of mitochondrial biogenesis and function, is involved in energy homeostasis and metabolism. Resveratrol (RSV) is a polyphenolic compound with pleiotropic properties produced in a variety of plant species, such as red grapes, mulberries, peanuts, and wines. RSV eliminates excessive ROS when cellular homeostasis is disturbed. Recent studies have indicated that the beneficial effects of RSV, including anti-apoptotic, anti-inflammatory, and antioxidant activities [3-7]. It has been shown that RSV can cross the blood-brain barrier (BBB) and be used to treat neuroinflammatory and neurodegenerative diseases [8-10]. Several studies have indicated that RSV could regulate mitochondria ROS homeostasis and enhance PGC-1α expression [11]. RSV can abrogate cardiac oxidative stress and mitochondrial dysfunction in diabetes [12-14]. Various studies demonstrate that RSV can improve the activation of PGC-1α and its downstream signaling pathways [15]. However, the exact mechanisms by which RSV regulates mitochondrial biogenesis and function in SAH have not been fully elucidated. Thus, we designed this study to confirm the hypothesis that RSV could exert a neuroprotective effect by improving mitochondrial biogenesis and function, via activation of the PGC-1α signaling pathway. Materials and methods Animal Ethical Approval Adult male Sprague-Dawley rats weighing 270 to 320 g were used in the study. The rats were maintained under standard conditions of temperature ((22±1 °C), relative humidity (30%), and lighting (a 12/12-hr light/dark cycle). Drinking water and food were given ad libitum. All experimental procedures and protocols were conducted by the Animal Care and Use Committee of Hainan Medical University (Haikou, China) and conformed to the Guide for the Care and Use of Laboratory Animals set by the National Institutes of Health (NIH). SAH Model The experimental SAH model in this study was used to create the SAH model as described previously [16]. Briefly, rats were anesthetized with inhalation anesthesia with isoflurane (2% in oxygen gas, 300 ml/min), subsequently placed in a stereotactic frame. Under the stereotactic guidance, fresh autologous non-heparinized arterial blood (0.3 mL) from the femoral artery was injected aseptically into the prechiasmatic cistern in 20 s with a syringe pump in the SAH group. The wound was then sutured. The sham group was injected with 0.3 mL artificial cerebrospinal fluid. The animals were allowed to recover in a head-down prone position for 45 min post-SAH. After surgery, all animals were returned to their cages, and food and water were supplied freely. The SAH grade was blindly assessed with a grading system before euthanasia as previously described before [17]. Rats with a grade less than eight were excluded for having no obvious brain damage. Experimental design All rats were randomly assigned to one of the following groups: Sham, SAH, SAH + vehicle (SAH+V), and SAH + resveratrol (SAH + RSV). Resveratrol (Sigma, St. Louis, USA) was dissolved in an equal amount of DMSO solution (0.1% dimethylsulfoxide in 0.9% saline) just before injection. Rats in the SAH + RSV group were treated with an intraperitoneal injection of 60 mg/kg resveratrol at 2 and 12 h post-SAH [18]. SAH vehicle-treated group were injected equal volumes of the vehicle at the corresponding time point. All the animals were euthanized at 24 h post-SAH[6, 19]. Neurobehavioral Evaluation and Brain Water Content The neurological scores were performed at 24 h and 72 h after SAH using the scoring methodology, including appetite, activity, and neurologic deficits [16]. All neurobehavioral evaluations were conducted by two veterinarians blinded to the grouping. Brain water content was measured by the wet/dry ration method [20]. In brief, the rat brain was quickly obtained 24 h after SAH. And the temporal cortical tissue was weighed immediately to acquire the wet weight (WW). The brain samples were dried for 72 h at 80 °C and weighted to obtain the dry weight (DW). Brain water content was calculated as [(WW – DW) /WW] × 100%. Tissue processing The rats were sacrificed at 24 h after SAH and perfused transcardially with PBS. The same part of the inferior basal temporal lobe was harvested. Then, the brain samples were analyzed for brain water content, western blot, and biochemistry indexes. For immunofluorescence analysis and TUNEL staining, the whole brain tissue was harvested, then immersed in 4% buffered paraformaldehyde for the night. Isolation of mitochondria Mitochondria from the inferior basal temporal lobe were extracted following the instructions of the manufacturer with the use of Mitochondrial Isolation Kit for Tissue (Beyotime Institute of Biotechnology, Nantong, China). In brief, the temporal lobe was grounded in the ice-cold isolation buffer and then centrifuged at 1200 g for 3 min. The supernatants were collected and centrifuged at 14 000 g for 14 min at 4°C to precipitate the mitochondria. Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) Staining TUNEL staining was determined by using a TUNEL detection kit (ISCDD, Boehringer Mannheim, Germany) according to the manufacturer’s instructions. Quantitative analysis of TUNEL-positive cells in the temporal cortex was performed in six random non-overlapping fields (400 ×) under epifluorescence microscopy. The extent of brain injury was assessed by the apoptotic index, defined as the average number of TUNEL-positive neurons in each section. The positive cells were conducted by two investigators blinded to the experiment. Western blot analysis Protein extraction was performed by using a Total Protein Extraction Kit, Nuclear-Cytosol Extraction Kit, and Tissue Mitochondria Isolation Kit (Beyotime Biotech, Nantong, China) following the protocol provided by the manufacturer. Protein concentrations were determined by the Bradford method. Protein samples (50 ug/lane) were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel, electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 2 h at room temperature and then incubated overnight at 4°C with the appropriate primary antibody respectively using the following dilution ratio: P53 (1:400, CST), PGC-1α (1:1000, CST), NRF1 (1:1000, CST), TFAM (1:1000, CST), cytochrome c (1:1000, Abcam), Bax (1:200, Santa Cruz), Bcl 2 (1:200, Santa Cruz), COX Ⅳ (1:1000, CST), H3 (1:1000, CST), β-actin (1:5000, Bioworld), cleaved caspase 3 (1:1000, CST). Subsequently, the membranes were incubated with horseradish peroxidase-linked IgG for 2 h. The protein bands were visualized by enhanced chemiluminescence (ECL) western blot detection reagents (Millipore, Billerica, MA, USA). The band relative density was evaluated using the Image J software. Immunofluorescence staining Immunofluorescence staining was conducted following the procedures of a previous immunostaining protocol as follows: Briefly, sections (6 μm) were incubated in blocking buffer (5% normal fetal bovine serum in PBS containing 0.1% Triton X-100) for 2 h followed by overnight incubation with primary antibody at 4 °C. Then, the sections were washed for three 10 min with PBS and incubated with proper secondary antibodies (Alexa Fluor® 594,1:200) for 1 h at room temperature. After sections were washed with PBS again, the sections were counterstained with DAPI (1: 1000, Sigma) for 2 min. Following the washes with PBS, the sections were coverslipped with anti-fade mounting medium. Fluorescence microscopy imaging was conducted using ZEISS HB050 inverted microscope system and analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, USA). All the brain sections were evaluated in six temporal cortical microscopic fields (400×) by two investigators blind to the experiment. Reactive Oxygen Species (ROS) Content Intracellular ROS contents were conducted using the fluorescence probe dihydroethidium (DHE) according to the manufacturer’s instructions (Sigma-Aldrich, St Louis, USA). Briefly, the frozen sections were incubated in DHE, and then treated with PBS for 30 minutes at 37°C in the dark. The sections were washed three 5 min in PBS (PH7.4). All the immunofluorescence images were performed using ZEISS HB050 inverted microscope system. The DHE-fluorescence intensities (representing the intracellular ROS levels) were measured using the Image-Pro Plus 6.0 software. The production of mitochondrial ROS was detected using MitoSOX™ Red mitochondrial superoxide indicator (Thermo Fisher, Waltham, USA). Fresh temporal cortex tissues weighed at 10 mg were dispersed to single-cell suspension by a pipettor. The cells were washed twice with PBS and diluted the 5 mM MitoSOX™ reagent stock solution in HBSS/ Ca/Mg buffer to make a 5 μM MitoSOX reagent working solution. 1.0 ml of 5 μM MitoSOX reagent working solution was applied to incubate the cells for 10 minutes at 37°C in the dark. Fluorescence was detected by the microplate reader at an emission wavelength of 510 nm and an excitation wavelength of 580 nm. Nissl staining For Nissl staining, the sections of paraffin-embedded brain tissue (5-μm thick) were conducted using cresyl violet according to a previous study [21]. Neurons in the normal structure had large nuclei, round, with one or two, located in the central soma and abundant cytoplasm. On the contrary, positive cells had irregular neuronal cell bodies, shrinking and hyperchromatic nuclei, and dried-up cytoplasm with vacuoles. Then, histological examination was used to evaluate the ratio of positive cells by two investigators blinded to the experiment. Mitochondrial MDA and SOD Content The brain tissue was homogenized in 2 ml of 10 mM phosphate-buffer (PH 7.4). Following centrifugation at 12,000 