A total of 64 specific-pathogen-free (SPF) mixed sex Wistar rats (male to female ratio, 31:33; age, 10–12 weeks; weight, 250–280 g) were purchased from the Experimental Animal Center of Shandong University (Jinan, China) and maintained under SPF conditions, with a maintained temperature of 22°C and a 12-h light/dark cycle at 60% humidity. All animals were fed a normal diet with free access to food and water until 2 days prior to surgery. All experimental protocols were approved by the Committee for Laboratory Animal Care and Use of Cangzhou People's Hospital (Cangzhou, China).

FA (purity ≥98%) was purchased from ALB Materials, Inc. (Henderson, NV, USA). MCAO was performed according to the intraluminal suture method, as described previously (16,17). Wistar rats (weighing 250–280 g) were anesthetized with an intraperitoneal injection of chloral hydrate (300 mg/kg). Body temperature was monitored and maintained at 37.0±0.5°C during surgery. Briefly, after making an incisions in the midline of the ventral cervical skin, the left common carotid, external carotid and internal carotid were exposed. A 4-0 nylon monofilament coated with polylysine was introduced into the internal carotid artery through the common carotid artery to occlude the origin of the middle cerebral artery. The intraluminal suture was carefully withdrawn to establish reperfusion following 90 min of ischemia to establish a transient MCAO model. Laser Doppler flowmetry (Periflux system 5000; Perimed AB, Datavägen, Sweden) was used to monitor cerebral blood flow. Rats that succumbed to ischemia were excluded from the study. Following MCAO induction for 7 days at 37.0±0.5°C, rats in each group were anesthetized with an intraperitoneal injection of chloral hydrate (300 mg/kg) and were sacrificed by cervical dislocation. Brain tissues and blood samples were collected for the subsequent experiments. The criteria for the successful establishment of the model was that rat local cortical blood flow decreased by 15±5% of the baseline following filament insertion, and could quickly be restored by reperfusion.

Rats were randomly divided into four groups (n=16 per group): i) Control (Ctrl) group (healthy rats); ii) FA group [healthy rats treated with FA (50 mg/kg/day) by intraperitoneal injection for 7 successive days]; iii) MCAO group (rats without treatment were subjected to MCAO); and iv) MCAO+FA group [rats subjected to MCAO were treated with FA (50 mg/kg/day) by intraperitoneal injection for 7 successive days following surgery].

The neurological scores were measured every 24 h from day 1 to 10 following MCAO according to the Zea-Longa neurological deficit scores (18). The scoring criteria were as follows: 0, normal, no neurological signs; 1, cannot completely stretch contralateral forelimbs; 2, contralateral circling when walking; 3, contralateral fall when walking; and 4, cannot walk and lowering of consciousness.

Brain tissues isolated from rats were fixed with 4% formaldehyde solution for 40 min at room temperature (25±0.5°C), embedded in paraffin and cut into 4 µm sections with a microtome. TUNEL staining was conducted using an in situ cell death detection kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's protocol. Briefly, following dewaxing and rehydration, sections were incubated with 20 µl/ml proteinase K for 15 min at 37°C. Sections were subsequently immersed in equilibration buffer for 10 min at room temperature (25±0.5°C) and incubated with TdT and dUTP-digoxigenin, followed by incubation with 2% anti-digoxigenin-peroxidase solution for 1 h at 37°C. Afterwards, sections were stained with 3% diaminobenzidine-H₂O₂ solution for 10 min at room temperature (25±0.5°C). The number of positive cells in each section were counted under a light microscope (magnification, ×400; Olympus Corporation, Tokyo, Japan).

