Myricetin (purity ≥98%) was purchased from Chengdu Technology and Market Co., Ltd., (Chengdu, China). Rat superoxide dismutase (SOD), rat malondiadehyde (MDA), rat glutathione (GSH), rat glutathione disulfide (GSSG), rat IL-1β, rat IL-6 and rat TNF-α kits was purchased from Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China). In Situ Cell Death Detection kit, POD was purchased from Roche Diagnostics GmbH (Mannheim, Germany). Anti-protein kinase B (AKT) antibody produced in rabbit, anti-phospho-AKT (pSer¹²⁹) antibody produced in rabbit, anti-nuclear factor (NF)-κB p65 antibody produced in rabbit, anti-phospho-NF-κB p65 (pSer281) antibody produced in rabbit, anti-p38 MAPK antibody produced in rabbit, and anti-p38 MAPK phospho (pT180/pY182) antibody produced in rabbit were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). 2,3,5-triphenyltetrazolium chloride staining (TTC) and the other chemicals and reagents were purchased from Sigma-Aldrich (Merck KGaA).

Adult Sprague-Dawley rats (230–280 g) were purchased from Jinan Pengyue Experimental Animal Breeding Co. Ltd. (Zhangqiu, China: http://www.cndss.net/company-jinanpengyueshiyandongwufanyu923.html). All experimental procedures were approved by the Institutional Animal Care and Use Committee of the National Institute Pharmaceutical Education and Research (Linyi, China).

Transient focal cerebral ischemia was induced in the rats via the left middle cerebral artery (MCA) occlusion technique, as previously described (7,9). The experiments were performed as follows: Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, following a ventral midline neck incision, the right common carotid artery (CCA), the external carotid artery, and the internal carotid artery (ICA) were carefully exposed and dissected away from adjacent nerves. Microvascular aneurysm clips were applied to the right CCA and the ICA. A filament rounded by gentle heating near a flame was introduced through a small incision into the CCA and then into the ICA. The suture was inserted 18–20 mm from the carotid bifurcation, which was verified by mild resistance, to occlude the MCA. The sham-operated rats underwent the same procedure, except that the filament was not inserted. During the surgery, body temperature was kept at 37°C with a heating pad and a warm light. Following incision closure, the rats were returned to standard housing conditions and allowed free access to food and water.

The 48 rats were randomly divided into five groups for administration of myricetin by oral gavage: Sham group (n=12), model group (n=12), low (L)-myricetin group (1 mg/kg myricetin + model, n=6), medium (M)-myricetin group (5 mg/kg myricetin + model, n=6), and high (H)-myricetin group (25 mg/kg myricetin + model, n=12). The sham group received normal saline intra-gastrically (i.g.) once per day for one week, and the sham-operation (the sham-operated rats underwent the same procedure, except that the filament was not inserted) was performed 1 h following the last administration of normal saline. The model group received normal saline i.g. once per day for one week, and the cerebral ischemia model operation was performed 1 h following the last administration of normal saline. The three myricetin groups received myricetin i.g. once per day for one week, and the operation was performed 1 h following the last administration of myricetin. Note: TTC staining required the whole brain, so the brain tissue could not be tested for other indicators (except for evaluation of neurological deficit) following TTC staining. TTC staining and evaluation of neurological deficit occurred only in the L-myricetin and M-myricetin groups, so the number of animals in these two groups was 6. However, in addition to the TTC staining and evaluation of neurological deficit, there were other tests [including terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, western blot assay, and assay of oxidative stress and inflammatory factors] in the other three groups (sham, model and H-myricetin groups), therefore the number of animals in these three groups was 12.

Following 1 day (24 h) of ischemia, evaluation of the neurological deficit scores was performed by an examiner blinded to the experimental groups. Neurological findings were scored on a five-point scale: No neurological deficits=0; failure to extend the right forepaw fully=1; circling to the right=2; falling to the right=3; failure to walk spontaneously with depressed levels of consciousness=4. The greater the neurological deficit score, the more severe the impairment (15). A total of 5 groups (sham group, model group, L-myricetin group, M-myricetin group and H-myricetin group) were used for the analysis of neurological deficit.

