Isoliquiritigenin alleviates cerebral ischemia-reperfusion injury by reducing oxidative stress and ameliorating mitochondrial dysfunction via activating the Nrf2 pathway

Abstract

Cerebral ischemia-reperfusion injury (CIRI) refers to a secondary brain injury that occurs when blood supply is restored to ischemic brain tissue and is one of the leading causes of adult disability and mortality. Multiple pathological mechanisms are involved in the progression of CIRI, including neuronal oxidative stress and mitochondrial dysfunction. Isoliquiritigenin (ISL) has been preliminarily reported to have potential neuroprotective effects on rats subjected to cerebral ischemic insult. However, the protective mechanisms of ISL have not been elucidated. This study aims to further investigate the effects of ISL-mediated neuroprotection and elucidate the underlying molecular mechanism. The findings indicate that ISL treatment significantly alleviated middle cerebral artery occlusion (MCAO)-induced cerebral infarction, neurological deficits, histopathological damage, and neuronal apoptosis in mice. In vitro, ISL effectively mitigated the reduction of cell viability, Na⁺-K⁺-ATPase, and MnSOD activities, as well as the degree of DNA damage induced by oxygen-glucose deprivation (OGD) injury in PC12 cells. Mechanistic studies revealed that administration of ISL evidently improved redox homeostasis and restored mitochondrial function via inhibiting oxidative stress injury and ameliorating mitochondrial biogenesis, mitochondrial fusion-fission balance, and mitophagy. Moreover, ISL facilitated the dissociation of Keap1/Nrf2, enhanced the nuclear transfer of Nrf2, and promoted the binding activity of Nrf2 with ARE. Finally, ISL obviously inhibited neuronal apoptosis by activating the Nrf2 pathway and ameliorating mitochondrial dysfunction in mice. Nevertheless, Nrf2 inhibitor brusatol reversed the mitochondrial protective properties and anti-apoptotic effects of ISL both in vivo and in vitro. Overall, our findings revealed that ISL exhibited a profound neuroprotective effect on mice following CIRI insult by reducing oxidative stress and ameliorating mitochondrial dysfunction, which was closely related to the activation of the Nrf2 pathway.

1. Introduction

Ischemic stroke (IS) is a leading account of cerebrovascular disease and is commonly caused by obstruction or stricture of the cerebral arteries. This change may provoke a cascade of neuronal damage and subsequent irreversible neurological impairments, including cerebral palsy, epilepsy, and limited cognitive functions [1,2], which can drastically compromise the sufferer's quality of life. Furthermore, along with the aging of the population, the prevalence of IS has been expected to increase worldwide, particularly in developing countries [3,4]. Presently, mechanical thrombectomy and intravenous thrombolysis with t-PA are the primary clinical strategies in the clinical setting. Nevertheless, few patients can benefit from the above strategy due to the narrow therapeutic time window and potential hemorrhage risks [5,6]. In addition, cerebral ischemia-reperfusion injury (CIRI) induced by thrombolysis will further aggravate brain injury [7]. Therefore, unraveling the underlying mechanisms and developing novel treatment modalities for preventing CIRI is urgently needed.

The pathogenic process of CIRI involves numerous underlying mechanisms, such as energy metabolism disorder, glutamate excitotoxicity, mitochondrial dysfunction, oxidative stress, neuro-inflammation, and apoptosis [8,9]. Emerging evidence reveals that excessive oxidative stress plays a pivotal role in the pathogenesis of ischemic brain injury [10,11]. The high levels of reactive oxygen species (ROS), products of lipid peroxidation and oxidatively modified proteins have been identified in IS patients [[12], [13], [14]]. Increased ROS levels may trigger an accumulation of excitatory amino acid in the synapse, which further causes an increase in Ca²⁺ influx and mitochondrial dysfunction. As the central site of energy metabolism, the integrity of mitochondrial structure and function is necessary for maintaining the balance among mitochondrial biogenesis, mitochondrial fusion-fission, and mitophagy [15,16]. However, excessive production of mitochondrial ROS induced by ischemic insult may lead to decreased ATP production, descent of mitochondrial membrane potential (MMP), and compromised mitochondrial homeostasis control. Meanwhile, an excess of cytochrome C (Cyt-C) and apoptosis inducing factor (AIF) released from impaired mitochondrial is suggested to engage in a cellular cascade process, subsequently triggering DNA damage and neuronal apoptosis, aggravating an array of cerebral ischemic injuries [17,18]. As a result, the preservation of redox homeostasis and optimal mitochondrial function are essential for facilitating the remission of CIRI.

Previous studies [[19], [20], [21]] have demonstrated that the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-derived factor 2 (Nrf2) system is a crucial defense mechanism of the cellular response to oxidative stress and mitochondrial dysfunction. Specifically, Nrf2 levels are tightly regulated and under physiological conditions, it is anchored in the cytoplasm and forms Keap1-Nrf2 heterodimer with Keap1, which is a cysteine-rich protein in the cytoplasm and serves as a vital sensor for oxidative stress [22]. The highly reactive Cys residues of Keap1 are easily modified by electrophilic or oxidative signals, resulting in the conformation change of Keap1, thus preventing Nrf2 ubiquitination and degradation and then translocating into the nucleus, subsequently activating antioxidant response element (ARE) [20,23]. Significantly, high levels of Nrf2 in the nucleus improve the transcription and production of downstream antioxidant enzymes, such as heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), and superoxide dismutase (SOD), thereby alleviating oxidative stress injury and maintaining mitochondrial homeostasis [24,25]. Consequently, ameliorating mitochondrial dysfunction via targeting activation of the Nrf2 signaling pathway is a promising therapeutic chance for treating ischemia-mediated cerebral impairment.

Isoliquiritigenin (ISL), a flavonoid component derived from the Chinese “homology of medicine and food” herb Glycyrrhiza uralensis (G. uralensis), has shown extensive pharmacological activities, such as anti-inflammatory, antioxidant, anti-apoptotic, anti-allergic, and detoxicate properties [26]. Previous evidence [[27], [28], [29], [30]] has demonstrated that ISL exerts potent neuroprotective effects in nerve system diseases, including traumatic brain injury, intracerebral hemorrhage, and cognitive disorders diseases, which is closely associated with its ability to inhibit oxidative stress and neuroinflammation, as well as mitigate mitochondrial dysfunction. Also, reports indicate that ISL can activate the Nrf2-mediated antioxidant pathway by facilitating the translocation of Nrf2 into the nucleus, subsequently initiating the expression of a series of target genes and exhibiting prominent neuroprotective properties [27,31]. Previous pharmacological investigations have preliminarily revealed that ISL can alleviate cerebral ischemic injury in rats by improving energy metabolism and antioxidant enzyme activities [32]. However, the underlying molecular mechanisms by which ISL confers neuroprotection against ischemic brain injury remain to be fully elucidated. Thus, in the present study, we sought to further investigate the potential neuroprotective effects of ISL on CIRI in mice. Additionally, whether the neuroprotective mechanism of ISL is involved in activating the Nrf2 signaling pathway and ameliorating mitochondrial dysfunction following ischemic insult was investigated.

2. Materials and methods

2.1. Experimental animals

ICR mice (26 ± 2 g) were acquired from the Experimental Animal Center of Ningxia Medical University, Yinchuan, China (Permit number: SCXK Ningxia 2015-0001). Mice were housed at a constant temperature (22–24 °C) with 12 h light/dark cycles, with ad libitum access to food and water. All animal experimental procedures were conducted according to the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines and the Institutional Animal Ethics Committee of Ningxia Medical University guidelines. Every effort was made to minimize the number of animals used and to reduce their suffering.

2.2. Experiment groups and drug administration

Isoliquiritigenin (Fig. 1A), a light yellow powder with a purity of ≥98.0 %, was purchased from Shanghai Yuanye Biotechnology Co., Ltd., China, and dissolved in 0.5 % CMC-Na solution before administration. All experimental mice were randomly assigned to the following groups: Sham group (with 0.5 % CMC-Na), MCAO group (with 0.5 % CMC-Na), MCAO + ISL group (with 5, 10, 20 mg/kg ISL, respectively), and MCAO + ISL + Bru (Brusatol, the inhibitor of the Nrf2) group (with 20 mg/kg ISL and 1 mg/kg Bru). Mice were treated with drugs or vehicles by intragastric administration once a day for seven days, respectively. The experimental design is shown in Fig. 1B.

Fig. 1.

