Male Sprague-Dawley rats (n=60; age, 8 weeks; weight, 270–280 g) were supplied by the Experimental Animal Centre of Guangzhou University of Chinese Medicine (Guangzhou, China). All rats were raised in a specific-pathogen free room under controlled temperature (24 ± 1°C) and humidity (55–70%) with a 12-h light-dark cycle, and were given free access to food and water. All experimental procedures were carried out according to the guidelines of the Administrative Panel on Laboratory Animal Care of Guangzhou University of Chinese Medicine (Guangzhou, China). After 1 week of adaptive housing and feeding, animals were randomly divided into six groups (n=10 each group): i) Sham surgery (control); ii) MCAO-R surgery (model); iii) MCAO-R + rapamycin (Rapa); iv) MCAO-R + 5 g/kg BHD; v) MCAO-R + 10 g/kg BHD; and vi) MCAO-R + 20 g/kg BHD.

A transient focal cerebral ischemia model (MCAO-R model) was established as previously described (18,35). Briefly, rats were anesthetized with 4% isoflurane and maintained with 1.5% isoflurane via an isoflurane vaporizer (RWD Life Science). A midline neck incision was then performed to expose the right common carotid artery, external carotid artery (ECA) and internal carotid artery (ICA). A 4-0 silicone rubber-coated nylon monofilament (cat. no. MSRC43B280PK100; RWD Life Science) was inserted into the ECA and then gently advanced into the ICA, 18–19 mm from the carotid bifurcation, to occlude the beginning of the MCA. After 2 h of occlusion, the monofilament was gently removed to restore blood flow. Throughout the surgery, the body temperature of all rats was maintained at ~37°C. Rats were anaesthetized by 1% sodium pentobarbital (40 mg/kg) and euthanized by cervical dislocation on day 5 following surgery.

BHD was prepared according to previously described and the quality control was also achieved (Fig. S1) (36). Briefly, the powdered sample of BHD (143 g) was mixed with Radix astragali, Radix angelicae sinensis, Radix paeoniae rubra, Rhizoma ligustici chuanxiong, Flos carthami, Semen persicae and Lumbricus at a 120:6:5:3:3:3:3 ratio. All ingredients were purchased from Guangzhou Zhixin Chinese Herbal Medicine Co. Ltd. and verified by the Department of Pharmacy, Guangzhou University of Chinese Medicine. The decoction was made by boiling the mixture in 10 times the amount of distilled water at 100°C for 60 min. The drug solution was removed for use and the residue was boiled once more. The two solutions were combined and concentrated on a rotary evaporator at ~60°C. The concentrated medicinal solution was vacuum-cooled and dried twice to form a powder and dissolved in distilled water and the final concentration was 2.0 g/ml (equivalent to the dry weight of the raw material).

All groups of rats were treated with an intraperitoneal injection of hydroxychloroquine (20 mg/kg; cat. no. S4430; Selleck Chemicals) 30 min after surgery (37). BHD powder and Rapa (cat. no. S1039; Selleck Chemicals) were dissolved with 0.9% saline. At the onset of reperfusion (38), the treatment groups, including the MCAO-R + BHD and MCAO-R + Rapa groups, were treated with BHD (5, 10 and 20 g/kg) by gavage and Rapa (10 mg/kg) via intraperitoneal injection, respectively.

The modified neurological severity score, which uses different scores to evaluate motor, sensory, reflex and balance functions, was used to assess neurological deficits on day 5 following surgery, as previously described (39–41). The motor test used a six-point scale to assess the movement and walking ability of the rats (muscle state, abnormal motion and tail lifting test). The sensory test used a two-point scale to assess superficial and deep sensations in the rats (vision, touch and proprioception). The reflex test used a four-point scale to assess shallow and deep reflection in rats. The balance functions test used a six-point scale to assess the movement of rats on the balance beam. The neurological function scores ranged between 0–18 points, and were graded as follows: Mild damage (1–6), moderate damage (7–12) and severe damage (13–18).

