Rosmarinic acid (RA, Molecular Formula: C21H20O10; CAS NO.: 32769-01-0, purity >98.3%,) was provided by the State Key Laboratory of Long-acting Extended-Release and Targeting Drug Delivery System, Yantai, PR China.
Human neuroblastoma (SH-SY5Y) cells were obtained from the Shanghai cell bank of the Chinese Academy of Sciences. SH-SY5Y cells were cultured and maintained in F12 + DMEM (1:1, v/v) media, supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were kept at 37°C in a humidified 5% CO2/95% O2 incubator. The dissociated cells were seeded in poly-L-lysine-coated plates at a density of 5 × 105/cm2 and cultured in DMEM (Shanghai lifetechnologies, PR China), supplemented with 10% (v/v) horse serum, 5% (v/v) fetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin. The fresh medium was changed twice weekly. After eight days of culture, the SH-SY5Y cells were randomly divided into seven groups: a normal group (no oxygen-glucose deprivation), an oxygen-glucose deprivation group (control group) and oxygen-glucose deprivation + RA groups (1, 3, 9, 27 and 81 μM). RA was dissolved in saline. For oxygen-glucose deprivation, the SH-SY5Y cells were washed twice in glucose-free balanced salt solution (BSS) containing 130 mM NaCl, 5.5 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, and 20 mM HEPES (pH 7.4), and incubated in BSS (no glucose) in a humidified chamber filled with 95% N2/5% CO2 for 3 h at 36.5°C. After oxygen-glucose deprivation, the cultures were replaced into neurobasal medium and incubated with RA (1, 3, 9, 27 and 81 μM) in a CO2 incubator for 12 h.
Determination of cell viability, lactic dehydrogenase (LDH) leakage and apoptosis
After oxygen-glucose deprivation for 3 h followed by 12 h incubation with or without RA, cell viability was assessed using a 3-(4, 5)-dimethylthiahiazo (−z-y1)-3, 5-di-phenytetrazoliumromide (MTT) assay. LDH, an indicator of cell injury, was detected according to the description of the LDH assay kit (Beijing Zhongsheng Bioreagent, Beijing, PR China). LDH leakage rate (%) = Ae/At*100%. Ae indicated extracellular lactic dehydrogenase (LDH, cells culture fluid), At indicated intracellular and extracellular LDH (cells lysate). Apoptotic cells were evaluated using an Annexin-V fluorescein isothiocyanate (FITC) apoptosis detection kit (Haimen Beyotime Biotechnology Institute, Jiangsu, PR China. In brief, cells were harvested, washed and incubated at 4°C for 30 minutes in the dark with annexin-V FITC and propidium iodide, then analyzed on a FACS Vantage SE flow cytometer (Beckman Coulter, Fullerton, CA, USA).
Adult male Sprague–Dawley rats were obtained from Shandong Luye Pharmaceutical Company (Yantai, PR China). All animals were housed individually at 22 ± 2°C and a relative humidity of 50 ± 10% with a 12-h light/dark cycle and had free access to chow and water. All procedures (including rat cerebral ischemia study protocol) were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) revised 1996.
Streptozotocin (STZ)-induced diabetes animal model and rat cerebral ischemia study protocol
Two hundred and eighty rats (fasting for 20 h) were induced by single intraperitoneal injection (i.p.) of STZ at a dose of 45 mg/kg. STZ was diluted in citrate buffer 0.1 M (pH 4.0). After STZ injection for three weeks, rats with a glycemia value between 11.0 to 22.0 mM were used. The diabetic rats were anesthetized with chloral hydrate (350 mg/kg, i.p.). Rectal temperature was recorded and maintained at 37°C throughout the surgical procedure. The operation of MCAO was carried out according to previous procedures with minor modifications . The adequacy of the anesthesia was monitored by the level and stability of the mean arterial pressure (MAP) and absence of corneal reflex, and adequate levels of anesthesia and analgesia were ensured with supplemental i.p. injection of pentobarbital sodium given as required. The left common carotid artery was occluded, and the branches of the external carotid artery were dissected and divided. The internal carotid artery was followed rostrally and a 4–0 filament (Beijing Shadong Biology Company, Beijing PR China. A filament (with a diameter of 0.25, and a tip diameter of 0.34 mm to create a globular stopper) was introduced into the internal carotid artery and advanced until resistance was felt. The filament was removed after 40 minutes. The rats were kept under control temperature (24°C to 25°C) conditions for the first 24 h after surgery.
For dose–response studies, 70 rats were randomly divided into seven groups of 10 rats each plus 10 rats as control (no diabetic). RA at doses of 0, 12.5, 25, 50, 100 or 200 mg/kg was administered by intravenous bolus injection into the tail vein 30 minutes after reperfusion. Diabetic or vehicle-treated rats were administered with saline. Rectal temperature was determined once every 3 h for a total of eight times. Neurological deficits were determined at 24 h after ischemia followed by brain infarct volume examination.
For therapeutic time-window studies, 50 rats were randomly divided into five groups of 10 rats each. Rats received a dose of 50 mg/kg by intravenous bolus injection into the tail vein 1 h, 3 h, 5 h and 7 h after reperfusion. Vehicle-treated rats were administered with saline. Neurological deficits were determined 24 h after ischemia followed by brain infarct examination.
For anti-inflammatory mechanism studies, 45 rats were randomly divided into three subgroups of 15 rats each, plus 15 rats as control (no diabetic). Rats received doses of 50 mg/kg intravenous bolus injections into the tail vein 30 minutes after reperfusion. Diabetic or vehicle-treated rats were administered with saline. The above three groups were evaluated for Evans blue extravasation, Western blots analysis, and histopathological damage by NeuN staining.