ISL alleviated MCAO-induced brain injury, neurological deficit, and histopathological damage in mice. (A) The chemical information of ISL. (B) Flow chart of the experimental design. (C) Representative TTC staining images of coronal brain sections (the sections were presented “rostral to caudal”) at 24h after CIRI. (D) Quantitative analysis of brain infarct volume. (E) Monitoring images of CBF. (F) Quantitative analysis of the reduced rate of CBF in the ischemic brains. (G) Neurological deficit scores at 24 h after CIRI. (H and I) Representative HE and Nissl staining images of the histopathological changes in the ischemic cerebral cortex ( × 400, scale bar = 20 μm), hippocampus CA1 ( × 400, scale bar = 20 μm), and hippocampus CA3 ( × 200, scale bar = 50 μm) regions. Neurologic deficit scores are expressed as median with interquartile range, and normally distributed data are presented as the mean ± SD (n = 6). ^###P < 0.001 versus sham group; ∗∗P < 0.01, ∗∗∗P < 0.001 versus MCAO group; ⁺⁺P < 0.01 versus MCAO + ISL (5 mg/kg) group; ^&&P < 0.01 versus MCAO + ISL (10 mg/kg) group.

2.3. Establishment of the MCAO model

After seven days of pretreatment with reagent or vehicle, mice were subjected to 2 h left middle cerebral artery occlusion (MCAO) and 24 h reperfusion. Briefly, according to the instructions of the small animal anesthesia equipment, the mice were deeply anesthetized by inhalation of isoflurane, and surgical procedures commenced once the righting reflex disappeared. A small incision was made in the midline of the mice's neck. The muscle tissues, nerves, and veins were isolated, and the left common carotid arteries were exposed. A 4−0 monofilament nylon suture was inserted into the internal carotid artery, and then it was advanced to block the left middle cerebral artery. Two hours after the occlusion (the mortality rate among the mice ranges from 15 % to 25 % during this period, which has been excluded from the experiment), the filament was dismantled carefully until the tip reached the external carotid artery, following the reperfusion for 24 h. Sham-operated mice were exposed to the same surgical procedure without conducting the arteries occlusion. A heating pad controlled the mice's body temperature at 37 ± 0.5 °C during the whole procedure. After the surgery, all mice were housed individually in dry, sterile cages with sufficient food and water provisions. Following a 24 h reperfusion period, the mice were euthanized by cervical dislocation, and then the brain tissue was removed quickly by decapitation for further experiments.

2.4. Neurological deficit score

Neurological deficits were evaluated after 24 h of reperfusion with a 5-point scale as follows [33]: 0 = no neurological deficit; 1 = failure to stretch the contralateral forelimb fully; 2 = circling to the contralateral side; 3 = falling to the contralateral side; 4 = no spontaneous locomotor activity and depressed levels of consciousness. The measurement and analysis were done by investigators blinded to experimental groups.

2.5. Laser speckle contrast imaging

Laser speckle blood flowmetry (PeriCam PSI NR, No. PSIN-01117, Perimed AB, Sweden) was used to monitor cerebral cortical blood flow in mice following MCAO. Briefly, a midline incision was made over the skull to expose the calvaria of the mice following ether inhalation anesthesia, and the laser probe was positioned 10 cm above the detected cortex region. The raw speckle images and cerebral blood flow (CBF) were then continuously measured for 60 s. The analytical results of the pictures and the relative CBF changes were recorded using Perisoft software (PeriCam PSI NR, Perimed AB, Sweden).

2.6. Measurement of the infarct volume

After 24 h of reperfusion, the experimental animals were anesthetized and then decapitated, subsequently removed the brain tissues for immediate measurement of infarct volume. Briefly, the brain tissues were sliced into five 2-mm-thick consecutive coronal brain slices, immersed in a 2 % 2,3,5-triphenyltetrazolium chloride (TTC) solution at 37 °C for 30 min, and then fixed in 4 % paraformaldehyde overnight. Brain infarct volumes were quantified with ImageJ analysis software (Image-Pro Plus, USA) and determined by an edema correction formula: Infarct volume (%) = [(contralateral hemisphere volume − ipsilateral hemisphere volume + infarct volume of the ipsilateral hemisphere)/contralateral hemisphere volume] × 100 %.

2.7. Histopathological analyses

Mice were anesthetized with ether inhalation at 24 h after ischemic reperfusion and perfused with 0.9 % normal saline, followed by 4 % paraformaldehyde solutions until the body stiffened. The mice brains were quickly removed and submerged in 4 % paraformaldehyde overnight at 4 °C. Each brain tissue was gradient dehydration in ethanol and xylene, embedded in paraffin, followed by cut into 5-μm-thick coronal sections for subsequent staining. The sections were dried at 70 °C for 2 h, deparaffinized and rehydrated for Hematoxylin & eosin (HE) and Nissl staining, respectively. The images of the cortex and hippocampal regions were captured by light microscopy (Olympus BX-51, Japan) at magnifications of × 200, and the analyzer was indistinct to the experimental groups.

2.8. TUNEL assay

Neuronal apoptosis was visualized using the Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling (TUNEL) reagent kit (Roche, Basel, Switzerland). Briefly, the brain coronal slices were deparaffinized, rehydrated, permeated with the proteinase K working solution for 20 min at 37 °C, and then washed thrice with PBS. Subsequently, the slices were co-incubated with NeuN antibody and TUNEL reaction mix in the dark for 1.5 h at 37 °C, then covered with DAPI at room temperature. Finally, the TUNEL-positive neurons in 6 different fields of each group were photographed with a fluorescence microscope (Olympus FV1000, Japan). The rate of neuronal apoptosis was calculated as follows: apoptotic neuron amount/total neuron amount × 100 %.

2.9. Cell culture, oxygen-glucose deprivation (OGD) model establishment and drug administration

PC12 cell lines were cultured in RPMI-1640 medium containing 10 % fetal bovine serum and 100 U/mL penicillin/streptomycin, and the cells were incubated in a 5 % CO₂ incubator at 37 °C until they reached 80 % confluency, followed by subjected to OGD intervention to simulate hypoxic-ischemic conditions in vivo. Briefly, the culture medium was replaced with Earle's balanced salt solution (EBSS), then placed the cells in a hypoxic condition (5 % CO₂ and 95 % N₂) for 4 h at 37 °C. After that, the EBSS solution was replaced by the RPMI-1640 medium, and the cells were cultured in a normal condition to undergo reperfusion for 24 h at 37 °C. During the period of re-oxygen and re-glucose, the cells were treated with (2, 4, and 6 μM, respectively) or brusatol (5 nM) for 24 h. The cells in the control and vehicle groups were cultured with normal medium conditions.

2.10. Cell viability assay

The viability of PC12 cells was measured by the CCK-8 assay. The cells were seeded in a 96-well plate and cultured until they reached 80 % confluency, followed by subject to OGD conditions for 4 h, and then administrated with drug or vehicle for 24 h. After that, the medium was removed, adding 100 μL of 10 % CCK-8 reagent to each well and then incubated for 1–4 h at 37 °C. Finally, the absorbance values were recorded at 450 nm using the microplate reader (Thermo, USA), and relative cell viability was calculated compared with the control group.

2.11. Determination the activities of Na ⁺ -K⁺-ATPase and MnSOD

PC12 cells were seeded at a density of 5 × 10⁵ in a 60 mm dish and harvested at 24 h after treatment with the reagent. The Na ⁺ -K⁺-ATPase and MnSOD activities were conducted spectrophotometrically according to the manufacturer's instructions (Beyotime Biotechnology, China).

2.12. Determination of DNA damage

The degree of DNA damage in the PC12 cells was performed by alkaline comet assay as previously described [34]. Images were captured by a fluorescence microscope, and the degree of DNA damage was evaluated by using the CASP image analysis software randomly, according to the tail area, tail length, tail DNA, percentage of tail DNA, tail moment, and olive tail moment of cells.

2.13. Ca²⁺ concentration assay

The concentration of intracellular Ca²⁺ in the PC12 cells was measured by F03 staining according to the manufacturer's instruction (HR0612, Beijing Baiao Laibo Technology, China). The F03 free ligand is nearly non-fluorescent, but upon binding with intracellular calcium ions, it substantially increases fluorescence intensity and emits strong green fluorescence when excited. In our experiment, cells were seeded at a density of 1 × 10⁵ per well in 24-well plates and treated with drug for 24 h, followed by washed thrice with PBS and incubated with the F03 working solutions for 40 min at 37 °C in the dark. After that, removed the solution and incubated with HBSS for 20 min at 37 °C. The images were captured with a confocal laser-scanning microscope, and the fluorescence intensity was calculated to evaluate the content of intracellular Ca²⁺.

