Angong Niuhuang Pill (ANP) was obtained from Guangzhou Baiyunshan Zhongyi Pharmaceutical Company Limited (Guangzhou, Guangdong, China). Aspirin effervescent tablet was obtained from AstraZeneca Pharmaceutical Co Ltd (Wuxi, Jiangsu, China). Rat IL-1β, TNFα, S100B, MBP, GST-a, clusterin ELISA kits were obtained from Wuhan Elabscience Biotechnology Co., Ltd (Wuhan, Hubei, China). The nitric oxide test kit and GSH assay kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Anti-IL-1β antibody (ab9787) and Anti-TNFα antibody (ab6671) were obtained from Abcam (Cambridge, UK). Blood biochemical reagents (ALT, AST, ALP, TP, ALB, GLB, UREA, Crea, GLU, CHOL, TBil, CK, LDH, P, GGT, TG, Cystatin C, TBA, CA) were obtained from Zhejiang Erkn Biological Technology Co., Ltd (Wenzhou, Zhejiang, China). Blood biochemical reagents (Na+, K+, Cl-) were obtained from MEDICA (USA). PT, APTT, Fbg and TT test kits were obtained from Siemens (Germany). Hematology Reagents (EPK, FFD, FFS, SLS, FBA, RED) were obtain from Sysmex (Japan). Chemical Urinalysis Strips were obtained from URIT (Guilin, Guangxi, China).
Seven-week-old SPF grade male Sprague-Daweley (SD) rats (weighing 240-300 g) were provided from Hunan SJA Animal Company. Rats were housed in SPF animal room of Guangzhou General Pharmaceutical Research Institute Company (GPRI) Center for Drug Non-Clinical Evaluation and Research. The housing environment was maintained at 20-26 ℃, humidity 40-70%. Rats were kept in a 12 h light/12 h dark cycle. Rats were given free access to food and water. At the end of study, all animals were euthanized. All animal welfare and experimental procedures were in strict accordance with the Guide for the Care and Use of Laboratory Animals (ethical code number: IA-PD2017005-01, IA-AP2015010-02M; approval date: 04/05/2017, 12/01/2015).
MCAO model preparation and reperfusion
Rats were anesthetized by using intraperitoneal (IP) injection of chloral hydrate. Transient focal cerebral ischemia was induced through middle cerebral artery occlusion . A midline neck incision was made, and then the right common carotid artery (CCA) and its branches were isolated. A paraffin-coated nylon monoﬁlament was inserted from CCA to internal carotid artery (ICA) and sent toward the origin of middle cerebral artery (MCA) to produce focal cerebral ischemia. The middle cerebral artery was blocked for 2 hours and reperfusion was conducted for 3 hours.
Neurological function evaluation
Neurological deficit scores (NDS) were assessed according to the ZeaLonga method  at 3 h, D7, D14, D21, D28 after ischemia/reperfusion. Using a 5-point scale as follows, score 0, no neurological deficit; score 1, mild focal neurological deficit (with contralateral forelimb flexion); score 2, moderate focal neurological deficit (circling to the contralateral side); score 3, severe focal neurological deficit (falling to the contralateral side); score 4, no spontaneous activity with a depressed level of consciousness or death. Only the rats with score of 1-3 at 3 h were considered successful models.
Group and dose
The model rats were randomly divided into 6 groups as followed: Sham group, model group, positive drug aspirin 25 mg/kg (7 days) group, ANP 270 mg/kg (1 day, 4 days and 7 days) groups. And animals were fed for 30 days.
Cerebral infraction volume analysis
Rats were anesthetized with 20% urethane. Six rats in each group were randomly selected to collect brain to do 2,3,5-triphenyltetrazolium chloride (TCC) staining. Brain was frozen at -20 °C for 30 minutes and cut into 2 mm thick slices using metal brain matrix. And then TCC staining was done to analyze cerebral infraction area. Slides were incubated in 1% TTC in PBS for 10 minutes at 37 °C in dark. The infarct region appeared in white color while the normal brain appeared in red color. Infarct size (mm2) was determined by digital planimetry of the slices using Image-Pro software (Image-Pro Plus 6.0) (Media Cybernetics Inc.). Infarct volume (mm3) was corrected by section interval thickness. Percentage of cerebral infarction volume = infarct volume / total brain volume × 100%.
Brain histology analysis
Brains were immersed in 4 % paraformaldehyde and stained with hematoxylin-eosin (HE). The tissue was embedded in paraffin after dehydration and coronal slices were sectioned. Tissue structure was observed under common microscope and scored according to the degree of injury. “-” there was no edema in the cerebral cortex, and the nerve cells were normal; “±” there was edema in the cerebral cortical nerve cells and small fraction of neuronal necrosis, and the infarction area was not more than 1/6 of the left cortex area; “+” there was edema in the cerebral cortical nerve cells and small fraction of neuronal necrosis, and the infarction area was not more than 1/3 of the left cortex area; “++” there was edema in the cerebral cortical nerve cells and large fraction of neuronal necrosis, and the infarction area was not more than 1/3–2/3 of the left cortex area; “+++” there was edema in the cerebral cortical nerve cells and large fraction of neuronal necrosis, and the infarction area was more than 2/3 of the left cortex area.
Serum cytokine detection: The whole blood was incubated for 2 hours at room temperature and then centrifuged for 15 minutes (4 ºC, 3000 rpm). The serum content of IL-1β, TNFα and NO were detected using the ELISA kits or biochemical kits. Detection was done in accordance to the kit manufacturer recommendations. The absorbance was measured at 450 nm (IL-1β, TNFα) or 550 nm (NO) wavelength using an ELx800 type microplate reader (BioTek, America).
Brain cytokine detection: brain slices were done with immunohistochemistry assays. Slides were deparaffinized, rehydrated, and incubated with 3% H2O2 for 10 minutes to inactivate endogenous peroxidases. Slides were drop normal goat serum sealing fluid for 30 minutes. Then 1:50 dilution of rabbit anti-IL-1β and 1:100 dilutions of rabbit anti-TNFα polyclonal antibodies (abcam) were added at 4°C overnight. After washing, the slices were immersed in poly-peroxidase-anti-mouse/rabbit IgG (Elabsicence) for 30 minutes at 37°C. Finally, slices were incubated with DAB solution (Elabsicence) at 20-37°C for 10–15 minutes. Images were obtained using a Leica DM 4000B photo microscope. The IL-1β and TNFα staining intensity were scored as 0 (negative), 1+ (weak positive), 2+ (mild positive), 3+ (medium positive) and 4+ (strong positive).
ANP used in pharmacology study was up to 7 days; therefore, 30 days repeated dose toxicity study was designed to assess the drug safety. For detail, refer to ICH S4 “duration of chronic toxicity testing in animals”.
Rats were randomly divided into 4 groups according body weight, control group, ANP (550 mg/kg, 1640 mg/kg and 4910 mg/kg) groups. 4 weeks and 6 weeks for recovery were followed. Detection indicators were as follows: appearance, behavior, excrement character were observed every day. Body weight was measured weekly. Food intake was measured weekly. Hematology, blood biochemistry, urine, bone marrow, and histopathology were detected. Biomarkers such as TBA, GSTα, Cystatin C, clusterin, GSH, S-100B and MBP were also detected.
Statistical analysis was performed using SPSS software (18.0 versions, SPSS, Chicago, IL, USA), all the data are presented as mean ± SD or mean ± SEM. Statistical significance was calculated using a Dunnett’s test. One-way ANOVA was used to compare multiple sets of data. Ranked ordinal data used Ridit test and u-test. The significance level was set at P < 0.05.