Rats were purchased from Beijing Huafukang Biotechnology Co., Ltd. and housed at the Animal Experimental Center of North China University of Technology at 20±2˚C with 55±5% humidity, 12-h light/dark cycles, and access to food and water ad libitum. All rats were acclimatized for a week with food and water before experimental manipulation. The present study was approved by the Animal Ethics Committee of North China University of Technology (approval no. LX201901).

A total of 58 adult male Sprague-Dawley rats (age, 50-60 days; weight, 250-280 g) were randomly divided into four groups: i) Control; ii) sham operation, iii) I/R; and iv) drug treatment. The I/R group was further divided into four subgroups: 3, 6, 12 and 24 h post-reperfusion, for sample collection. The drug treatment group was further divided into three subgroups: i) I/R + negative control (NC); ii) I/R + miRNA-124 agonist (agomiRNA-124); iii) and I/R + miRNA-124 antagonist (antagomiRNA-124). Rats were used for the following experiments: 2-3-5 triphenyl tetrazolium chloride (TTC) staining (n=14; sham (n=4), NC (n=3), agomiRNA-124 (n=3) and antagomiRNA-124 groups (n=4)); 30 were used for western blotting and reverse transcription-quantitative PCR (RT-qPCR; n=30; sham (n=4), control (n=4), I/R (n=12) and drug (n=10) groups); and immunohistochemistry (n=14; sham (n=4) and drug (n=10) groups). Rats in the I/R and drug groups were subjected to CIRI, rats in the sham operation group underwent the same surgical operation but without an inserted suture and rats in the control group were not subjected to surgical operation. MCAO was performed in the left hemisphere to induce CIRI as previously described (19,20). Briefly, rats were anesthetized with an intraperitoneal injection of 2% sodium pentobarbital (30 mg/kg) (21). Subsequently, the common carotid artery (CCA) was separated from the nerves and tissue to gently expose the internal carotid artery (ICA) and the external carotid artery (ECA). The ECA was clipped and the ECA stump was stretched to align it with the ICA. A length of 3.0 cm plugging-up suture (cat. no. 3600AAA; Guangzhou Jialing Biological Technology Co., Ltd.) with its rounded tip was inserted to cut off the origin of the middle cerebral artery. Finally, the microvascular clip was removed and the thread was fastened. The length of insertion was 18.5-19.5 mm from the CCA bifurcation. Following ischemia for 2 h, the monofilament was removed to induce CIRI.

At 2 days prior to MCAO establishment, rats received an intracerebroventricular injection of 20 nM miRNA-124 agonist (5 µl; Guangzhou RiboBio Co., Ltd.), 20 nM miRNA-124 antagonist (5 µl; Guangzhou RiboBio Co., Ltd.) or 20 nM miRNA-124 negative control (5 µl; Guangzhou RiboBio Co., Ltd.) for 5 consecutive days. All experiments were performed under anesthetic and the lateral ventricle was located [coordinates from the bregma: Anterior-posterior (AP)=-0.8 mm, lateral (L)=-1.5 mm, ventral (V)=-4.8 mm] based on a stereotaxic atlas. Animals with hind limb paralysis or paresis following surgery were excluded from the study to rule out the effects of anesthetics and operative failures. Three rats died from operation (sham operation group excluded two rats in TTC straining and immunohistochemistry respectively; control group excluded one rat).

Neurological deficits were scored by one investigator after the rats awakened. The scores were evaluated using the 5-point scale described by Longa et al (19) as follows: 0, no neurological deficit; 1, failure to adequately extend the left forepaw; 2, circling to the left; 3, falling to the left; 4, depressed level of consciousness without spontaneous walking; and 5, death. Rats subjected to MCAO without detectable neurological deficits or death were removed from the following experiments to exclude the interference arising from operative failures (Three rats that died were excluded).

Total RNA was extracted from brain tissues using the Total RNA Purification kit (Suzhou GenePharma Co., Ltd.). Total RNA was reverse transcribed into cDNA using an miRNA RT primer and MMLV reverse transcriptase (contained in Hairpin-it™ miRNA RT-PCR Quantitative kit; cat. no. E22007; Suzhou GenePharma Co., Ltd.), at 25˚C for 30 min, 42˚C for 30 min and 85˚C for 5 min, then stored at 4˚C, according to the manufacturer's protocol. Subsequently, qPCR was performed using the QuantStudio 6 Flex Detection system (Thermo Fisher Scientiic, Inc.) and a rno-miRNA-124 probe Hairpin-it™ miRNA and U6 snRNA Normalization RT-PCR Quantitation kit (cat. no. E22007; Suzhou Jima Gene Co., Ltd.), according to the manufacturer's protocol. The following primers were used for qPCR: miRNA-124 forward, 5'-TGTCTCTAAGGCACGCGGT-3' and reverse, 5'-TATGGTTGTTCACGACTCCTTCAC-3'; and miRNA-124 probe, 5'-ATGCC+GTCT+GTATGGTTGGATAGGGA-3', U6 forward, 5'-CGCTTCGGCAGCACATATACTAA-3' and reverse, 5'-TATGGAACGCTTCACGAATTTGC-3'. The following thermocycling conditions were used for qPCR: Initial denaturation at 95˚C for 3 min; followed by 40 cycles of 12 sec at 95˚C and 40 sec at 62˚C; the fluorescence signal was collected after 40 cycles. miRNA expression levels were quantified using the 2^-ΔΔCq method and normalized to the internal reference gene U6(22).