2.14. Mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (MPTP) assay

MMP assay kit and MPTP assay kit were used to determine the structural integrity and function of mitochondria. All procedures were conducted according to the manufacturer's instructions, respectively. (C2001S, C2009S, Beyotime Biotechnology, China). For the MMP assay, cells in each group were washed twice with HBSS and then incubated with the tetramethylrhodamine ethyl ester (TMRE) working solutions for 35 min at 37 °C. After that, removed the solution and incubated with DAPI for 7 min at 37 °C, followed by photographed the images. Furthermore, for MPTP detection, cells were incubated with the Calcein AM working solutions for 40 min at 37 °C, followed by incubated with medium for 30 min at 37 °C in the dark. After washed trice with PBS, added detection buffer and captured the images with a fluorescence microscope, the fluorescence intensity was analyzed using the Image J software.

2.15. Mitochondrial ROS detection

The content of mitochondrial ROS was evaluated by a MitoSOX Red Mitochondrial Superoxide Indicator (M36008, Thermo Fisher, China). After intervention with drugs, the cells were reacted with a 5 μM MitoSOX working solution at 37 °C for 30 min in the dark, and then washed with PBS 3 times, subsequently incubated with DAPI for 10 min. Fluorescence intensity was measured under a fluorescence microscope and then calculated using Image J software.

2.16. Immunofluorescence staining

For immunofluorescence staining, the brain tissue sections were dried at 70 °C for 2 h, followed by deparaffinization and rehydration. The brain slices were then immersed in boiled citrate buffer for 10 min for antigen retrieval. After the temperature returned to room temperature, the sections were blocked with goat serum for 1 h at 37 °C and then co-incubated with Nrf2 antibody (1:200, 80593-1-RR, Proteintech) and NeuN antibody (1:200, 66836-1-1g, Proteintech), or co-incubated with Cyt-C antibody (1:200, 10993-1-AP, Proteintech) and NeuN antibody (1:200, 66836-1-1g, Proteintech) overnight at 4 °C. Subsequently, the sections were washed with PBS, followed by incubated with Goat Anti-Rabbit fluorescence-conjugated secondary antibody (TRITC, 1:50, SA00007-2, Proteintech) and Goat Anti-mouse fluorescence-conjugated secondary antibody (FITC, 1:50, RGAM002, Proteintech) for 1 h at 37 °C. Next, the slides were washed with PBS and covered with DAPI for fluorescence observation. The Cyt-C/NeuN positive staining of images was calculated using Image J software. Moreover, at 24 h of OGD injury in PC12 cells, cells were fixed with 4 % paraformaldehyde and then incubated with 5 % BSA, followed by AIF antibody (1:50, 17984-1-AP, Proteintech) and Nrf2 antibody (1:200, Ab137550, Abcam) overnight at 4 °C. The cells were then incubated with proper species-corresponding secondary antibodies, followed by covered with DAPI at room temperature. Finally, the immune-positive cells in different fields of each group were captured with a confocal fluorescence microscope (Olympus FV1000, Japan).

2.17. Electrophoretic mobility shift assay (EMSA)

The nuclear protein was prepared using the nuclear and cytoplasmic extraction kits as per the manufacturers' instructions. The Nrf2 anti-oxidant response element (ARE) sequence was 5′- TCTAGAGTCACAGTGACTTGGCAAAATCTGA-3′ and 5′- TCAGATTTTGCCAAGTCACTGTGACTCTAGA-3′, and it was end-labeled with biotin. After the binding reaction was performed at room temperature for 30 min, the DNA–protein complex was separated by 6.5 % polyacrylamide gel electrophoresis in 0.5 × TBE buffer at 150 V for 60 min, followed by transferred onto a membrane at 300 mA for 30 min and then crosslinked under the X-ray for 20 min. Finally, Nrf2 antibody diluent was added to the binding reaction for 30 min, and subsequently, the binding signals were visualized using ECL reagents.

2.18. Co-immunoprecipitation (Co-IP)

The cells were collected and washed trice with PBS and then lysed by immunoprecipitation (IP) lysis buffer for 25 min on the ice, followed by detecting the protein concentration using the BCA protein assay method. A sufficient Keap1 antibody or IgG negative control was added to the protein supernatant and then incubated overnight at 4 °C, and subsequently, incubated with 80 μL of protein A/G agarose beads for 2 h at 4 °C. The immunoprecipitation mixture was washed using IP lysis buffer for four times and then collected by centrifuging at 2000 g for 5 min at 4 °C to obtain the supernatant, followed by Western blotting analysis.

2.19. Western blotting analysis

Brain tissues or cell samples were collected and homogenized in 1:10 (w/v) cooled lysis buffer, followed by centrifuging at 12,000 g for 5 min at 4 °C, and then obtained the supernatant. Protein concentrations were quantified using the BCA protein assay kit (P0010, Beyotime Biotechnology, China). Approximately 40 μg of protein sample was separated by 10 % or 12 % SDS-PAGE electrophoresis gel and transferred onto a polyvinylidene fluoride (PVDF) membrane at 200 mA for 2 h. The membranes were then blocked with 5 % skim milk powder for 2 h at room temperature, followed by incubation with primary antibodies at 4 °C overnight. The antibodies used were as follows: Nrf2 antibody (1:1000, 80593-1-RR, Proteintech), HO-1 antibody (1:1000, 10701-1-AP, Proteintech), NQO1 antibody (1:800, Ab28947, Abcam), Bax antibody (1:2000, 50599-2-Ig, Proteintech), Bcl-2 antibody (1:1000, 68103-1-Ig, Proteintech), cleaved-caspase-3 antibody (1:500, 25128-1-AP, Proteintech), AIF antibody (1:1000, 17984-1-AP, Proteintech), Cyt-C antibody (1:2000, 10993-1-AP, Proteintech), NRF1 antibody (1:1000, 12482-1-AP, Proteintech), PGC1α antibody (1:500, AF5395, Proteintech), TFAM antibody (1:500, 22586-1-AP, Proteintech), MFN2 antibody (1:1000, 12186-1-AP, Proteintech), FIS1 antibody (1:1000, 10956-1-AP, Proteintech), OPA1 antibody (1:1000, 27733-1-AP, Proteintech), Drp1 antibody (1:1000, DF7037, Proteintech), PINK1 antibody (1:1000, 23274-1-AP, Proteintech), Parkin antibody (1:1000, HY-P80779, MedChemExpress), LC3 antibody (1:1000, 14600-1-AP, Proteintech), P62 antibody (1:2000, 18420-1-AP, Proteintech), GAPDH antibody (1:5000, 10494-1-AP, Proteintech), β-actin antibody (1:5000, 20536-1-AP, Proteintech). After washing thrice with PBST, the membranes were incubated with secondary antibody (1:5000, SA00001-2, Proteintech) at room temperature for 2 h. The protein bands were visualized using ECL reagents, and the signal intensities were analyzed by Quantity One software (Bio-Rad, USA).

2.20. Molecular docking

The crystal structure of the Keap1 protein (PDB ID: 8XGK) was obtained from RCSB Protein Data Bank, and the 3D chemical structure of ISL was obtained from PubChem. The Keap1 protein was prepared using Schrodinger Maestro 11.5, followed by removing water, adding hydrogen atoms, and optimizing energy. Upon the preparation of the protein structure, a grid model was generated, and the binding site centered on the Kelch domains of Keap1. Subsequently, the conformational ensemble of ISL was docked to the prepared protein structure employing the Glide module with standard precision. Finally, the interactions between ISL and Keap1 were visualized using PyMOL.

2.21. DIA-Based Quantitative Proteomic analysis

DIA-Based Quantitative Proteomic analysis was used to distinguish different proteins in the ischemic side brain tissues of mice after MCAO insult. According to the protocol, brain samples obtained from the ischemic side were homogenized in liquid using lysis buffer, followed by ultrasonication on ice for 30 min. After centrifugation at 12,000×g for 30 min at 4 °C, the supernatants were collected, and the protein concentrations were determined using the Bradford protein assay kit. The desalted peptide samples obtained using enzymatic hydrolysis were dissolved in acetonitrile in 0.1 % formic acid and separated using an EASY-nLC1200 HPLC system. Each sample peptide was scanned using DIA (data-independent acquisition) analysis under the mass spectrometry conditions, equipped with a Thermo Scientific Q Exactive TM HF-X system. The identification and quantification of the proteins were both finished by DIA-NN (v1.8.1). Peptide Spectrum Matches (PSMs), with a credibility of more than 99 %, were identified as PSMs. The primary parameter iRT regression type was set as non-linear regression. All results were filtered by a Q value cutoff 0.01 (with FDR no more than 1.0 %). The protein, with the difference of protein abundance ratio >1.5 times between experimental and control groups and the p-value <0.05 analyzed by T-test between four biological repetitions of each sample, was defined as the differentially expressed proteins. Finally, bioinformatic analysis, including GO and InterPro (IPR) functional analyses, KEGG enrichment, and protein-protein interactions, was conducted to filter out the pivotal differential proteins and related signaling pathways.

