Quality control of AGNHW
Chemicals and materials
AGNHW was obtained from Beijing Tong Ren Tang Co., Ltd., (Z11020959, 3 g/pill). The Millipore Milli Q-Plus system was used to prepare the deionized water (Merk, Mini Q ZQ7000). VWR supplied the ethanol, and DUKSAN Ltd supplied the acetonitrile (HPLC gradient). Geniposide, wogonoside, wogonin and berberine with the purity of 98% were purchased from Chengdu Ruifensi Biotechnology Co., Ltd.; Baicalein, palmatine hydrochloride, epiberberine and coptisine with the purity of 98% were obtained from Nanjing Yuanzhi Biotechnology Co., Ltd.; Baicalin with the purity of 95% was purchased from Sigma. Additional file 1 lists the chemical structures of these reference compounds.
We compared various extraction methods for AGNHW using various solvents. The entire AGNHW pill was placed in the grinding crucible and homogeneously pulverized using liquid nitrogen. AGNHW (10 mg) was accurately weighed and ultrasonically extracted for 1 h at power 250 W and frequency 45 kHz with 1 ml solvent (distill water, or 30% ethanol, or 50% ethanol, or 70% ethanol, or 100% ethanol). After cooling in a water bath for 5 min, the samples were centrifuged at 10,000 rpm and the supernatant was filtered (0.2 µm). For HPLC analysis, a 100 μL aliquot of was diluted with 900 μL of 70% Ethanol. The final concentration was 1 mg/ml of AGNHW. We chose the 70% ethanol extract for quality control of AGNHW and in vitro study based on the extraction efficiency results.
Quality control analysis with ultra high-performance liquid chromatography (UPLC)
Quality control for AGNHW was performed by using ultra-high performance liquid chromatography (Thermo-Scientific, Ultimate-3000) system, controlled by Thermo Scientific Chromeleon 7.2 software. The separation of 9 analytes was accomplished using an ACE-C 18 HPLC column (4. 6 mm × 250 mm, 5 μm) at temperature 30 °C. As shown in Additional file 2, the gradient elution is optimized. The injection volume is 10 µl and the nine analytes were monitored simultaneously at 240 nm. We first validated the method's precision, reproducibility, accuracy, stability, and linearity before quantitatively analyzing the AGNHW extract. Six replicate injections of samples were used to assess precision. Six independently prepared samples were analyzed to determine reproducibility. he stability was determined by analyzing the same sample every 3 h for five times at room temperature. The recovery test was used to determine the method's accuracy by using marker compounds. The average percent of recoveries was calculated by dividing the detected amount by the added amount. To detect linearity, 70% ethanol stock solutions of nine reference compounds were diluted to a series of concentrations, 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 µg/ml, for the construction of calibration curves. To create the standard curve, each concentration was examined in triplicate. These standard curves were used to analyze a total of 9 compounds in AGNHW samples.
ONOO− scavenging assay
We tested whether AGNHW extract could react with ONOO− directly by using UPLC analysis. First, we recorded the HPLC chromatograms of AGNHW extract (1 mg/ml). The AGNHW extract solution was then treated with various concentrations of ONOO− (50 µM, 100 µM, 200 M, 400 µM, 800 µM, 1600 µM). We quantitatively identified the compounds reacting with ONOO− by comparing the retention time and UV absorbance with the standard compounds, demonstrating the dose-dependent reduction of peak after adding various concentrations of ONOO− solution.
Middle cerebral artery occlusion (MCAO) model
Male Sprague–Dawley (SD) rats (290–310 g) were obtained from the Laboratory Animal Unit, The University of Hong Kong. The Committee on the Use of Live Animals in Teaching and Research at The University of Hong Kong approved all animal experimental protocols. Experiments were carried out in accordance with national and institutional ethics and biosafety guidelines. The rats were kept in humidity and temperature-controlled environment with a 12-h light/dark cycle. To mimic a prolonged stroke, rats were subjected to 5 h of middle cerebral artery occlusion (MCAO) followed by 19 h of reperfusion . Following isoflurane anesthesia, the skin of rat’s neck was opened to expose the common carotid artery (CCA), external carotid Silicon (ECA) and internal carotid artery (ICA). From the ECA to the ICA, a silicone coated suture (Doccol, Redlands, CA, USA) was inserted until it occluded the origin of the middle cerebral artery (MCA). The same procedure was performed on sham control rats but without MCA occlusion. The duration of anesthesia was recorded (Additional file 3A). Rats’ body temperature was monitored during the surgery and recovery using an infrared thermometer (Additional file 3B). To allow for reperfusion, the suture was removed 4.5 h after MCAO. To confirm the success of the brain infarct, a thin slice of brain tissue was stained with 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) .