g for 20 min, the contents of mitochondrial MDA and SOD in the supernatant were quantitated spectrophotometrically according to the manufacturer’s instructions (Nanjing Jiancheng Biochemistry Co., Nanjing, China). The protein concentrations were determined using the Bradford method. Quantification of mtDNA Total mtDNA was extracted from the inferior basal temporal lobe using a rapid animal genomic DNA isolation kit according to the manufacturer’s instructions (BioVision Inc). The mtDNA copy number was determined by SYBR green quantitative real-time PCR using specific primers for the mtDNA coded NADH-dehydrogenase subunit 1 (ND1) gene, and the nuclear DNA coded β-globin gene. The primer sequences are as follows: ND1: Forward 5′-TCGACGTTAAAGCCTGAGACT-3′; Reverse 5′-TTAATCCCCGCCTGACCAATA-3′; β-globin: Forward 5′-GTCTACCCTTGGACCCAGAG-3′, Reverse 5′-CCAAGTGTTTCAGGCCATCA-3′. Relative transcript abundance was calculated using the ΔΔCt method. Mitochondrial membrane potential (MMP) measurement To evaluate mitochondrial viability and function, we monitored changes in mitochondrial membrane potential (MMP) by using 5,5,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1) dye [22, 23]. The fluorescence intensity was detected on a flow cytometer (FACScan; BD Biosciences) and analyzed in FACSuite software. The emitted green and red fluorescence was measured through 585nm (FL2) and 530 nm (FL1) bandpass filters. The FL1–FL2 compensation was approximately 4% and the FL2–FL1 compensation was 10.6%. Bivariate plots of FL2 versus FL1 were applied to assess MMP. Intracellular ATP level measurement The intracellular ATP level was analyzed by using a Bioluminescent Somatic Cell Assay Kit according to the manufacturer’s instructions (Sigma-Aldrich, Germany) [24]. Briefly, viable mitochondria were dissolved to liberate the intracellular ATP, then mixed with substrate and luciferase enzyme, and transferred into a 96-black well plate. Finally, luminescence analysis was conducted using a luminometer (Berthold, Germany). Statistical analysis Statistical analysis was conducted using the GraphPad Prism 8.0 (GraphPad Software Inc., La Jolla, CA, USA) software. Values are expressed as the mean ± SEM. Statistical comparisons between groups were analyzed with one-way ANOVA and Tukey's test. A p-value less than 0.05 was considered statistically significant. 3. Results Resveratrol restored the neurological function and alleviated cerebral edema following SAH. To evaluate the neuroprotective role of RSV on neurological function, the neurological scores were recorded at 24 h and 72 h after SAH as described above. All rats were trained 24 hours before SAH. Within 3 days after SAH, there was no statistically significant change between time points in the sham group. No obvious difference was observed between the SAH group and the SAH+V group at 24 h and 72 h (P =0.095, Fig. 1C). However, RSV treatment significantly improved motor performance compared with the vehicle-treated rats at 24h after SAH (P = 0.0023, Fig. 1D). Nevertheless, RSV could not improve motor performance compared to that of the vehicle-treated rats at 72h following SAH. Therefore, 72 h group after SAH was not used for the following studies, such as sacrifice, brain extraction, measurements, and histology. The finding suggested that 24 h time frame might be an optimal time point for studying RSV treatment for SAH. Thus, 24 h group after SAH was used in the subsequent studies. Brain water content was further applied to explore the protective role of RSV. As shown in Figure 1B, the brain water content was markedly increased in the SAH group when compared with the sham group. No significant difference was observed between the vehicle-treated group and the SAH group (P =0.1151). Consistent with the neurological scores, RSV was successful in ameliorating the SAH-induced brain edema than the vehicle-treated group (P =0.021). Resveratrol ameliorated neuronal apoptosis following SAH To evaluate whether RSV administration could attenuate apoptosis in the inferior temporal cortex 24 h after SAH, TUNEL analysis was employed to assess neuronal apoptosis. As shown in Fig. 2, only scattered TUNEL-positive cells were observed in the sham group, whereas the SAH and SAH + V groups displayed numerous apoptotic cells in the temporal cortex up to nearly 36%. However, the apoptosis index in the SAH + RSV group fell to about 22% (Fig. 2E and 2F). We detected the expression of Cleaved caspase 3, P53, and Bcl-2 in the protein level. The results indicated that the levels of the antiapoptotic protein Bcl-2 were markedly decreased, and the levels of the proapoptotic protein P53 and cleaved caspase 3 were notably increased, compared with the sham group, whereas these effects were reversed by RSV administration (Figures 2 A-D). Collectively, these