2.22. Statistical analysis

Data analysis was performed using GraphPad Prism 8.0 (San Diego, CA, USA). Bioinformatics analysis was performed with R (version 4.2.1) (http://www.R-project.org/). Results were analyzed using Prism 8.0 software (GraphPad, San Diego, CA). Normally distributed data are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) followed by least significant difference (LSD) tests was conducted to assess differences among multiple groups. Neurologic deficit scores are expressed as median with interquartile range and were assessed using the Mann-Whitney U test due to the non-normal distribution of the data. Statistical significance was set at P < 0.05.

3. Results

3.1. ISL treatment alleviated MCAO-induced brain injury, neurological deficit, and histopathological damages

To evaluate the therapeutic potential of ISL on MCAO-triggered brain injury, we first pretreated mice with ISL at a dosage of 5, 10, or 20 mg/kg for 7 days, as described in previous study [32]. Results of the TTC-staining (Fig. 1C and D) revealed that the brain infarct volume in the MCAO group was significantly increased compared to the sham group (P < 0.001). In contrast, the infracted areas showed a notably dose-dependent decrease in the ISL group (P < 0.01, P < 0.001), especially 20 mg/kg ISL reduced the percentage of infarct volume to 19.97 ± 2.31 %, which is equivalent to Nimodipine group (positive control). Thus, we selected 20 mg/kg as the ISL dose for the following experiments. Laser Speckle Contrast Imaging results (Fig. 1E and F) indicated no significant differences in the relative CBF between normal and ischemic side hemispheres of mice. Compared with the sham group, relative reduced CBF in the ischemic region was significantly increased in the MCAO group (P < 0.001), while post-treatment with ISL notably decreased the relative CBF compared with the MCAO group (P < 0.001). At the same time, we observed that the score of neurological deficit in the MCAO group was sensibly increased when compared to the sham group (P < 0.001), whereas ISL administration significantly decreased neurological impairment than that in the MCAO group (P < 0.01) (Fig. 1G). These results revealed that ISL could ameliorate MCAO-induced brain damage in mice.

HE and Nissl staining was performed to assess the histopathological changes in neurons. As shown in Fig. 1H and I, the neurons in the cortex, hippocampal CA1, and hippocampal CA3 regions were abundant and well-arranged, and the staining of the cytoplasm and nucleus was uniform in the sham group. By contrast, in the MCAO group, the number of neurons was remarkably decreased, along with disorder neuronal arrangement, karyopyknosis, and interstitial swelling, even the neuronal structure and Nissl's body disappeared. Compared with the MCAO group, ISL pre-treatment apparently alleviated the conditions of neuronal damage, characterized by a relatively low degree of degeneration and a high integrity of neurons, as well as an increase in neuronal amounts and Nissl's body. Thus, these findings indicated that ISL alleviated the histopathological damage following CIRI.

3.2. ISL improved the cell viability and prevented PC12 cells from OGD-induced DNA damage

Further confirmation of the neuroprotective effect of ISL on CIRI was conducted in vitro. Specifically, the cell viability, Na⁺-K⁺-ATPase activity, MnSOD activity, and the integrity of DNA were observed at 24 h after OGD injury in the PC12 cells. In the MTT assay (Fig. 2A), the viability of PC12 cells was significantly decreased in the OGD group than that in the control group (P < 0.001), ISL administration dose-dependently enhanced cell viability than that of the OGD group, particularly 6 μM ISL treatment increased cell viability by approximately 25 % (P < 0.001). Similarly, the results of the Na⁺-K⁺-ATPase and MnSOD activities (Fig. 2B and C) were significantly increased by ISL compared to the OGD group (P < 0.001, P < 0.001), which is consistent with that of cell viability. In addition, we further detected the DNA damage in PC12 cells, which is a key indicator of cellular apoptosis. Comet assay results (Fig. 2D–J) revealed that the cellular DNA was intact, with a clear structure in comet images, and the comet tail was almost undetectable in the control group. However, apparent DNA damage was observed in the OGD group, evidenced by a high ratio of comet tail area and tail DNA, longer tail length, and increased tail moment and olive tail moment. Compared with the OGD group, treated with ISL exhibited significant amelioration of DNA damage, as demonstrated by a lower concentration of tail DNA (P < 0.001) and reduction in tail area (P < 0.001), tail length (P < 0.001), tail moment (P < 0.001), and olive tail moment (P < 0.001). These results revealed that ISL exhibited neuroprotection on the OGD-induced PC12 cell damage, which was in accordance with our in vivo experiment consequence.

Fig. 2.

ISL prevented OGD-induced cell death and the consumption of antioxidase activities and alleviated DNA damage in PC12 cells. (A) Cell viability assay (n = 6). (B) Na⁺-K⁺-ATPase activity assay (n = 3). (C) MnSOD activity assay (n = 3). (D) Representative fluorescence images of comet assay in evaluating the degree of DNA damage, with a magnification of 400 × , scale bar = 20 μm. (E–J) Quantitative analysis of comet tail area, tail DNA, tail DNA (%), tail length, tail moment, and olive tail moment, respectively (n = 10). All values are expressed as the mean ± SD. ^###P < 0.001 versus control group; ∗∗P < 0.01, ∗∗∗P < 0.001 versus OGD group.

3.3. ISL regulated redox homeostasis and mitochondrial function

To elucidate the underlying mechanism of ISL in neuroprotection, we exploited proteomic analysis to analyze the differentially expressed proteins between the sham, MCAO and ISL treatment group in mice. As shown in Fig. 3A, a total of 770 proteins were significantly changed in the MCAO group compared with the sham group, of which 161 proteins were upregulated and 609 were down-regulated. Totally 218 differential proteins between the MCAO and the ISL group were identified, including 112 upregulated proteins and 106 downregulated proteins after ISL pre-treatment at the concentration of 20 mg/kg. Meanwhile, 90 overlapping differentially expressed proteins exist between the sham/MCAO group and the MCAO/ISL group. KEGG cluster analysis results showed that mitochondrial biogenesis and oxidative phosphorylation pathway was enriched in both sham/MCAO group and MCAO/ISL group, as illustrated in Fig. 3B. Specifically, the top 50 differential proteins between the MCAO and ISL group were displayed in Fig. 3C. According to the GO enrichment analysis and KEGG pathway analysis (Fig. 3D and E), we found mitochondrial biogenesis pathway, oxidative phosphorylation pathway and reactive oxygen species pathway were enriched in the ISL treatment group. The protein expression levels were significantly upregulated after ISL treatment, including Mapk10, Snx25, Timmdc1, Crtc1, Mtch1, Tomm6, and Atp5mpl, which is closely related to improving mitochondrial biogenesis and promoting oxidative phosphorylation pathway. In addition, ISL decreased the protein levels of Hdac3, Cryab, Cyb5r4, and Nab2, which are involved in the production of reactive oxygen species, as marked in Fig. 3F. Thus, we deduced that ISL might improve the cellular redox homeostasis via decreasing oxidative stress and improving the mitochondrial function.

Fig. 3.

DIA-Based quantitative proteomic analysis results (n = 4). (A) Statistical chart and Venn diagram of different protein changes in the sham vs. MCAO and MCAO vs. ISL groups. (B) Cluster analysis of the differential expressed proteins in the sham vs. MCAO and MCAO vs. ISL groups. (C) Heatmap of the top 50 differential expressed proteins in the MCAO and ISL group. (D) GO enrichment of those commonly selected differential proteins in the MCAO and ISL group, including the molecular function (blue), cellular component (orange), and biological process (green). (E) Bubble diagram of KEGG pathway analysis. (F) Volcano map of the differential expressed proteins in the MCAO and ISL group. All data values of |log2(Fold Change)| >0.585, Q-value <0.05.

Subsequently, several biomarkers related to oxidative stress and mitochondrial function were detected to further confirm the protective role of ISL. Ca²⁺ content was evaluated by F03 staining to assess the effects of ISL on PC12 cells, which is a substantial indicator of mitochondrial oxidative stress injury. Results (Fig. 4A) showed that cells in the OGD group had a much higher level of Ca²⁺ fluorescence intensity than that in the control group (P < 0.001), while treatment with ISL for 24 h significantly decreased the Ca²⁺ fluorescence intensity (P < 0.001). Besides, ROS production was visualized by a MitoSOX staining (Fig. 4B). In accordance with our expectation, the MitoSOX relative fluorescence in the OGD group was markedly increased relative to that in the control group (P < 0.001), while ISL administration significantly reduced the red fluorescence intensity (P < 0.001). Meanwhile, the accumulation of ROS and Ca²⁺ in mitochondria are two crucial factors that exacerbate mitochondrial dysfunction. Therefore, the level of MMP and the penetration of MPTP were further conducted to evaluate the mitochondrial function in the present study. TMRE staining results (Fig. 4C) confirmed that the MMP level was significantly decreased in the OGD group than that in the control group. In contrast, treatment with ISL apparently enhanced the MMP fluorescence intensity (P < 0.001). Similarly, the fluorescence tendency of the Calcein AM was consistent with that of MMP, as illustrated in Fig. 4D. In addition, the expression of AIF was also conducted to reflect the mitochondrial integrity and function. Results showed that the immunofluorescence intensity and nuclear translocation phenomenon of AIF were distinctly observed in the OGD group compared to the control group. However, ISL treatment obviously decreased the AIF-immunofluorescence intensity and inhibited the release of AIF from the mitochondria to the nucleus (Fig. 4E). Interestingly, brusatol administration hampered these positive effects of ISL. These findings confirmed that ISL incubation improved redox homeostasis and mitochondrial function following OGD injury in PC12 cells.