Following euthanasia, rat brains were rapidly sliced using a rat brain matrix (RWD Life Science) to measure the cerebral infarct size. Continuously cut 5 sections of each brain tissue, 2 mm each, were made (n=4). The sections were stained with 2, 3, 5-triphenyltetrazolium hydrochloride (TTC; cat. no. T8877; MilliporeSigma) at 37°C for 15 min (42). Normal tissues stained red and ischemic tissues, white. Image-Pro Plus 6.0 (Media Cybernetics, Inc.) image analysis software was used for image analysis.

The tissue of the ischemic hemisphere of the rats was chosen for the oxidative stress kit testing (n=3). The brain tissues were homogenized with ice-cold saline and centrifuged at 14,000 × g for 10 min at 4°C. The supernatant was then used to detect the levels of CAT (cat. no. A007-1-1; Nanjing Jiancheng Bioengineering Institute), MDA (cat. no. A003-1-2; Nanjing Jiancheng Bioengineering Institute) and GSH-PX (cat. no. A005-1-1; Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's instructions. Absorbance was measured using a microplate reader with the wavelength of 532, 405 and 412 nm.

The tissue of the ischemic hemisphere of the rats was chosen for H&E and Nissl staining testing (n=3). Brain paraffin-embedded sections were deparaffinized and rehydrated in xylene and gradient alcohol. The sections were then washed in PBS (Beyotime Institute of Biotechnology) and underwent H&E (Beyotime Institute of Biotechnology) or Nissl (Nanjing Jiancheng Bioengineering Institute) staining for 10 min at 37°C. The sections were then washed with PBS. Images were captured using a light microscope (Leica Microsystems, Inc.). Image-Pro Plus 6.0 (Media Cybernetics, Inc.) software was used for image analysis.

The tissue of the ischemic hemisphere of the rats was chosen for western blotting (n=3). Brain tissue was homogenized in ice-cold RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) and centrifuged at 12,000 × g for 10 min at 4°C. The supernatant was then extracted to determine the total protein concentration using a bicinchoninic acid protein assay (cat. no. P0012S; Beyotime Institute of Biotechnology). Next, the appropriate volume of loading buffer (cat. no. BL511B; Biosharp Life Sciences) was added, followed by boiling for 10 min at 100°C. Proteins samples (30 µg per well) were separated using 8, 10 and 12% SDS-PAGE gels and transferred onto a PVDF (cat. no. ISEQ00010; cat. no. IPVH00010; MilliporeSigma) membrane. The membrane was blocked with 5% skimmed milk (cat. no. 1172GR500; BioForxx) at 37°C for 1 h. Then, incubated with primary antibodies against SIRT1 (cat. no. ab189494; 1:1,000; Abcam), LC3 (cat. no. 2775; 1:1,000; Cell Signaling Technology, Inc.), beclin 1 (cat. no. 3738, 1:1,000; Cell Signaling Technology, Inc.), p62 (cat. no. 39749; 1:1,000; Cell Signaling Technology, Inc.), doublecortin on the X chromosome (DCX; cat. no. ab18723; 1:1,000; Abcam), β-actin (cat. no. 58169; 1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight and incubated with goat anti-rabbit IgG (cat. no. S0001; 1:3,000; Affinity Biosciences) or goat anti-mouse IgG (S0002; 1:3,000; Affinity Biosciences) at 37°C for 1 h. ECL reagent (cat. no. WBKLS0500; MilliporeSigma) was added to the membrane for visualizing the target bands. Digital images of the blots were visualized using Image Lab 3.0 software (Bio-Rad Laboratories, Inc.).

The tissue of the ischemic hemisphere of the rats was chosen for immunofluorescence testing (n=3). Rat sections (10 µm each) were blocked with 5% BSA (Beyotime Institute of Biotechnology) and incubated with primary antibodies for nestin (cat. no. 4760; 1:300; Cell Signaling Technology, Inc.), LC3 (cat. no. 2775; 1:300; Cell Signaling Technology, Inc.), brain-derived neurotrophic factor (BDNF; cat. no. ab108319; 1:300; Abcam) and DCX (cat. no. ab18723; 1:300; Abcam) overnight at 4°C. The slices were incubated with fluorescence-coupled secondary antibody, anti-mouse IgG (cat. no. 4408; 1:1,000; Cell Signaling Technology, Inc.), anti-mouse IgG (cat. no. 4409; 1:1,000; Cell Signaling Technology, Inc.) or anti-rabbit IgG (cat. no. 4412; 1:1,000; Cell Signaling Technology, Inc.) for 2 h at 37°C. Following rinsing, sections were incubated with DAPI (cat. no. P0131; Beyotime Institute of Biotechnology). Fluorescence was detected using a laser scanning confocal microscope (Carl Zeiss AG). Image-Pro Plus 6.0 (Media Cybernetics, Inc.) image analysis software was used for image analysis.