The rats were euthanized 24 h following permanent middle cerebral artery occlusion (pMCAO). The brains (whole brain) were quickly removed, sliced, fixed, stained, and photographed as described previously (9). The specific experimental operations were as follows: Rats were sacrificed and brains were quickly removed and placed at −20°C for 5 min. Following 5 min of freezing, the brains were sliced into three 2 mm-thick coronal sections. The sections were incubated in 1% TTC at 37°C for 30 min, and the sections were turned over in 15 min. Normal brain tissues were dyed bright red, and the unstained area (white area) was defined as the ischemic lesion. Infarct areas were measured using Photoshop CS6 image analysis software (Adobe Systems Europe, Ltd., Maidenhead, UK). To compensate for brain edema, the corrected volume was calculated as follows: Percentage hemisphere lesion volume (% HLV)=[total infarct volume-(left hemisphere volume-right hemisphere volume)]/right hemisphere volume ×100%.

The brains were harvested 24 h following the operation and stored at −70°C for later analysis. The homogenates extracted from the cortex of the ischemic brain hemispheres were analyzed for biochemical parameters. According to the manufacturers' protocol, the cortexes of the ischemic brain hemispheres were homogenized in 10% homogenate solution, and the supernatant was collected for biochemical marker analyses.

The levels of SOD activity, MDA, GSG/GSSH ratio, IL-1β, IL-6 and TNF-α levels in brain tissue were determined using a rat SOD kit (cat. no. ml540172), rat malondialdehyde (MDA) kit (cat. no. ml016824), rat T-GSH/HSSG kit (cat. no. ml022385), rat interleukin 1β (IL-1βa) kit (cat. no. ml037361), rat interleukin 6 (IL-6) kit (cat. no. ml102828) and rat TNF-α kit (cat. no. ml002859) following the manufacturer's protocol (Enzyme-linked Biotechnology Co., Ltd.). Briefly, the standards and sample diluents were added and incubated for 30 min at 37°C, followed by washing 5 times, and then horseradish peroxidase-conjugate reagent was added and incubated for 30 min at 37°C. The samples were washed 5 times, and Chromogen solutions A and B were added, incubated for another 30 min at 37°C, and then the stop solution was added and the absorbance was measured at the corresponding wavelength ranges within 15 min, and calculated.

The degree of brain cell apoptosis was determined using TUNEL assay as previously described (9). Briefly, the brain tissue was fixed with 10% neutral-buffered formalin for 48 h at room temperature (about 25°C). Then the brain tissue was embedded in paraffin (cat. no. YA0015; Beijing Solarbio Science and Technology Co., Ltd., Beijing, China), following deparaffinization and gradient ethanol rehydration the sections were treated with proteinase K (10 mM) for 15 min at 37°C, and the slides were immersed in the TUNEL reaction mixture for 60 min at 37°C in a humidified atmosphere in the dark. Next, the slides were incubated in a converter-POD (50 µl) for 30 min at 37°C, resulting in the characteristic blue nuclear staining, and were then analyzed using an Eclipse Ti2 inverted microscope (Nikon Corporation, Tokyo, Japan). To evaluate the apoptosis index of TUNEL-stained brain tissues, the TUNEL index (%) was calculated as the ratio of the number of TUNEL-positive cells divided by the total number of cells. For each sample, the average was calculated from the counting of eight randomly selected areas of TUNEL-stained slices at ×400 magnification.