Brain tissues were sampled and homogenized with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China) containing 1% phenylmethylsulfonyl fluoride (Thermo Fisher Scientific, Inc., Waltham, MA, USA) on the ice. Homogenates were centrifuged at 12,000 × g for 20 min at 4°C and the supernatant was collected. Protein concentration was measured with a bicinchoninic acid protein assay (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Following this, 20 µg protein extracts were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking with 5% bovine serum albumin (Bio-Rad Laboratories, Inc.) for 1 h at room temperature, membranes were incubated with the following primary rabbit monoclonal antibodies: Caspase-3 [cat. no. 9662; 1:1,000; Cell Signaling Technology, Inc. (CST)], caspase-9 (cat. no. 9502; 1:1,000; CST), Nrf2 (cat. no. 12721; 1:1,000; CST), glutathione-s-transferase (GST) (cat. no. 2624; 1:1,000; CST), NAD(P)H quinone dehydrogenase 1 (Nqo1) (cat. no. 3187; 1:1,000; CST), protein kinase RNA-like ER kinase (PERK) (cat. no. 5683; 1:1,000; CST), phosphorylated (phospho)-PERK (cat. no. 3179; 1:1,000; CST), phospho-inositol-requiring enzyme 1 (p-IRE1α) (cat. no. ab48187; 1:1,000; Abcam), IRE1α (cat. no. ab37073; 1:200; Abcam), C/EBP homologous protein (CHOP) (cat. no. 2895; 1:1,000; CST), B-cell lymphoma 2 (BCL-2) (cat. no. 15071; 1:1,000; CST) and β-actin (cat. no. 4970; 1:1,000; CST) at 4°C overnight. Following rinsing with Tris buffered saline with 5% Tween 20 (TBST) three times, membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (cat. no. ab6721; 1:2,000; Abcam) for 1 h at room temperature. The immunoreactive bands were observed using an enhanced chemiluminescence reagent kit (Bio-Rad Laboratories, Inc.) and analyzed using ImageJ software (version 1.42; National Institutes of Health).

Following centrifugation at 3,000 × g for 15 min at 4°C, serum was collected in order to measure the degree of oxidative stress. Superoxide dismutase (SOD), malonaldehyde (MDA) and glutathione (GSH) expression in serum were detected using corresponding commercial detection kits (SOD assay kit, cat. no. 20170316; MDA assay kit, cat. no. 20170314; and GSH assay kit, cat. no. 20170310; all purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The level of GSH and glutathione disulfide (GSSG), and the GSH:GSSG ratio was measured by High-performance liquid chromatography (HPLC) as previously described (19,20). The experiment was conducted strictly according to the manufacturer's instructions.

All data are expressed as the mean ± standard deviation. Comparisons of data among groups were performed by two-way analysis of variance followed by Tukey's post-hoc test, using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The structure of FA was illustrated in Fig. 1A. To explore the effects of FA on focal cerebral ischemic injury, survival rate and neurological scores were measured. As presented in Fig. 1B, the survival rate was markedly elevated in the MCAO+FA group compared with MCAO group. At the end of the study, 4 (out of 16) and 11 rats (out of 16) succumbed in the MCAO+FA and MCAO group, respectively. No difference was observed in the FA treatment group when compared with the Ctrl group. However, the neurological deficit scores were elevated in the MCAO model group, and FA administration significantly decreased the neurological deficit scores at 3–10 days in MCAO rats (Fig. 1C). These results demonstrated that focal cerebral ischemic injury was alleviated by FA treatment.

Apoptotic cells in brain tissues were analyzed by TUNEL staining (Fig. 2A). The results indicated that no significant levels of apoptosis were detected in rats treated with FA alone. An increased proportion of apoptotic cells was detected in the MCAO group when in comparison with the Ctrl group. However, FA administration significantly reduced the percentage of apoptotic cells (Fig. 2B). To further confirm the effects of FA in alleviating the cell apoptosis caused by MCAO, the expression levels of the apoptosis markers caspase-3 and −9 were measured (Fig. 2C). The results demonstrated that the elevated expression of caspase-3 and −9 induced by MCAO was significantly suppressed by FA treatment (Fig. 2C).

To further determine whether FA was directly involved in oxidative stress, Nrf2 signaling pathway proteins were detected by western blotting. As illustrated in Fig. 3A, FA significantly attenuated the MCAO-induced decrease in Nrf2, Nqo1 and GST expression. In addition, FA administration markedly reduced the serum levels of MDA, and significantly elevated SOD and GSH level in the MCAO rat model when compared with the untreated MCAO group (Fig. 3B). Furthermore, the GSH:GSSG ratio was decreased to 10:1 in the MCAO model group, compared with the Ctrl group. FA treatment notably counteracted the decrease caused by MCAO and increased the ratio by 7.5 times (Fig. 3B).

To detect MCAO-mediated ER stress, ER stress markers, including PERK, phospho-PERK, phospho-IRE1α, IRE1α, CHOP and BCL-2, were measured in brain tissues by western blot analysis (Fig. 4). Levels of phospho-PERK/PERK, phospho-IRE1α/IRE1α and CHOP were significantly reduced following FA treatment in the MCAO rat model when compared with the untreated MCAO group. In addition, Bcl-2 expression was markedly elevated in the MCAO+FA group compared with the untreated MCAO group (Fig. 4).