A total of 24 male adult Sprague-Dawley rats (age, 8–10 weeks; weight, 240–260 g) were provided by the Lab Animal Center of The Fourth Military Medical University (Xi'an, China) and housed in polypropylene cages in an air-conditioned room (24°C, 50% humidity) with a 12-h dark:light cycle, allowing free access to pelleted diet and water. There were three groups in total, each with 8 rats. All experiments were performed in accordance with the Guidelines for Animal Research of The Fourth Military Medical University. The study was approved by the Ethics Committee of Medical Ethics and the Human Clinical Trial Committee of Xijing Hospital (approval no. XJYYLL-2015694).

Sanguinarine was purchased from Shaanxi Huike Botanical Development Co., Ltd (Xi'an, China). Triphenyl tetrazolium chloride (TTC) was purchased from Amresco, LLC (Solon, OH, USA) and was dissolved in phosphate buffered saline (PBS) at 1% (m/v). All ELISA kits for IL-1β (cat no. CSB-E08055r), IL-6 (cat no. CSB-E04640r) and TNF-α (cat no. CSB-E11987r) were purchased from Cusabio Biotech Co., Ltd. (Wuhan, China). All antibodies were purchased from Proteintech (Wuhan, China). The enhanced chemiluminescence kit was purchased from EMD Millipore (cat no. WBKLS0100; Billerica, MA, USA).

The MCAO model was established in male Sprague-Dawley rats as previously described (18,19). Rats were anesthetized using 10% chloral hydrate (Dalian Meilun Biotech Co., Ltd., Dalian, China) throughout the surgery. MCAO was maintained for 2 h, followed by reperfusion for 24 h (20). Sanguinarine was suspended in 0.5% carboxymethyl cellulose (CMC)-Na (Tianguan, Guangzhou, China) and rats were administered a dose of 15 mg/kg intragastrically. For the vehicle-treated group, equal volumes of 0.5% CMC-Na were administered. In the vehicle-treated group, the same volume of 0.5% CMC-Na was administered 1 h prior to MCAO. Rats were anesthetized with chloral hydrate and decapitated 24 h after MCAO.

Modified neurological severity score (mNSS) was determined 1 day after MCAO by two independent observers. All assessments were performed in triplicate. This scoring included evaluation of the balance, sensory, reflex and motor of rats, and scores were graded on a scale of 0–18 (21). A higher score represents a more severe injury (normal = 0; maximal deficit = 18).

Rats were anesthetized with 10% chloral hydrate and decapitated following reperfusion for 24 h. Brains were rapidly and carefully removed, and sliced into 2-mm continuous slices using a metallic brain matrix (Xinruan Informatlon Technology Co. Ltd., Shanghai, China). The slices were stained with 1% TTC at 37°C for 15 min in the dark, then fixed with 4% formaldehyde in PBS. The unstained waxy area of the brain slice was defined as infarction, and the infarct volume ratio was measured and calculated as described previously (22).

The rats treated by ischemia reperfusion after MCAO and sustained for 24 h, then anesthetized with 10% chloral hydrate, and processed by transcardial perfusion with PBS and then with 10% neutral-buffered formalin. Next, the brains were removed and immersed in 10% fresh neutral-buffered formalin for 24 h and embedded in paraffin. The brains were then cut into 2 mm thick sections, dewaxed, rehydrated with xylene and ethanol and stained with 0.5% (m/v) Toluidine Blue (Sigma-Aldrich, St. Louis, MO, USA). Nissl bodies were visualized with an Eclipse Ti-U Inverted Microscope System (Nikon Corporation, Tokyo, Japan) and counted using Image-Pro Plus version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