results demonstrate that RSV treatment possibly abrogated neuronal apoptosis following SAH. Resveratrol Exerted Protective Role in the Mitochondria. To further elucidate the neuroprotective role of RSV in the mitochondria, the expression of mitochondrial apoptosis-related proteins Bax and cytochrome c was examined following SAH. The results showed that the level of mitochondrial Bax protein was increased after SAH, whereas mitochondrial cytochrome c levels were decreased, relative to the sham group (Fig. 3C-D). However, RSV treatment suppressed mitochondrial translocation of Bax and subsequent cytosolic release of cytochrome c. To explore the antioxidative effect of RSV after SAH, we detected the production of mitochondrial MDA and SOD in the mitochondria. Our results underlined that the activity of SOD was significantly decreased after SAH when compared with the sham group. However, RSV treatment elevated the activity of SOD (P = 0.012; Fig. 3F). In contrast, SAH insult increased mitochondrial MDA level and RSV administration could restore the content of MDA. (P = 0.016, Fig. 3G). For a better understanding of the comprehensive effects of RSV on EBI following SAH, neuronal survival was further evaluated at 24 h post-SAH. As shown (Fig. 3A-B), RSV significantly increased the proportion of surviving neurons. Taken together, these results suggested that RSV could improve mitochondrial function and promote neuronal survival in the temporal cortex following SAH. Resveratrol administration ameliorated ROS induced by SAH To determine whether the protective role shown by RSV is associated with its antioxidant property, we examined the effect of RSV on ROS production. ROS were detected in the form of superoxide anions following SAH. The relative content of intracellular ROS production was detected by DHE fluorescence. As shown in Figure 4, SAH insult significantly (P = 0.0061) increased ROS levels showed a significantly higher DHE fluorescence intensity compared with that in the sham group. Interestingly, the total ROS level was significantly reduced by RSV treatment (Fig. 4A-B). And then, the mitochondrial ROS content was analyzed by MitoSOX staining. Our results showed that RSV treatment significantly ameliorated the relative level of mitochondrial ROS content (Fig. 4C), consistent with the result of DHE fluorescence. To further testify the beneficial role of RSV, we detected the expression of 8-Hydroxy-2 deoxyguanosine (8-OHdG) by immunofluorescence staining in or around the nucleus following SAH. Our observation showed that numerous 8-OHdG positive cells dyed as red were obvious in the SAH and vehicle-treated groups, whereas RSV treatment significantly alleviated the number of 8-OHdG positive cells in the temporal cortex at 24 h after SAH (Fig. 4D). Resveratrol promoted PGC-1α Nuclear Translocation The data obtained demonstrated that RSV markedly increased the activities of antioxidant enzymes induced by SAH. However, the underlying molecular mechanisms remain elusive. PGC-1α plays an essential role in the restoration of mitochondrial function. Accordingly, it was reasonable to hypothesize that RSV might activate PGC-1α, thereby enhancing the restoration of mitochondrial function. We measured the expression of nuclear PGC-1α and total PGC-1α. Our results suggested that RSV administration promoted PGC-1α nuclear translocation compared with the vehicle-treated group (Fig. 5A). Additionally, the RSV treatment significantly increased the relative protein level of nuclear PGC-1α, while the total PGC-1α level was also upregulated in the SAH+RSV group. This finding suggested that RSV promoted PGC-1α nuclear translocation, thus enhancing its binding activity to promote mitochondrial biogenesis and function. This effect was also confirmed by the immunofluorescence staining of PGC-1α. As shown in Fig. 5, only a few PGC-1α immunoreactive neurons were displayed in the sham group, whereas numerous PGC-1α immunoreactive neurons were exhibited in the temporal cortex following SAH. These observations demonstrated that SAH promotes PGC-1α nuclear translocation. However, the number of PGC-1α immunoreactive neurons was remarkably increased by RSV treatment compared with the vehicle-treated group. Together, these results suggested that RSV promotes PGC-1α translocation, thereby enhancing the binding ability to downstream proteins. Resveratrol upregulated the expression of PGC-1α downstream factors Precious evidence obtained indicated that RSV could activate PGC-1α and improve mitochondrial biogenesis and function after SAH. Henceforth, we speculated that RSV might also regulate the downstream factors in the PGC-1α pathway. We measured the expression of NRF1 and TFAM. Our data demonstrated that NRF1 and TFAM were both upregulated following SAH (P = 0.0031 and P = 