For long-term studies, 30 rats were randomly divided into two groups of 15 rats each. Rats received doses of 50 mg/kg by intravenous bolus injection into the tail vein 30 minutes after reperfusion. The vehicle-treated rats were administered with saline. Neurological deficits were determined at the 3rd, 7th and 14th day after I/R. Fourteen days after I/R, seven rats in each group were randomly selected for brain infarct examination according to a previous method .
Evaluation of neurological deficits
Neurological deficits were evaluated using a modified six-point scoring method  by an investigator who was blinded to each experimental group. The scale is 0, no neurological deficits (normal); 1, failure to extend left forepaw fully (mild); 2, circling to the left (moderate); 3, falling to the left (severe); 4, no spontaneous walking with a depressed level of consciousness (very severe), and 5, death.
Evaluation of infarct volume and brain water content
After 23 h of reperfusion, rats were anesthetized with sodium pentobarbital (40 mg/kg) through intraperitoneal injection, and the brain was quickly removed. Total wet weight of the brain was measured accurately. Brain was cut at the forebrain 3 mm, and each brain was sliced into five coronal sections of 2-mm thickness each, then stained with a 2% solution of tetrazolium chloride (TTC, Sigma, Shanghai, PR China) in saline at 37°C for 20 minutes, and photographed. The images were digitized, and infarct volume was calculated with a Compix system computer (C imaging 1280 system, Compix Inc., Cranberry. Township, PA, USA). Afterwards, brain water content was determined as an indicator of cerebral edema using a wet/dry method.
Evaluation of blood–brain barrier (BBB) leakage with Evans blue extravasation
Determination of Evans blue extravasation was based on a previous method  with minor modifications. After reperfusion, 0.1 ml of 4% Evans blue (Urchem, Shanghai, PR China) in 0.9% saline was intravenously administered. Twenty-three hours after I/R, rats were anesthetized with chloral hydrate (350 mg/kg, i.p.), then perfused with 20 ml 10 U/ml heparinized saline to wash out the blood, the brain was then isolated, weighed and homogenized in 50% solution of trichloroacetic acid. After centrifugation at 400 × g for 20 minutes, the supernatant was spectrophotometrically measured at 595 nm. Cerebral Evans blue was quantified as micrograms of dye per gram of wet weight.
NF-κB binding assay
SH-SY5Y cells (5 × 106) were pre-incubated with RA 9 μM for 22 h, then incubated with TNF-α (20 ng/ml) for 1 h or 2 h, then washed once with phosphate-buffered saline (PBS), scraped cells into 1 mL cold PBS, and pelleted by centrifugation. Nuclear extracts were prepared as described previously . The DNA binding activity of NF-κB (p50/p65) was determined using an ELISA kit (Solar Biology Technology Company, Shanghai, PR China).
Western blots analysis
For the experiment of TNF-α stimulated SH-SY5Y cell lines in vitro, SH-SY5Y cells (5 × 106) were pre-incubated with RA (9 μM) or HMGB1 inhibitor, glycyrrhizin (100 μM) for 120 minutes, then incubated with TNF-α (20 ng/ml) for 30 minutes and cultured in a CO2 incubator for 12 h. Cells were washed twice with ice cold PBS on ice and lysed in NP40 lysis buffer (Biosource, Camarillo, CA, USA) (50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 1% NP-40 and 0.02% NaN3) supplemented with 1 mM PMSF and 1 × protease inhibitor cocktail (Sigma, Saint Louis, MO, USA).
For Western blot analysis of collected brain tissues, the tissues were defrosted and immersed in ice-cold NP40 lysis buffer. Equal amounts of cell protein (50 μg) were separated by SDS-PAGE and analyzed by Western blot using specific antibodies to HMGB1, IκB, phosphor-IκB-α, phosphor-NF-κB and proliferating cell nuclear antigen (PCNA, loading control). All antibodies were purchased from Beijing Biosynthesis Biotechnology Company (Beijing, PR China). Optical densities of the bands were scanned and quantified with a Gel Doc 2000 (Bio-Rad laboratories, Milan, Italy). Data were normalized against those of the corresponding PCNA bands. Results were expressed as fold increase over control or sham group.
Myeloperoxidase (MPO) activity assay
The remaining slices of the brain, not used for Western blot analysis, were used to determine MPO activity as described previously . For each individual rat, the ischemic and non-ischemic hemispheres (from the six slices) were pooled separately for the assay, and wet weight was then recorded. The tissues were homogenized (1: 20, wt/vol) in 5 mmol/L phosphate buffer (pH 6, 4°C) and centrifuged at 30,000 g for 30 minutes (4°C). MPO activity was determined according to the kit instructions (Xitang Biology Technology Company, Shanghai, PR China). MPO activity for each tissue sample is normalized on the basis of grams per wet weight of tissue.
The left hemisphere of the brain was cut coronally into three blocks from the level of the optic chiasm and the infundibular stem of the hypophysis. The middle block was further cut into three sub-blocks. The middle sub-block (0.1 × 0.1 cm2) was embedded in paraffin. NeuN-immunolabeling was carried out according to the previous method . NeuN-immunopositive cells were counted in three randomly selected fields (400 ×). The number of NeuN-immunopositive cells was calculated by averaging the three counts.
Neurological deficit scores between groups were analyzed using a non-parametric test. Quantitative data from the experiments were expressed as mean ± standard deviation (S.D.); significance was determined by one-way analysis of variance (ANOVA) followed by Dunnett's test (SPSS 16.0. IBM Company,Chicago, USA). In all cases, differences were considered significant if P <0.05.