The injured areas of the brain tissue were removed 24 h after ischemia/reperfusion (I/R) injury, washed in cold PBS and placed into a homogenate tube. Appropriate volume of PBS were added into the tubes at 4°C, to allow for the tissues to be ground into 10% homogenate. The supernatant was collected following centrifugation at 4,000 × g for 15 min. The protein levels of TNF-α (cat no. CSB-E11987r), IL-6 (cat no. CSB-E04640r) and IL-1β (cat no. CSB-E08055r) were determined using ELISA kits (Cusabio Biotech Co., Ltd.) according to the manufacturer's instructions, which included the addition of 100 µl of appropriately diluted samples to each well. Standards (triplicates) and blanks were run with each plate to ensure accuracy

The injured area of the brain tissue was homogenized in cold radioimmunoprecipitation assay buffer (Sigma-Aldrich). The concentrations of whole cell extracts were determined by bicinchoninic acid assay (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 20 µg total proteins were loaded into each well. Protein extracts were subjected to electrophoresis on a 10% Bis-Tris protein gel (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The membranes were incubated with the following primary antibodies: Rabbit anti-Bax polyclonal antibody (1:500; cat no. 50599-2-Ig), rabbit anti-Bcl-2 polyclonal antibody (1:500; cat no. 12789-1-AP), mouse anti-GAPDH monoclonal antibody (1:1,000; cat no. 60004-1-Ig). The horseradish peroxidase-conjugated goat anti-mouse/rabbit IgG were used to detect the primary antibodies. Subsequently, enhanced chemiluminescence reagent was added. The respective densities of western blots were analyzed using Quantity One 1-D Analysis Software (version 4.4; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Data are expressed as the mean ± standard error. Multiple group comparisons were performed using one-way analysis of variance followed by Dunnett's test in order to detect inter-group differences. The difference between the mean values of two groups was assessed using a non-paired Student's t-test. SPSS version 12 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA) were used to perform all statistical analyses. All experiments were independently repeated three times. P<0.05 were considered to indicate a statistically significant difference.

Fig. 1 presents representative brain slices stained with TTC 24 h after reperfusion in vehicle- and sanguinarine-treated rats. Infarct volumes were expressed as a percentage of the intact contralateral hemisphere, and were found to be significantly lower in sanguinarine-treated rats (15.03%), when compared with the infarct volumes of vehicle-treated animals (38.76%; P<0.05) in the transient ischemic model of stroke.

Nissl staining was also investigated in brain samples from the three groups. The number of Nissl bodies was significantly reduced following MCAO (vehicle group), when compared with the sham group (189.33±29.41 vs. 506.00±30.83, respectively; P<0.01; Fig. 2). Treatment with sanguinarine significantly increased the number of Nissl bodies (328.00±45.77) when compared with that in the vehicle-treated rats (189.33±29.41; P<0.05).

Prior to MCAO, the neurologic scores were normal in all animals (score = 0 in all groups). High-grade behavioral deficits (scores >12) were recorded in all animals 60 min after MCAO (Fig. 3). Vehicle-treated animals exhibited severe behavioral impairments throughout the survival period including circling to the right, and the inability to balance and walk successfully on the beam. Treatment with sanguinarine significantly improved the neurologic score in comparison with the vehicle-treated rats 24 h after reperfusion (P<0.05; Fig. 3).

ELISA was performed to detect the contents of TNF-α, IL-6 and IL-1β in injured brain tissue. Compared with brain tissues obtained from sham-operated animals, global cerebral I/R brains had a significant higher expression level of TNF-α, IL-6 and IL-1β proteins (Fig. 4). Pre-treatment with sanguinarine significantly attenuated the increase in the TNF-α, IL-6 and IL-1β protein expression levels following global cerebral I/R (Fig. 4).

To investigate apoptotic signaling, the expression of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax was investigated. The ratio of Bcl-2/Bax is presented in Fig. 5. Global cerebral I/R injury resulted in a significant reduction in Bcl-2 protein expression in comparison with the sham-operated group. In addition, treatment of sanguinarine significantly increased the reduced Bcl-2 expression (P<0.05). By contrast, Bax protein expression following global cerebral I/R was significantly increased in comparison with sham-operated animals. This increase in Bax protein expression was significantly ameliorated by pre-treatment with sanguinarine.