Fig. 4.

ISL alleviated the oxidative stress damage and ameliorated the mitochondrial function after OGD injury in PC12 cells. (A) Representative F03 staining images of Ca²⁺ content in PC12 cells and statistical histogram, green fluorescence indicates the intensity of intracellular calcium ion fluorescence. (B) Representative MitoSOX staining images of mitochondrial ROS and statistical histogram, MitoSOX (red) represents the ROS contents in mitochondria, and DAPI (blue) was used to label the nuclei. (C) Representative TMRE staining images of MMP and statistical histogram, and red represents the fluorescence intensity of the TMRE cationic probe. (D) Representative Calcein AM staining images of MPTP and statistical histogram, and green represents the fluorescence intensity of the Calcein AM probe. (E) Representative immunofluorescent-stained images of AIF in the PC12 cells at 24 h after OGD injury, AIF (green) represents the expression of AIF-positive cells, and DAPI (blue) was used to label the nuclei. All the representative images are presented with a magnification of 400 × , scale bar = 20 μm. Data are expressed as the mean ± SD (n = 6). ^###P < 0.001 versus control group; ∗∗∗P < 0.001 versus OGD group; ⁺P < 0.05, ⁺⁺P < 0.01 versus OGD + ISL group.

3.4. ISL alleviated oxidative stress damage by activating Nrf2/HO-1 signaling pathway

Nrf2 is well known as a critical transcription factor sensitive to redox reaction, regulating numerous antioxidant defense gene expressions, such as HO-1 and NQO-1, to protect from oxidative stress injury and mitochondrial dysfunction. To elucidate whether the Nrf2 pathway was involved in the neuroprotective effects of ISL, immunofluorescence and Western blotting analysis were performed to determine the protein expressions related to the Nrf2 pathway in the ischemic brain tissues after MCAO insult. As shown in Fig. 5A–C, only a few Nrf2 immuno-positive neurons were observed in the cortex, hippocampus CA1, and hippocampus CA3 regions in the sham group. In contrast, the immunofluorescence intensity of Nrf2 was enhanced in the MCAO group compared to the sham group, and a partial nuclear translocation of Nrf2 was also observed. Inturingly, ISL treatment further increased the Nrf2 immuno-positive neurons and promoted the translocation of Nrf2 from the cytoplasm to the nucleus, especially in hippocampus CA1 and CA3 regions. Nevertheless, the brusatol administration partly reversed this favorable effect of ISL in promoting Nrf2 nuclear translocation. In addition, Western blot analysis was performed to evaluate the expression of Nrf2 and the downstream antioxidant enzymes, including HO-1 and NQO-1. Results (Fig. 5D–F) showed that ISL treatment significantly increased the Nrf2 expression compared with the MCAO group (P < 0.001). Meanwhile, we further observed that ISL obviously lifted the protein expressions of these Nrf2-target genes, as illustrated by the up-regulation of HO-1(P < 0.001) and NQO1 (P < 0.001). In contrast, the positive effects of ISL were weakened by brusatol. These results suggested that promoting the activation of the Nrf2/HO-1 signaling pathway is crucial for ISL exerts its neuroprotective effect after CIRI in mice.

Fig. 5.

ISL treatment promoted the nuclear translocation of Nrf2 and the downstream antioxidant enzyme protein expressions at 24 h following ischemic insult in mice. Representative fluorescence images showed the expression of Nrf2 immuno-positive cells and nuclear translocation in the ischemic cerebral cortex (A), hippocampus CA1 (B), and hippocampus CA3 (C) regions after CIRI, with a magnification of 400 × , scale bar = 20 μm. Nrf2 (green) represents the expression of Nrf2-positive cells, NeuN (red) was used to label the neuron, and DAPI (blue) was used to label the nuclei. Representative Western blotting bands and quantification results of Nrf2 (D), HO-1 (E), and NQO1 (F) protein expression in ischemic brain tissues. Data are presented as the mean ± SD (n = 6). ^#P < 0.05, ^###P < 0.001 versus sham group; ∗∗∗P < 0.001 versus MCAO group; ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001 versus MCAO + ISL group.

3.5. ISL restored the mitochondrial function by ameliorating mitochondrial biogenesis, mitochondrial fusion-fission balance, and mitophagy

Mitochondrial biogenesis and mitochondrial dynamics play an essential role in maintaining the number of mitochondria and regulating cellular bioenergetics. As reported, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1 (Nrf1), and mitochondrial transcription factor A (TFAM) widely participated in the process of mitochondrial biogenesis. Western blot analysis (Fig. 6A–C) displayed that the protein levels of PGC-1α, Nrf1, and TFAM were markedly reduced in the MCAO group compared to that in the sham group (P < 0.01, P < 0.001, P < 0.001). Interestingly, ISL treatment significantly raised the PGC-1α, Nrf1, and TFAM protein expressions relative to the MCAO model group (P < 0.001, P < 0.001, P < 0.01). Therefore, ISL administration significantly promoted mitochondrial biogenesis following ischemic brain injury in mice. Besides, our further investigation found that ischemic insult disrupted normal mitochondrial fission/fusion balance. Results (Fig. 6D and E) showed the key regulatory proteins of mitochondrial fission, dynamin-related protein 1 (Drp1) and mitochondrial fission protein 1 (Fis1), was significantly up-regulated in the MCAO group than that in the sham group (P < 0.001, P < 0.001), while ISL treatment dramatically decreased the protein expressions of Drp1 and Fis1 (P < 0.001, P < 0.001). Moreover, the levels of mitochondrial fusion protein optic atrophy 1 (OPA1) and mitofusin-2 (MFN2) showed a pronounced decrease in the MCAO group (P < 0.001, P < 0.001), whereas ISL intervention evidently improved the protein levels of both OPA1 and MFN2 at the concentration of 20 mg/kg (P < 0.001, P < 0.01) (Fig. 6F and G). To investigate whether mitophagy is involved in the neuroprotective effect of ISL, we next detected the protein expressions of mitophagy-related protein. As presented in Fig. 6H–K, the expression of mitophagy-related protein PTEN-induced putative kinase 1 (PINK1) was slightly increased but didn't reach statistical significance in the MCAO group, while the expression of p62 was markedly downregulated (P < 0.01). Meanwhile, substantial Parkin and LC3 were accumulated in the MCAO group compared to the sham group (P < 0.05, P < 0.001). Interestingly, treatment with ISL further elevated the protein levels of PINK1 (P < 0.01), Parkin (P < 0.001), and LC3 Ⅱ/LC3 Ⅰ ratio (P < 0.01) while decreasing the expression of p62 than that in the MCAO group (P < 0.01). Nevertheless, the regulating effects of ISL were partly blocked by brusatol. Overall, cerebral ischemia triggered unbalanced mitochondrial fission/fusion and abnormal biogenesis process, while ISL treatment significantly improved the mitochondrial dynamics, mitochondrial biogenesis and mitophagy in mice.

Fig. 6.

ISL improved the mitochondrial biogenesis, mitochondrial fusion-fission balance, and mitophagy. Representative Western blotting bands and quantitative analyses depicted the PGC1α (A), Nrf1 (B), TFAM (C), Drp1 (D), Fis1 (E), OPA1 (F), MFN2 (G), PINK1 (H), Parkin (I), p62 (J), LC3 (K) protein expression. Data are presented as the mean ± SD (n = 6). ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus sham group; ∗∗P < 0.01, ∗∗∗P < 0.001 versus MCAO group; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001 versus MCAO + ISL group.