Statistical analysis was performed using SPSS version 17 (SPSS, Inc.). Data are presented as the mean ± standard deviation. One-way ANOVA was applied to analyze differences in data for the biochemical parameters among the different groups, followed by Dunnett's post hoc test, and an unpaired Student's t-test was also used to determine statistical differences. P<0.05 was considered to indicate a statistically significant difference.

The design of the present study is shown in Fig. 1A. First, the infarct volume was measured. TTC staining demonstrated that the infarct volume in the MCAO-R + BHD group was markedly decreased in a dose-dependent manner compared with that in the MCAO-R group (Fig. 1B and C). The neurological scores following MCAO-R in two BHD groups were improved (Fig. 1D), particularly in the 20 g/kg BHD group. A dosage of 20 g/kg BHD was selected for the next experiments. These data demonstrated that BHD could effectively ameliorate infarction and reduce neurological scores following MCAO-R.

To determine whether BHD exerted protective effects against oxidative stress, the level of MDA and the activity of CAT and GSH-PX were detected next (Fig. 2). Compared with the sham group, the MDA level in the MCAO-R group was significantly increased and the activity of CAT and GSH-PX was decreased. However, after the oral administration of BHD, the MDA levels in MCAO-R rats were significantly reduced and the activity of CAT and GSH-PX were significantly increased. These data demonstrated that BHD could relieve neuronal oxidative stress damage following MCAO-R.

As shown in Fig. 3A and B (H&E and Nissl staining, respectively), large areas of neuronal necrosis were induced by cerebral I/R. The MCAO-R group exhibited extensive neuronal death accompanied by the disappearance of cytoplasmic bodies, swelling of cell bodies, nuclear condensation and sparse Nissl bodies. Conversely, sham group neurons exhibited clear and large cell nuclei and bodies, abundant Nissl bodies and strong staining. Notably, BHD reversed these changes. The expression of BDNF (Fig. 3C and D) was further determined by immunostaining. MCAO-R downregulated BDNF expression. Post-surgical treatment of MCAO-R rats with BHD caused a significant increase in BDNF. Collectively, these data demonstrated that BHD could protect against neuronal death following MCAO-R.

As shown in Fig. 4, the expression of DCX, a protein expressed by neural precursor cells, was detected using immunostaining and western blot analysis. MCAO-R downregulated DCX. Post-surgical treatment with BHD caused a significant increase in DCX expression in MCAO-R rats. Thus, these results supported that BHD promotes neurogenesis in MCAO-R rats.

In order to determine whether the SIRT1/autophagy pathway was involved in the protective effect of BHD, SIRT1 and autophagic markers (LC3, p62 and beclin 1) were detected using western blot analysis (Fig. 5A-E). The results demonstrated that the expression of SIRT1 in the MCAO-R and MCAO-R + BHD groups was elevated. SIRT1 expression was significantly increased in the MCAO-R + BHD group, compared with that in the MCAO-R group. The expression of LC3-II and beclin 1 was increased, while that of p62 was slightly decreased in the cerebral peri-ischemic area of rats in the MCAO-R group. Post-surgical treatment with BHD markedly increased autophagy, when compared with the MCAO-R group. Post-surgical treatment with Rapa had a similar effect to that of BHD treatment. In addition, the distribution pattern of nestin and LC3 was elucidated using tissue immunostaining. The expression pattern of LC3 was similar to that observed following western blot analysis (Fig. 6A-C). The expression of nestin, a protein marker for neural stem cells, was also elevated by BHD. Therefore, these data suggested that the neuroprotective effect of BHD might be associated with the SIRT1/autophagy pathway.