0.00056, respectively;). However, RSV treatment further promoted the expression of PGC-1α downstream proteins compared with the vehicle-treated group ((P = 0.0043 and P = 0.00025, respectively; Fig. 6A-B). These results demonstrated that RSV promoted the expression of PGC-1α downstream factors at the protein level, by activation of the PGC-1α signaling pathway. Mitochondrial membrane potential (MMP), Intracellular ATP content, and mtDNA copy number The MMP and intracellular ATP content values decreased in the SAH and SAH + vehicle groups, and increased notably after RSV treatment (Fig. 6C-D). Furthermore, the mtDNA copy number in the SAH+V group increased compared to the Sham group (P = 0.035). However, compared to the SAH+V group, the mtDNA copy number significantly decreased in the SAH+RSV group (Fig. 6E). These findings presented above indicated that RSV improved mitochondrial biogenesis in EBI following SAH. Discussion Mitochondria are fundamental in the regulation of the function of neurons. Neurons, which are susceptible to oxidative stress, have fewer endogenous antioxidants than other cells. Extensive evidence demonstrates mitochondrial dysfunction plays a key role in the development of secondary injury induced by SAH [2]. SAH increases ROS levels and impairs mitochondrial function. Superfluous ROS in the mitochondria causes mitochondrial injury, leading to increased ROS synthesis, decreasing adenosine triphosphate synthesis, and resulting in the endogenous apoptotic pathway activation. A recent analysis indicated that mitochondrial dysfunction could be a hallmark for SAH. Consequently, improving mitochondrial biogenesis and function may be a novel beneficial therapeutic target for the treatment of SAH. In the current study, we investigated the effects of RSV treatment on mitochondrial biogenesis and function via activation of the PGC-1α signaling pathway in EBI following SAH. The main findings of this study are as follows: (1) RSV treatment attenuated the mitochondria-dependent apoptosis following SAH. (2) RSV administration reduced redundant ROS production induced by SAH; (3) The PGC-1α signaling pathway was activated by RSV treatment and the modulation of PGC-1α nuclear shuttling was promoted following SAH; and (4) SAH-induced oxidative damage, brain edema, and neurological impairment were ameliorated by RSV treatment. These findings indicated for the first time that RSV could promote mitochondrial biogenesis and function against SAH-induced oxidative stress and apoptosis, via activating the PGC-1α pathway. RSV, a natural phytoalexin product found in grapes and other plants [25], is well known for its phytoestrogenic and antioxidant properties [26]. Recently, studies have demonstrated that RSV could be a promising therapeutic ingredient against various central nervous system diseases[18, 27]. Previous studies have revealed that RSV has pleiotropic effects, such as antioxidant, anti-apoptotic, anti-inflammatory, and vasodilatory properties [5, 28-31]. RSV treatment can mitigate neuronal apoptosis in numerous brain injury models, such as cerebral ischemia, SAH, and spinal cord injury [6, 32]. In the current study, we observed that RSV treatment significantly improved the expression of apoptosis-related proteins, such as P53, Bcl2, and cleaved caspase3, and also promoted neuronal survival in EBI after SAH. RSV treatment alleviated MDA levels and improved the activity of SOD. These results indicated that RSV could attenuate neuronal apoptosis and oxidative damage in EBI following SAH. These findings extend those of previous studies [6, 33]. However, the molecular mechanisms of RSV in EBI have yet to be elucidated. Neurons appear particularly vulnerable to mitochondrial dysfunction [34]. A recent analysis indicated that mitochondrial dysfunction plays an essential role in the pathophysiological process of SAH. Preventing mitochondrial damage and dysfunction may be a new approach for the treatment of SAH. We speculated that the neuroprotective effects of RSV may be associated with improving mitochondrial biogenesis and function, but the exact mechanism remains obscure. The oxidative damage by SAH caused the opening of mitochondrial permeability transition pores (PTPs), leading to increased ROS release to the cytoplasm, and resulting in mitochondrial dysfunction. Mitochondria injury causes the overproduction of ROS [35], contributing to mitochondrial dysfunction and upsetting the normal balance of endogenous oxidant and antioxidant mechanisms [35]. Mitochondrial dysfunction leads to oxidative modification of proteins, lipids, and DNA, terminating in excessive ROS release. In the current study, we assessed the potential effects of RSV on ROS scavenging after SAH. We found that RSV treatment could notably reduce ROS release in the temporal cortex. Therefore, the