3.6. ISL promoted the dissociation of Keap1/Nrf2 and the binding of Nrf2 with ARE

Since our results demonstrated that the neuroprotective effects of ISL involved the Nrf2/HO-1 signaling pathway in vivo, we next explored the molecular mechanism of ISL in PC12 cells at 24 h following OGD injury. As shown in Fig. 7A, Co-IP assay results showed that the level of Keap1/Nrf2 complex in the ISL group was observably decreased compared with the OGD group, suggesting that ISL accelerated the dissociation of Nrf2 from the Keap1/Nrf2 heterodimer. Similar results of immunofluorescence staining demonstrated that the number and fluorescence intensity of Nrf2-positive cells were relatively low in the OGD group. In contrast, ISL treatment significantly increased the amounts of Nrf2-positive cells and promoted the Nrf2 translocate to the nucleus from the cytoplasm (Fig. 7B). Meanwhile, Western blot analysis (Fig. 7C and D) confirmed that the expression of Nrf2 in the cytoplasm have no significant differences in each group (P > 0.05), while the level of nuclear-Nrf2 was remarkably increased in the ISL group than in the OGD group (P < 0.01). In addition, the binding activity of Nrf2 and ARE was confirmed by EMSA, as shown in Fig. 7E. The binding activity of Nrf2 with ARE in the OGD group was inferior, whereas ISL treatment increased the binding affinity between Nrf2-ARE, as illustrated by the increased shift band gray value. In contrast, brusatol administration evidently hampered this positive effect of ISL. These findings supported our hypothesis that ISL facilitated the dissociation of Keap1/Nrf2 and enhanced the translocation of Nrf2 to the nucleus, meanwhile promoting the binding activity of Nrf2 with ARE.

Fig. 7.

ISL promoted the dissociation of Keap1/Nrf2 and the binding of Nrf2 with ARE in PC12 cells. (A) Co-IP assay showing the formation of Keap1/Nrf2 complex in each group. (B) Representative immunofluorescent-stained images of Nrf2 in PC12 cells following OGD injury at a magnification of 400 × , scale bar = 20 μm. Nrf2 (red) represents the expression of Nrf2-positive cells, and DAPI (blue) was used to label the nuclei. Western blot analysis and quantification of Nrf2 in cytoplasmic cell lysates (C) and nuclear (D) after ISL administration. (E) An EMSA image of the binding activity between Nrf2 and ARE. (F) The binding modes of ISL with residue in Kelch domain in the binding site of Keap1 (PDB:8XGK) and 2D interaction diagram. Data are presented as the mean ± SD (n = 6). ^#P < 0.05 versus control group; ∗∗P < 0.01 versus OGD group; ⁺⁺P < 0.01 versus OGD + ISL group.

In an effort to illustrate the binding mode between ISL and Keap1 protein, molecular docking was carried out. In our docking model, we examined the interaction of ISL with the Kelch domain of Keap1 (PDB:8XGK). As shown in Fig. 7F, the hydroxyl groups of ISL form hydrogen bonds with residues Asn382, Asn414, Ser602, and Tyr575 within the pocket of the Keap1-Kelch domain, with a docking score of −6.193 kJ/mol. These interaction modes may demonstrate the solid binding affinity between ISL and the Kelch domain of Keap1, which accounts for the destabilization of the Keap1 dimer and causes the release of Nrf2 from the Keap1/Nrf2 complex.

3.7. ISL inhibited neuronal apoptosis via activating the Keap1/Nrf2 pathway and improving mitochondrial function in mice

The Keap1/Nrf2 antioxidant pathway is an important signaling pathway in regulating cell growth and apoptosis in the process of IS. In our experiment, the potential anti-apoptotic activities of ISL were conducted in vivo. As shown in Fig. 8A–F, rarely TUNEL-positive cells in the cortex, hippocampus CA1, and hippocampus CA3 regions were observed in the sham group, whereas the number of TUNEL/NeuN-positive cells in the MCAO group significantly increased (P < 0.001, P < 0.001, P < 0.001). Interestingly, treatment with ISL substantially reduced the apoptotic cells compared with the MCAO group (P < 0.001, P < 0.001, P < 0.001). Moreover, immunofluorescence staining (Fig. 8G and H) revealed that the fluorescence intensity of Cyt-C was higher in the MCAO group than that in the sham group (P < 0.001), whereas ISL administration markedly decreased the Cyt-C immuno-positive levels compared with the MCAO group (P < 0.001). Finally, the levels of apoptosis-related proteins in the ischemic brain tissues were determined by immunoblotting assay. As illustrated in Fig. 8I–N, the protein expressions of Cyt-C, AIF, Bax, and cleaved caspase-3 in the MCAO group were significantly higher than those in the sham group (P < 0.001, P < 0.001, P < 0.001, P < 0.001), but Bcl-2 level was apparently downregulated (P < 0.001). Compared with the MCAO group, ISL evidently inhibited the pro-apoptotic protein levels of Cyt-C, AIF, Bax, and cleaved caspase-3 (P < 0.001, P < 0.001, P < 0.001, P < 0.001), at the same time, decreased the ratio of Bax/Bcl-2 (P < 0.001). In contrast, all the above favorable effects of ISL were obviously weakened by brusatol intervention. Together, these findings indicated that ISL remarkably suppressed MCAO-induced neuronal apoptosis, partially due to the activation of the Keap1/Nrf2 pathway and ameliorating mitochondrial function in mice (see Fig. 9).

Fig. 8.

ISL inhibited neuronal apoptosis via activating the Keap1/Nrf2 pathway and improving mitochondrial function in mice. Representative images of TUNEL/NeuN double staining in the ischemic cerebral cortex (A), hippocampus CA1 (B), and hippocampus CA3 regions (C) at a magnification of 400 × , scale bar = 20 μm. TUNEL (green) was used to label dying cells, NeuN (red) was used to label the neuron, and DAPI (blue) was used to label the nuclei. Quantification of TUNEL/NeuN-positive cells in the ischemic cerebral cortex (D), hippocampus CA1 (E), and hippocampus CA3 regions (F) (n = 4). Representative fluorescence photographs (G) of the Cyt-C and the quantitative analysis (H) of Cyt-C fluorescence intensity (n = 6). Representative Western blotting bands and quantification results of Cyt-C (I), AIF (J), Bax (K), Bcl-2 (L), cleaved-caspase-3 (M) and Bax/Bcl-2 (N) protein expression in the ischemic brain tissues (n = 6). Data are presented as the mean ± SD. ^###P < 0.001 versus sham group; ∗∗∗P < 0.001 versus MCAO group; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001 versus MCAO + ISL group.

Fig. 9.

Schematic diagram of the underlying mechanism by which ISL exhibits a neuroprotective effect following CIRI. Ischemia-reperfusion induces the accumulation of Ca²⁺ and excessive generation of ROS, leading to mitochondrial structural disruption and dysfunction, an imbalance of mitochondrial fission/fusion, and cascade apoptosis reactions. ISL intervention promotes Nrf2 nuclear translocation and antioxidative gene expression, as well as enhances mitochondrial biogenesis, mitochondrial fusion-fission balance, and mitophagy. Ultimately, this ameliorates mitochondrial function and inhibits cellular apoptosis after cerebral ischemic insult.

4. Discussion

Cerebral ischemia is a severe and life-threatening disease of the central nervous system (CNS) characterized by inadequate blood supply to brain tissue. The pathogenesis of this condition has not yet been fully elucidated, and there are currently very limited treatment options available in clinical settings. ISL is a flavonoid compound isolated from the plant of Glycyrrhiza uralensis, which possesses anti-inflammatory, antioxidant, anti-apoptotic, and mitochondrial protective activities. It has been reported to exert potential neuroprotective effects on several CNS diseases, such as subarachnoid hemorrhage, ischemic brain injury, Alzheimer's disease, and Parkinson's disease [32,35]. However, the underlying mechanism of ISL exhibiting neuroprotection on cerebral ischemic injury remains unclear. Nevertheless, the precise mechanism through which ISL confers neuroprotection against cerebral ischemic injury remains elusive. In an effort to further elucidate the neuroprotective attributes of ISL and unravel its potential molecular mechanisms, an investigation was conducted.

Our present study provides substantial evidence that ISL exerts a neuroprotective effect on ischemic brain injury by ameliorating mitochondrial dysfunction and inhibiting neuronal apoptosis via the activation of the Nrf2 signaling pathway. The present findings indicated that ISL treatment alleviated the MCAO-induced neurological deficit and brain injury, accompanied by an increase in CBF and a reduction in brain infarct volume and histopathological damages. Secondly, the decrease in cell viability, Na⁺-K⁺-ATPase, and MnSOD activities, as well as the degree of DNA damage induced by OGD injury in PC12 cells, were effectively remedied by ISL treatment. Meanwhile, administration of ISL significantly improved redox homeostasis and restored mitochondrial function via inhibiting oxidative stress injury and ameliorating mitochondrial biogenesis, mitochondrial fusion-fission balance, and mitophagy. In addition, ISL facilitated the dissociation of Keap1/Nrf2 and enhanced the translocation of Nrf2 to the nucleus, meanwhile promoting the binding activity of Nrf2 with ARE. Finally, experiment evidence showed that treatment with ISL obviously inhibited neuronal apoptosis by activating the Nrf2 pathway and ameliorating mitochondrial dysfunction in mice. Nevertheless, inhibition of the Nrf2 pathway by using brusatol reversed the mitochondrial protective activities and anti-apoptotic effects of ISL following ischemic insult.