Total protein was extracted from fresh cerebral cortex in the ischemic penumbra. Total protein was quantified using a bicinchoninic acid assay kit (cat. no. PT0001; Beijing Leagene Biotechnology Co., Ltd.). Proteins (30 µg/lane) were separated via a 10% SDS-PAGE, transferred onto PVDF membranes and blocked with 5% BSA for 1 h at room temperature. Subsequently, the membranes were incubated at 4˚C overnight with the following primary antibodies: Anti-gasdermin D (1:500; cat. no. sc-393581; Santa Cruz Biotechnology, Inc.), anti-STAT3 (1:1,000; cat. no. 12640; Cell Signaling Technology, Inc.), phosphorylated (p)-STAT3 (1:1,000; cat. no. 52075; Cell Signaling Technology, Inc.), anti-pro-caspase-1 (1:1,000; cat. no. ab179515; Abcam), anti-caspase-1 p20 (1:1,000; 2225, Cell Signaling Technology, Inc.), anti-IL-1β (1:1,000, cat. no. 12242; Cell Signaling Technology, Inc.), anti-IL-18 (1:1,000; cat. no. ab71495; Abcam) or anti-β-actin (1:2,000; cat. no. T0023; BIOSS). Following primary incubation, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (1:5,000; ZB2307, Zsbio Commerce Store) for 2 h at 37˚C. The membranes were rinsed three times with TBS-0.1% Tween-20 for 10 min. Protein bands were visualized using EXPlus ECL reagent (Beijing Zoman Biotechnology Co., Ltd.) and the expression levels were quantified using ImageJ software (version 1.6.0; National Institutes of Health) with β-actin as the loading control.

The online target gene prediction software TargetScan (http://www.targetscan.org/vert_72/) was used to analyze the potential target genes of miRNA-124 and find the STAT3 is one of the target genes of miRNA-124. Dual-luciferase reporter plasmids containing the wild-type (WT) or mutant (MT) 3'-untranslated region (UTR) of STAT3 (pGL3-Promoter Vector, R0531, Promega Corporation) were constructed. PC12 cells (1x10⁵ cells/ml; Chinese Academy of Sciences Cell Bank) were co-transfected with plasmids (2 µg) and miRNA-124 mimics (50 nM) or miRNA-124 negative control (50 nM) using TurboFect transfection reagent (R0531, Thermo Fisher Scientific). The following cell groups were generated: i) WT plasmid + miRNA-124 negative control group (WT-STAT3 + NC); ii) WT plasmid + miRNA-124 mimic group (WT-STAT3 + miRNA-124); iii) MT plasmid + miRNA-124 negative control group (MT-STAT3 + NC); and iv) MT plasmid + miRNA-124 mimic group (MT-STAT3 + miRNA-124). At 48 h post-transfection, a dual-luciferase reporter assay system kit (cat. no. E1910; Promega Corporation) was used to determine the relative luciferase activity. Relative luciferase activity=Firefly luciferase/Renilla luciferase.

Animals were anesthetized with an intraperitoneal injection of 2% sodium pentobarbital (30 mg/kg) and the ascending aorta was immediately perfused with 4% paraformaldehyde. Subsequently, the brain tissue was fixed in 4% paraformaldehyde for 24 h at 4˚C and the infarct sections were embedded in paraffin and cut into 3 µm slices. Sections were blocked in 10% normal goat serum (cat. no. SP-9000; OriGene Technologies) for 2 h at room temperature and then incubated at 4˚C overnight with the following primary antibodies: Anti-gasdermin D (1:50; cat. no. sc-81868; Santa Cruz Biotechnology, Inc.), anti-caspase-1 (1:100; 2225, Cell Signaling Technology, Inc.), anti-IL-1β (1:100; cat. no. 12242; Cell Signaling Technology, Inc.) and anti-IL-18 (1:100; cat. no. ab52914; Abcam). Following primary incubation, the sections were incubated with Cy3-conjugated and fluorescein isothiocyanate-conjugated secondary antibodies (1:50; cat. no. ZF-0311; OriGene Technologies) for 1 h at room temperature. Stained sections were visualized using a DP-80 fluorescence microscope (Olympus Corporation) in ten randomly selected fields of view and the positive cell rate was calculated as follows: (Positive cells/total cells) x100%.