activity of MDA was decreased and SOD increased by the RSV treatment. As a consequence, the mitochondrial proapoptotic protein Bax accumulated in the cytoplasm while cytochrome c was liberated from the mitochondria, triggering the activation of the intrinsic apoptosis pathway [36] [37], resulting in activation of the mitochondrial-dependent apoptotic pathway and inducing the sequential activation of the caspase cascades [38]. This series of events causes the degradation of DNA and essential proteins and ultimately leading to neuronal apoptosis [39, 40]. A similar phenomenon was found in our study. Our data showed that RSV significantly ameliorated mitochondrial Bax and restored cytochrome c. These results indicated that the mitochondrial apoptotic pathway was activated following SAH. Moreover, RSV eliminated redundant ROS production generated by mitochondria, erasing the free radicals from the mitochondrial root, thus attenuating mitochondrial-dependent apoptosis and improving the antioxidative ability. We hypothesize that mitochondrial biogenesis might account for these findings. Mitochondrial biogenesis is a complicated process that requires the coordinated regulation of multiple proteins. Growing data indicate that PGC-1α, NRF-1, and TFAM, are responsible for mitochondrial biogenesis [41]. Neuronal mitochondrial biogenesis by itself has been poorly established. PGC-1α, a transcriptional coactivator with pleiotropic functions, plays a pivotal role in mitochondrial biogenesis and function [42]. However, the underlying mechanisms by which PGC-1α is activated and mitochondrial biogenesis is upregulated are poorly understood in the SAH rat model. In our study, we explored the expression of the PGC-1α signaling pathway. We found that the expression of PGC-1a was upregulated by RSV treatment following SAH. RSV treatment also promoted PGC-1α nuclear translocation. And its downstream genes (NRF1 and TFAM) were activated. NRF1, bound to the binding sites of the TFAM gene which regulates the transcription of the mitochondrial genome, together acts on the promoter within the D-loop region of mtDNA, initiating replication and transcription of the mitochondrial genome [43, 44]. The findings indicated that RSV activated PGC-1α and its downstream transcriptional factors (NRF1 and TFAM), which are closely linked to an increase in mitochondrial biogenesis. Furthermore, an increase in MMP following RSV treatment suggested the effect on mitochondrial biogenesis post-SAH. ATP levels were also analyzed, which were in accordance with alterations of MMP, providing further evidence for the protective effect of RSV on mitochondrial function. Importantly, RSV restored the mtDNA copy number to almost normal levels after SAH. These data suggested that RSV could reverse mitochondrial dysfunction, and improve mitochondrial biogenesis following SAH. Increasing evidence suggests that PGC-1α is a powerful regulator of ROS removal by increasing the expression of numerous ROS-detoxifying enzymes [45, 46]. We also found that the activity of the mitochondrial ROS-detoxifying gene SOD was elevated by RSV and activated PGC-1a. The RSV treatment eliminated the overproduction of ROS originating from mitochondria, improving SAH-mediated mitochondrial impairment and ROS activity. These results showed that the PGC-1α pathway was activated and involved in mitochondrial biogenesis in the early period after SAH. For a better understanding of these neuroprotective effects of RSV following SAH, we further explored SAH-induced brain edema and neurological function at 24 h after SAH. Our results indicated that RSV treatment could improve neural survival and neurological impairment. There are some limitations to this study. First, an in vitro model of SAH should be used to explore whether RSV is involved in the neuroprotection against neuronal apoptosis, oxidative stress, and PGC-1α nuclear translocation. Second, a PGC-1α gene knockout mice model should be used to identify the mitochondria-boosting property of RSV by activation of the PGC-1α pathway. Research in many other associated areas is warranted. Conclusion The current findings reveal that RSV treatment could provide neuroprotection in the SAH models by inhibiting mitochondrial-dependent apoptosis, attenuating mitochondria-related ROS release, alleviating the mtDNA copy number, and improving the antioxidative ability in the temporal cortex. Our data support a previous hypothesis that RSV could improve mitochondrial biogenesis and function after SAH by activating the PGC-1α signaling pathway. The PGC-1α signaling pathway may be a key therapeutic target for future neuroprotective strategies against SAH. Further study should establish the mitochondria-boosting property of RSV in the neuron SAH model in vitro and examine the underlying mechanisms.