An appropriate experiment model is valuable for determining the underlying pathophysiological mechanisms and the therapeutic interventions of cerebral ischemic injury. The MCAO model is commonly selected in CIRI studies, which causes moderate brain damage in experiment animals and is characterized by reliable and well-reproducible brain infarct volume. It is one of the models that most closely resembles human IS and is widely used in the research of neuroprotective agents [36]. In our experiment, we first evaluated cerebral infarction in mice via TTC staining, which is generally considered a gold indicator for assessing the effectiveness of candidates on cerebral ischemic injury [37]. Results revealed that the pale infarct areas in the MCAO group are stabilized at approximately 40 %, indicating that the MCAO model was successfully established in mice. Meanwhile, ISL treatment dose-dependently decreased the percentage of cerebral infarct volume compared to that in the MCAO group, especially at the dose of 20 mg/kg has an optimum effect. Besides, numerous studies have reported that the reduction of cerebral blood flow and neurological impairment is the typical characteristic of ischemic brain injury in rodents [[38], [39], [40]]. As expected, the CBF of the ischemic region was rapidly decreased after MCAO operation in mice, while pre-treatment with ISL markedly improved the blood flow in the cerebral cortex, suggesting that ISL can promote blood supply after CIRI. Also, the behavior assay demonstrated that ISL significantly ameliorated the neurological deficits in mice, as evidenced by the reduction of neurological deficit scores in the ISL group to that in the MCAO group. Consistent with the above results, HE and Nissl staining images displayed that the histopathological changes such as loss of neurons and Nissl bodies, disordered neuronal arrangement, karyopyknosis, and cellular vacuolation induced by CIRI insult in the cortex, hippocampus CA1, and CA3 regions were apparently relieved after ISL treatment at 20 mg/kg, further verified the neuroprotective effects of ISL on ischemic brain injury in mice.

In order to further validate the neuroprotective effect of ISL, PC12 cells were chosen in our in vitro experiment in terms of similar morphology, function, and phenotype with neurons. CCK8 assay showed that the cell viability has a notable decrease following OGD injury, indicating that the in vitro model was successfully established. As expected, ISL administration was beneficial for cellular survival rate, especially at the concentration of 6 μM. During the period of CIRI, inadequate supplies of oxygen and nutrients disrupted the energy balance and were mainly responsible for the decrease of Na⁺-K⁺-ATPase activity, which is a critical factor for maintaining neurons at normal conditions [41,42]. As an innate oxygen sensor, the Na⁺-K⁺-ATPase is highly sensitive to cerebral ischemia-hypoxia, and it has been observed to undergo rapid degradation due to the presence of mitochondria-generated ROS and the ubiquitin-conjugating system following hypoxic insult [43,44]. Thus, restoration of the activity of Na⁺-K⁺-ATPase in neurons has been considered as a potential strategy to relieve ischemic brain injury. Besides, as an essential antioxidant enzyme, MnSOD was reported to remove excessive reactive oxygen free radicals in mitochondria and alleviate oxidative stress damage in cells [45]. In the present study, incubation with ISL significantly improved the Na⁺-K⁺-ATPase and MnSOD activities in PC12 cells following OGD injury, which demonstrated the favorable antioxidant properties of ISL. Meanwhile, growing evidence has proposed that DNA fragmentation is a pivotal hallmark of cellular apoptosis and death, which was also involved in the process of ischemic brain injury [46,47]. Thus, detecting the percentage of DNA breaks was considered as a suitable approach in evaluating the degree of neuronal injury. Comet assay was conducted in the present study due to its high sensitivity and quantification in detecting DNA damage [34]. The findings revealed that OGD insult triggered a remarkable elevation in tail DNA, tail area, tail length, tail moment, and olive tail moment of the comet in PC12 cells, which are typical features of DNA damage. Interestingly, ISL administration restrained the extent of DNA fragmentation. Together, these findings further confirmed the neuroprotective effect of ISL in vitro.

To elucidate the underlying mechanism of ISL-induced neuroprotection, a proteomic analysis was conducted to examine the differential expression of proteins following CIRI in mice. Results of GO enrichment and KEGG pathway analysis hinted that the neuroprotective effect of ISL is closely involved in regulating redox homeostasis and mitochondrial biogenesis in mice. Thus, we evaluated the oxidative stress levels and mitochondrial function in the PC12 cells. As a major organelle in the body, the mitochondria act as a “cellular power center” and an intracellular calcium reservoir, which can maintain the dynamic balance of intracellular calcium ions and widely participate in the regulation of neuronal survival and apoptosis [48,49]. Increasing evidence revealed that cerebral ischemic injury is a common reason for intracellular calcium overload, which can cause a disruption in the mitochondrial electron transport chain and the generation of ROS, subsequently leading to redox homeostasis out of balance and exacerbating cellular oxidative damage [[50], [51], [52]]. Therefore, the abnormal accumulation of Ca²⁺ and the sharp outbreak of ROS in the mitochondria are typical features of mitochondrial oxidative damage. In the present study, ISL treatment significantly decreased the content of intracellular Ca²⁺ and excessive mitochondrial ROS levels than that in the OGD model group, indicating that ISL had a strong anti-oxidate potential to protect PC12 cells from OGD injury. Furthermore, the sharp reduction in oxygen and glucose contents in the neurons principally accounts for the mitochondrial impairment following OGD insult. In this study, the MMP level and MPTP permeability in PC12 cells were assessed after OGD injury at 24 h, which was considered a crucial indicator in evaluating mitochondrial integrity and function [53,54]. TMRE staining and Calcein AM fluorescence staining results showed that ISL treatment significantly prevented the loss of MMP and the excessive opening of MPTP induced by OGD intervention, which further demonstrated that the neuroprotective effects of ISL were closely related to the improvement of mitochondrial function.

The transcription factor Nrf2 has emerged as an essential modulator in regulating mitochondrial function and cellular redox homeostasis by inducing the expression of detoxifying and antioxidant enzyme genes, which widely participated in the process of IS [55,56]. Under the conditions of oxidative stress, the endogenous antioxidant factor Nrf2 dissociates from Keap1 and translocates into the nucleus from cytoplasm, subsequently binds with the ARE promoter regions, and then controls the transcription of downstream genes, regulating cellular activities, including the maintenance of redox homeostasis, energy metabolism, and detoxification reactions, among others [20,57]. In our present work, we found that pre-treatment with ISL evidently improved the expression of Nrf2 immuno-positive neurons in the cortex, hippocampus CA1, and hippocampus CA3 regions, at the same time, promoting the translocation of Nrf2 from cytoplasm to the nucleus in the ischemic brain tissues. Similarly, the protein expression level of Nrf2 was significantly raised by ISL treatment, as reflected by Western blot analysis. As Nrf2 target genes, the expression of HO-1 and NQO1 plays a pivotal role in maintaining mitochondrial function and inhibiting oxidative stress injury and cellular apoptosis [58]. Noteworthy, our experiment results showed that ISL markedly enhanced the protein levels of antioxidant enzymes HO-1 and NQO1. Nevertheless, the Nrf2 inhibitor brusatol effectively blocked the favorable effect of ISL, which suggests that the Nrf2 signaling pathway is involved in the anti-oxidative properties of ISL in CIRI therapy.