The frozen brain tissues (cortex) were ground with a mortar and pestle, and then were homogenized with total protein extraction lysis buffer (Beijing Solarbio Science and Technology Co., Ltd). The protein concentration was determined using a Bio-Rad protein assay (Bio-Rad Laboratories Inc., Hercules, CA, USA). The lysates (30 µg) were separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), then transferred to nitrocellulose membranes (GE Healthcare, Chicago, IL, USA), and blocked at room temperature for 2 h with 5% non-fat milk in Tris-buffered saline with Tween (TBST). Membranes were incubated with an antibody against rabbit anti-AKT (cat. no. SAB4300574), rabbit anti-phospho-AKT (pSer¹²⁹; cat. no. SAB4300259), rabbit anti-NF-κB p65 (cat. no. SAB4502610), rabbit anti-phospho-NF-κB p65 (pSer281; Cat. no. SAB4301496), rabbit anti-p38 MAPK (cat. no. SAB4500491) and rabbit anti-p38 MAPK phospho (pT180/pY182; cat. no. SAB5600057) all at dilution: 1:10,000 in 5% milk/TBST at 4°C overnight (15 h). The membrane was then washed with TBST and incubated with horseradish peroxidase-conjugated antibody (mouse anti-rabbit IgG cat. no. 93702; 1:5,000; Cell Signaling Technology Inc., Danvers, MA, USA) at room temperature (25°C) for 1 h. The blots were developed with an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and were exposed on Kodak radiographic film. The immunoblots were visualized with an EC3 Imaging System, and analyzed with VisionWorks^®LS Analysis Software (UVP, LLC, Phoenix, AZ, USA).

All experimental protocols were approved by the Institutional Animal Care and Use Committee of Lin Yi Central Hospital (Linyi, China). The methods were carried out in accordance with the approved guidelines.

Data are presented as the mean ± standard deviation from at least six independent experiments. Statistical differences were determined using analysis of variance followed by Bonferroni correction. The analyses were performed using SPSS version 22.0 software (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The area of brain infarction was measured with TTC staining. As presented in Fig. 1A and B, no infarct area was observed in the sham group. When compared with the sham group, the model group demonstrated a marked increase in brain infarct area (P<0.01). Pretreatment with M-myricetin and H-myricetin significantly decreased the brain infarct size induced by ischemia (P<0.05 and P<0.01). As presented in Fig. 1C, pMCAO resulted in a significant increase in the neurological deficit score of the model group (P<0.01), and a significant decrease was then observed in the M-myricetin and H-myricetin groups (P<0.05 and P<0.01). The results indicated that myricetin protected against ischemic brain injury, and that the neuroprotective effect of H-myricetin was increased compared with M-myricetin. Considering the significant neuroprotective effects observed with myricetin at 25 mg/kg body weight (H-myricetin), this concentration was used for the subsequent studies.

Previous studies have indicated that oxidative stress results in ischemic cerebral injury. Therefore, the GSH/GSSG ratio, MDA contents, and the SOD activity in the brain tissue were detected. As presented in Fig. 2, compared with sham group, the MDA content increased significantly, whereas the SOD activity and GSH/GSSG ratio decreased significantly in the model group (P<0.01). However, treatment with H-myricetin significantly reversed these alterations (P<0.01). Decreased MDA content and increased GSH/GSSG ratio and SOD activity were observed following treatment with H-myricetin.

It has previously been demonstrated that the inflammatory response is critical in ischemic cerebral injury, and that myricetin has an anti-inflammatory effect (13), therefore the present study detected the effects of myricetin on levels of inflammatory cytokines (IL-1β, TNF-α and IL-6) in brain tissue. As presented in Fig. 3, compared with the sham group, the levels of IL-1β, IL-6 and TNF-α increased significantly in the model group (P<0.01). Treatment with H-myricetin significantly reversed these alterations in levels of inflammatory factors. Levels of inflammatory cytokines were significantly decreased in the H-myricetin group (P<0.01) compared with the model group.

As presented in Fig. 4, there was almost no apoptosis in the sham group. pMCAO induced an increase in apoptosis in the model group (P<0.01, Apoptosis index: 65.94±4.89). Compared with the model group, treatment with H-myricetin substantially reduced the ischemia-induced neuronal apoptosis (P<0.01, apoptosis index: 38.79±3.12).

To study the possible mechanisms of myricetin's neuroprotective effects, the expression of proteins associated with apoptosis, inflammation, and oxidative stress (AKT, NF-κB/p65 and p38 MAPK, respectively) were measured via western blot analysis. As presented in Fig. 5, compared with the sham group, a decreased level of phosphorylated AKT and increased levels of phosphorylated p38 MAPK and NF-κB/p65 were observed in the model group (P<0.01). Compared with the model group, treatment with H-myricetin significantly increased the relative level of phosphorylated AKT and decreased the levels of phosphorylated p38 MAPK and NF-κB/p65.