Animals

Male C57BL/6 J mice (25 ± 2 g, 8–10 weeks old) were obtained from Guangdong Yaokang Biotechnology Co., Ltd (Guangzhou, China). The animal experiments were carried out in accordance with the experimental animal welfare and security system and were approved by the Animal Ethics Committee of Guangdong Medical University (ethical approval number: GDY2002071).

Transient middle cerebral artery occlusion (tMCAO) model and drug treatment

The tMCAO model was performed as previously reported [21]. Mice were anesthetized with an intraperitoneal injection of pentobarbital sodium and toluene thiazide. The right common carotid artery, external carotid artery, and internal carotid artery were exposed and separated, and a threaded plug (~ 2 cm) was then carefully inserted from the external carotid artery through the internal carotid artery, reaching into the middle cerebral artery. After 90 min of ischemia, reperfusion was initiated by carefully removing the plug. Mice were randomly divided into the following groups (n = 6): sham, tMCAO, and tMCAO + ozone (tMCAO + O3). Ozone saline(20 mg/mL, 1.5 mg/kg) was intravenously administered at the onset of reperfusion. The sham and tMCAO groups were injected with an equal volume of normal saline.

O3 preparation

The O3 was produced using ozone treatment apparatus(HUMAZON ProMedic, Germany) and injected into sterile saline immediately after extraction. The O3 concentration in ozone sterile saline was measured using a high-precision medical ozone meter (HUMAZON ProMedic, Germany). The concentration range analyzed ranged from 1 to 56 µg/mL, while the gas flow rate varied between 0 and 1,300 mL/min.

The 2,3,5-triphenyltetrazolium chloride (TTC) staining

The cerebral infarct volume was assessed using TTC staining 24 h after tMCAO. Following anesthesia, the brains of mice were swiftly removed, sliced into 2 mm tissue sections, stained with 2% TTC (Sigma-Aldrich, St. Louis, MO, USA) for 20 min, and then immersed in 4% formaldehyde for 24 h. The infarct area was delineated and analyzed using Image J.

Neurological scoring

A blinded investigator evaluated the neurological deficit (ND) score 24 h after reperfusion. The scoring system was as follows: (1) no ND (0 points); (2) forelimb weakness and torso turning to the ipsilateral side when held by the tail (1 point); (3) circling to the affected side (2 points); (4) inability to bear weight on the affected side (3 points); and (5) no spontaneous locomotor activity or barrel rolling (4 points).

Rotarod test

The mice underwent a 2-day training period prior to modeling. Following 24 h of reperfusion, the mice were placed on a suspended rod and the speed was increased from 4 to 40 rpm over 5 min. The trial concluded when the mice fell off the rotarod or after reaching 300 s. Each mouse was tested three times with a 5-min rest period between each test. The average latency was utilized for subsequent calculations.

Cell counting assay

SH-SY5Y cells (CL-0208, Porcell, Wuhan, China)were cultivated on 96-well plates to perform the cell counting assay. Following treatment with H2O2 and drugs, a fresh medium containing CCK8 (K009, Zeta Life, CA, USA) was added and incubated at 37 °C for 2 h. An enzyme marker was then used to measure the absorbance at 450 nm.

Flow cytometry

For apoptosis detection, SH-SY5Y cells were incubated with AnnexinV-PE (559763, BD Biosciences, New Jersey, USA) for 15 min, and 7AAD was added 5 min before detection. To detect ROS, dichloro-dihydro-fluorescein diacetate (DCFH-DA) (S0033S, Beyotime, Shanghai, China) was diluted in a serum-free medium at a final concentration of 10 µM. The cells were incubated in the cell incubator at 37 °C for 20 min. Subsequently,, they were washed three times in serum-free cell culture. For Ca2+ detection, cells were removed from the medium, washed with Hank's Balanced Salt Solution (HBSS) solution three times, and cultured at 37 °C for 15 min after adding 1 µM Fluo-3 AM probe (40703ES50, Yeasen, Shanghai, China) working solution. Detection was performed using a FACScalibur cytometer (BD Biosciences, New Jersey, USA), and data processing was done using FlowJo 10.8.1. The fluorescence signal was analyzed on at least 10,000 positive events.

Western blotting

In western blotting, an SDS-PAGE gel was utilized for electrophoresis. Once the bands reached the desired position, they were transferred to a membrane and blocked with 5% skim milk. A diluted primary antibody was then added and incubated overnight at 4 °C. On the following day, the bands were incubated with secondary antibodies specific to the species. The primary antibodies used are listed in Table 1.