Brain tissues were obtained following 12 h reperfusion and immediately stored at -20˚C for 30 min. Frozen brain tissue was cut into five coronal 2-mm thick sections. The sections were placed in TTC staining solution (cat. no. DK003; Beijing Leagene Biotech Co., Ltd.) to incubation at 37˚C for 30 min in the dark, and turned over at 15 min. TTC staining solution reacted with the dehydrogenase in the healthy brain tissue to stain the healthy brain tissue red. By contrast, decreased dehydrogenase activity in the ischemic brain tissue was indicated by a pale color, as the lack of dehydrogenase did not react with the TTC staining solution. Subsequently, TTC stained brain tissue sections were fixed in 4% paraformaldehyde at 4˚C for 24 h. ImageJ software (version 1.6.0; National Institutes of Health) was used to calculate the infarct area, which was expressed as a percentage (%) of the infarct area. The ischemic area ratio (%)=(sum of white ischemic areas)/(sum of brain slice areas) x100%.

Statistical analyses were performed using GraphPad Prism software (version 5.0; GraphPad Software, Inc.). Data are presented as the mean ± SD with at least three experimental repeats. Statistical differences among multiple groups were determined using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The western blotting results indicated that the expression levels of caspase-1 (Fig. 1A) and IL-18 (Fig. 1D) were significantly increased in the I/R groups compared with the control group, reaching a peak at 24 h following CIRI (P<0.05). In addition, the expression levels of gasdermin D (Fig. 1B) and IL-1β (Fig. 1C) were significantly increased in the I/R groups compared with the control group, peaking at 12 h but decreasing by 24 h following CIRI (P<0.05). There was no significant difference in expression levels of pyroptosis-associated proteins and proinflammatory cytokines observed between the sham operation and control groups (P>0.05; Fig. 1).

The RT-qPCR results revealed that there was no significant difference in miRNA-124 expression levels between the sham operation and control groups (P>0.05). The expression levels of miRNA-124 in the I/R groups were significantly increased compared with the control group (P<0.05). At 12 h following CIRI, the relative expression levels of miRNA-124 were ~7 times higher compared with the control group. Between 12 and 24 h following CIRI, the expression levels of miRNA-124 decreased; however, the expression levels of miRNA-124 at 24 h were higher compared with the expression levels at 6 h (Fig. 2).

The RT-qPCR results suggested that the expression levels of miR-124 in the NC group were significantly increased compared with the sham group (P<0.001; Fig. 3A). Furthermore, the expression levels of miRNA-124 in the agomiRNA-124 and antagomiRNA-124 groups were significantly increased (P<0.05; Fig. 3A) and decreased (P<0.001; Fig. 3A) compared with the NC group, respectively, which indicated that the drugs were effective. The neurological function scores revealed that there was no obvious neurological deficit in the sham group. The neurological function score of the agomiRNA-124 group was significantly decreased compared with the NC group (P<0.05; Fig. 3B), whereas the neurological function score of the antagomiRNA-124 group was significantly increased compared with the NC and agomiRNA-124 groups (P<0.05; Fig. 3B). TTC staining also indicated that the infarct size in the agomiR-124 group was significantly decreased compared with the NC group (P<0.001; Fig. 3C), whereas the infarct size in the antagomiRNA-124 group was significantly increased compared with the NC group (P<0.05; Fig. 3B). The results suggested that increased expression levels of miRNA-124 may significantly reduce CIRI.

TargetScan software identified a complementary binding site between miRNA-124 and the 3'-UTR of STAT3 (Fig. 4A). Subsequently, the dual-luciferase reporter assay demonstrated that the 3'-UTR of STAT3 bound to miRNA-124. Compared with the WT-STAT3 + NC group, the WT-STAT3 + miRNA-124 group displayed significantly reduced relative luciferase activity (P<0.05; Fig. 4B). The western blotting results also suggested that the expression levels of STAT3 and p-STAT3 were significantly decreased in the agomiRNA-124 group compared with the NC group (P<0.05; Fig. 4C).

Immunohistochemical staining was conducted to investigate the role of important proteins and proinflammatory cytokines involved in pyroptosis. No positive cells were observed in the sham operation group (Fig. 5), whereas the number of caspase-1- (P<0.05; Fig. 5A), gasdermin D- (P<0.05; Fig. 5B), IL-1β- (P<0.05; Fig. 5C) and IL-18-positive cells (P<0.05; Fig. 5D) were significantly increased in the NC group compared with the sham operation group. miRNA-124 agonist significantly decreased the expression levels of caspase-1, gasdermin D, IL-1β and IL-18 compared with the NC group, whereas antagomiRNA-124 displayed the opposite effect (P<0.05; Fig. 5E).