As indicated in the DIA proteomics analysis result, mitochondrial biogenesis and oxidative stress pathways were enriched in the ISL treatment group. Besides, growing evidence highlights the pivotal role of Nrf2 in promoting energy production and neuroprotection by regulating mitochondrial biogenesis, mitochondrial fusion-fission balance, and mitophagy [19,23]. To further confirm the regulatory effect of ISL on mitochondrial function following CIRI in mice, the critical protein levels related to mitochondrial biogenesis were assessed, including PGC-1α, Nrf1, and TFAM. Importantly, ISL obviously boosted the protein expressions of PGC-1α, Nrf1, and TFAM than that in the MCAO group, which was consistent with the results of proteomics analysis. In addition, recent studies point to the substantial effect of Nrf2 activation in maintaining mitochondrial dynamics. Excessive mitochondrial fragmentation resulting from augmented fission and decreased fusion is often a common event induced by oxidative stress, which has been associated with different disorders, such as IS and Alzheimer's disease [[59], [60], [61]]. As a motor protein, Drp1 emerges as a pivotal regulator of mitochondrial fission, with its dysregulation that can disrupt mitochondrial homeostasis and thereby exacerbate CIRI severity [62]. Subsequently, activated Drp1 will translocate to the outside of the mitochondrial membrane from the cytoplasm, thereby corresponding with Fis1 and further accelerating the fission of mitochondrion [63,64]. Unlike the process of mitochondrial fission, mitochondrial fusion is another process that inhibits the formation of mitochondrial fragmentation and maintains genomic balance within mitochondria, which was mainly regulated by OPA1 and MFN1/2 [65]. In our current study, ISL treatment dramatically decreased the protein expressions of Drp1 and Fis1, while the fusion-related protein OPA1 and MFN2 were evidently up-regulated by ISL. Furthermore, we found that ISL treatment promoted the expression of PINK1 and Parkin, which are the markers of mitophagy. As one of the selective autophagy, mitophagy has been demonstrated to be beneficial in the context of CIRI, as it promotes neuronal survival by eliminating dysfunctional mitochondria and maintaining a healthy mitochondrial network following ischemia-hypoxia [66,67]. On the other hand, a direct correlation exists between Nrf2 and mitophagy, as evidenced by the transcriptional regulation of various autophagic mediators induced by Nrf2 [23]. Activation of Nrf2 improves PINK1 expression via binding to the ARE sequences present in the PINK1 promoter [68]. Besides, the mutual activation is feasible due to the presence of a positive feedback loop between Nrf2 and the autophagy substrate p62 [69]. As one of the target genes of Nrf2, p62 interacts with the Nrf2 binding site of Keap1 to prevent Nrf2 degradation and facilitate its translocation to the nucleus to its target genes [70]. In the present study, we found that ISL treatment lifted the level of PINK1 and Parkin, accompanied by the ratio of LC3 II/LC3 I rose and the accumulation of p62 diminished, which suggested the neuroprotection of ISL on MCAO/R mice is related to the activation of mitophagy mediated by PINK1/Parkin. As expected, this positive regulation effect of ISL was markedly discounted by Nrf2 inhibitor brusatol. Collectively, the existing findings have unveiled that the neuroprotective effects of ISL on CIRI in mice are partially attributed to its enhancement of the equilibrium between mitochondrial fusion and fission, as well as its promotion of mitochondrial biogenesis and mitophagy, which is dependent on the activation of Nrf2 pathway.

The Keap1-Nrf2 interaction is indispensable for activating the ARE-induced antioxidant enzyme system, and also has been considered a critical therapeutic target for IS [25,56,71]. To validate the role of the Nrf2 pathway in mediating the neuroprotective effect exerted by ISL against CIRI in mice, we further investigated the levels of Keap1/Nrf2 complex and the protein expression of Nrf2 in the nucleus, as well as the binding activity of the Nrf2 with ARE in vitro. Co-IP assay results showed that the level of Keap1/Nrf2 complex in the ISL group was observably decreased compared with the OGD group, suggesting that ISL induced the dissociation of Nrf2 from the Keap1/Nrf2 complex. In addition, immunofluorescence and Western blot analysis found that ISL treatment evidently promoted the fluorescence intensity of Nrf2-positive cells in the nucleus and the protein expression of nuclear-Nrf2, which could be partly blocked by brusatol, indicating that ISL effectively promoted nuclear translocation of Nrf2 in PC12 cells following OGD injury. Accordingly, the previous study claimed that the accumulation of Nrf2 in the nucleus can bind with antioxidant response elements, thus regulating the expressions of downstream proteins and exerting biological effects [72]. In the present study, we found ISL treatment promoted the binding activity of Nrf2 with ARE, while brusatol intervention inhibited these positive effects. Considering previous understanding of the Keap1/Nrf2 domain, our docking results exhibited the composite pattern of ISL and Keap1, which further supported the regulating effects of ISL. Hence, our in vitro experimental results further revealed that ISL promoted the release of Nrf2 from the Keap1/Nrf2 dimer and enhanced Nrf2 expression and nuclear translocation, thereby exhibiting neuroprotection against OGD injury in PC12 cells.

It is widely accepted that the maintaining of redox homeostasis and mitochondrial function is a substantial mechanism in regulating cellular survival and apoptosis [73,74]. On the basis of our preliminary experiment results, we further evaluated the anti-apoptotic activity of ISL in mice following MCAO insult. Intriguingly, ISL apparently inhibited MCAO-induced neuronal apoptosis in the cerebral cortex, hippocampus CA1, and CA3 regions, as demonstrated by the decreased amounts of TUNEL/NeuN positive cells. Moreover, growing evidence highlighted that the increased permeability of the mitochondrial outer membrane induced by ischemic brain injury accounts for the release of mitochondrial Cyt-C and AIF, which subsequently activated the downstream caspase cascade reaction and triggered cellular apoptosis [75,76]. Besides, as an anti-apoptotic protein, Bcl-2 has been identified as a “main switch” in regulating the mitochondrial-mediated apoptosis pathway, which can prevent the release of Cyt-C in the cytoplasm and directly mirrors the integrity and function of mitochondrial [77]. In contrast, the accumulation of Bax in the mitochondrial outer membrane may destroy the integrity of the mitochondrial membrane under the stimulation of apoptotic-related signals, thereby triggering the activation of caspase-3 and leading to irreversible cellular apoptosis [78,79]. Therefore, the equilibrium between Bcl-2 and Bax is crucial to regulate cell survival and apoptosis. In the present study, ISL administration dramatically decreased the expression of Cyt-C, AIF, and Bax rather than that in the MCAO group, as well as restricted the proportion of Bax/Bcl-2 and the level of cleaved-caspase-3. Nevertheless, the anti-apoptotic effects exerted by ISL were apparently intercepted in the presence of Nrf2 inhibitor brusatol. Thus, we proposed that ISL treatment effectively inhibited neuronal apoptosis following CIRI insult in mice, which is closely related to the improvement of mitochondrial function induced by the activation of the Nrf2 pathway.

5. Conclusion

In conclusion, our current study has unequivocally demonstrated the neuroprotective effects of ISL on mice suffering from CIRI both in vivo and in vitro. The underlying mechanism is closely linked to its capacity to attenuate oxidative stress damage and ameliorate mitochondrial dysfunction through the activation of the Nrf2 pathway. These findings provided a substantial foundation for ISL as a promising candidate for the clinical treatment of cerebral ischemic injury.

CRediT authorship contribution statement

Xiaobing Lan: Conceptualization, Data curation, Formal analysis, Methodology, Software, Visualization, Writing – original draft, Writing – review & editing. Qing Wang: Conceptualization, Data curation, Investigation, Software, Validation, Writing – original draft. Yue Liu: Conceptualization, Formal analysis, Investigation, Methodology, Supervision. Qing You: Data curation, Investigation, Methodology, Software, Visualization. Wei Wei: Data curation, Formal analysis, Software, Validation. Chunhao Zhu: Data curation, Formal analysis, Software, Visualization. Dongmei Hai: Formal analysis, Investigation, Methodology, Software. Zhenyu Cai: Conceptualization, Funding acquisition, Investigation, Supervision. Jianqiang Yu: Conceptualization, Data curation, Funding acquisition, Investigation, Project administration, Supervision, Writing – review & editing. Jian Zhang: Conceptualization, Data curation, Funding acquisition, Supervision, Writing – review & editing. Ning Liu: Conceptualization, Formal analysis, Investigation, Project administration, Supervision, Visualization, Writing – review & editing.

Data availability

Data will be made available on request.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Abbreviations

AIF

apoptosis inducing factor

ARE

antioxidant response element

CBF

erebral blood flow

CIRI

cerebral ischemia-reperfusion injury

CNS

central nervous system

Co-IP

co-immunoprecipitation

Cyt-C

cytochrome C

DIA

data-independent acquisition

Drp1

dynamin-related protein 1

EBSS

Earle's balanced salt solution

EMSA

electrophoretic mobility shift assay

Fis1

mitochondrial fission protein 1

HO-1

heme oxygenase-1

IS

ischemic stroke

ISL

isoliquiritigenin

Keap1

kelch-like ECH-associated protein 1

MCAO

middle cerebral artery occlusion

Mfn1/2

mitofusin 1/2

MMP

mitochondrial membrane potential

MPTP

mitochondrial permeability transition pore

Nrf1

nuclear respiratory factor 1

Nrf2

nuclear factor erythroid 2-derived factor 2

NQO1

NAD(P)H quinone oxidoreductase 1

OGD

oxygen-glucose deprivation

OPA1

optic atrophy 1

PGC-1α

peroxisome proliferator-activated receptor gamma coactivator-1 alpha

PINK1

PTEN induced putative kinase 1

ROS

reactive oxygen species

SOD

superoxide dismutase

TFAM

mitochondrial transcription factor A

TMRE

tetramethylrhodamine ethyl ester

TTC

2,3,5-triphenyltetrazolium chloride