Table 1 Primary antibodies used in current studyMeasurement of Adenosine triphosphate (ATP) levels

The ATP Assay Kit (S0175, Beyotime, Shanghai, China) was employed to quantify the intracellular ATP levels. Cells were harvested, lysed, and then centrifuged at 4 °C at 12000 g for 5 min, after which the supernatant was collected. Subsequently, 100 µl of ATP working solution was added to the assay well, left at room temperature for 3–5 min, and then measured using a luminometer.

Cellular nicotinamide adenine dinucleotide (NAD+) analysis

The NAD+/NADH Assay Kit with WST-8 (S0175, Beyotime, Shanghai, China) was used to assess cellular NAD+ levels. Cells were harvested and suspended in 200 µl of NAD+/NADH extraction buffer. After centrifugation at 12,000 g for 10 min at 4 ℃, the supernatant was collected. To decompose NAD+, 100 µl of the sample was heated in a 60 ℃ water bath for 30 min, and 20 µl of the supernatant was utilized for testing. Alcohol dehydrogenase working solution was added, and the mixture was incubated in the dark at 37 ℃ for 10 min. Optical density at 565 nm was measured after 0 and 15 min using a spectrophotometer.

Mitochondrial membrane potential (JC-1) assay

The Mitochondrial membrane potential assay kit with JC-1 (C2006, Beyotime, Shanghai, China) was utilized to measure the expression of mitochondrial membrane potential. Cells were incubated with 1 ml of JC-1 staining solution at 37 ℃ for 20 min in the cell incubator. After removing the supernatant, cells were washed twice with JC-1 staining buffer. The mitochondrial membrane potential was observed using a laser confocal microscope (FV10i-DOC, Olympus, Japan).

Superoxide dismutase (SOD) activity detection

SOD activity was assessed using the total SOD Activity Detection Kit (WST-8 Method) (S0101S, Beyotime, Shanghai, China). The WST-8/enzyme working solution was added as per according to the instructions and then incubated at 37 ℃ for 30 min. The absorbance at 450 nm was measured using a microplate reader.

Hematoxylin–Eosin (HE) staining

After dewaxing and dehydrating, paraffin sections were stained with Harris hematoxylin for 3–8 min and then differentiated with 1% hydrochloric acid alcohol for several seconds. The sections were subsequently rinsed with tap water and returned to a blue color using 0.6% ammonia. Following rinsing with running water, the eosin dye solution was applied for 1–3 min. The sections were dehydrated, mounted, and examined under a microscope. Images were captured and analyzed.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay

The neuron apoptosis was detected using the Vazyme Biotech Co., Ltd. (Nanjing, China) kit via TUNEL staining following the provided instructions. Paraffin sections were dewaxed, dehydrated, and subjected to antigen retrieval before adding 50 μl of TUNEL working solution to the sample, which was then incubated in the dark at 37 ℃ for 60 min.

Immunofluorescence

For SH-SY5Y cells seeded on 12-mm glass coverslips, the process involved fixation with 4% paraformaldehyde and permeabilization with 0.1% TritonX-100 were followed by overnight incubation with the specified primary antibody at 4 ℃, after blocking with 5% bovine serum albumin (BSA) for 30 min at room temperature. Following incubation with fluorescent conjugated secondary antibodies and 4',6-diamidino-2-phenylindole (DAPI), the designated primary antibody was applied to paraffin sections. The sections were incubated in a sealing solution containing 0.5% Triton and 5% BSA and left to incubate at room temperature for 2 h. The fluorescence signal was visualized using an Olympus confocal microscope (FV10i-DOC, Olympus, Japan).

Transmission electron microscopy

Mice were systemically infused with 4% paraformaldehyde under deep anesthesia, followed by 5 ml of 2.5% glutaraldehyde. The brains of the mice were then extracted, and the cortex was sliced into 1–2 mm small pieces using a blade. The cortex pieces were immersed in 2.5% glutaraldehyde and fixed overnight. Following dehydration, the tissues were embedded in epon and ultra-thin sections were obtained using a microtome. These sections were placed on copper electron microscopy grids and subsequently stained with uranyl acetate and lead citrate. Micrographs were then captured and collected using a transmission electron microscope (Hitachi H7500 TEM, Tokyo, Japan).

Statistical analysis

Statistical analyses were conducted using GraphPad Prism 9. The Student's t-test was employed to assess differences between two groups. One-way analysis of variance (ANOVA) was utilized to analyze datasets comprising more than two groups with consistent sample sizes, while two-way ANOVA (with Turkey's multiple comparisons test) was used for datasets with varying sample sizes. All graphs depict the mean ± standard deviation (SD). A p-value less than 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).