Experimental designs and drug treatment
SD rats were randomly assigned to one of four groups: Sham, MCAO 5 h plus reperfusion 19 h (M5/R19), M5/R19 plus t-PA, M5/R19 plus t-PA and AGNHW treatment. Each group had at least ten rats. To simulate early intervention, AGNHW dissolved in saline (257 mg/kg) was administered orally 2 h after MCAO. By converting the body surface area of humans to rats, this dosage is equivalent to a daily dosage in human subjects (3 g/pill per day) . Saline was used as a vehicle control. The rats were given t-PA (10 mg/kg, Actilyse, Boehringer Ingelheim) or saline via the femoral vein at 4.5 h after MCAO ischemia, with a 10% bolus followed by a 90% slow infusion within 0.5 h, defining the infusion time at 5 h after MCAO. In this study, the outcome was measured by a researcher who was not aware of the grouping or surgical procedure.
Mortality rates and neurological deficit scores
We calculated the mortality rates 24 h after the onset of an ischemic stroke. The dead rats were not subjected to any further testing. The modified neurologic severity score (mNSS) test was used to assess each group's neurological deficit. The mNSS test comprises the motor, reflex, and balance tests described in our previous publication: the score ranges from 0 to 18, with 0 representing normal and 18 representing maximal neurological deficit .
Brain edema measurement
At 24 h after stroke, rats were deeply anesthetized with isoflurane and subjected to intracardiac perfusion with ice-cold PBS to remove circulating blood. After that, rat brains were collected and cut into 2-mm thick coronal slices. The brain slices were photographed digitally and then analyzed using Image J software (National Institutes of Health). A brain swelling index was calculated by dividing the ischemic side area by the non-ischemic side area .
Evans blue test for BBB leakage
The amount of Evans blue (EB) extravasation into ischemic brain indicates the severity of BBB leakage . Under anesthesia, rats were intravenously injected with 2% EB (3 ml/kg, Sigma); 1 h later, rats were transcardially perfused with 250 ml PBS to completely remove the circulating EB. The brain tissues were then harvested and cut into 2-mm thick coronal sections in a series. EB extravasation in brain tissues was digitalized and tissues were frozen for further study. To determine the amount of EB in brain tissue, we homogenized and sonicated the tissue PBS before adding the same volume of 50% trichloroacetic acid (Sigma). Supernatants were collected after 20 min of centrifugation at 15,000 rpm to determine the OD value at 620 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). The amount of EB was calculated using an EB standard curve, and extravagated EB dye was quantified as g/g brain tissue.
Hemorrhagic transformation measurement
The severity of hemorrhage was assessed using hemorrhagic scores and quantified using hemoglobin levels, as recommended by experts . Hemoglobin in the brain parenchyma represents the volume of extravasated red blood cells. After transcranial perfusion, each brain hemisphere was collected and homogenized with cold PBS and sonicated on ice for 30 s, followed by a 15-min centrifuge at 15,000g to collect the supernatant. The level of hemoglobin in the supernatant was determined using a hemoglobin assay kit (BioAssay Systems, Hayward, CA, USA), yielding an optical density value with a microplate reader at 400 nm (Bio-Rad). Each sample’s hemoglobin concentration was calculated as (OD sample − OD blank)/(OD standard − OD blank) 200 (mg/dL). To assess hemorrhagic scores, we cut rat brains into 2-mm coronal slices and photographed them digitally. We previously described how hemorrhages were macroscopically classified into five severity levels .
Peroxynitrite and superoxide detection in ischemic brain tissue
At 24 h after MCAO, brain tissues were collected and immediately frozen in embedding medium (Leica). To detect peroxynitrite, frozen brain sections (20 µm) were rapidly prepared and incubated with our specific fluorescent probe HKYellow AM . To detect superoxide separately, a commercially available superoxide probe HEt (Thermo Fisher Scientific) was used. At room temperature, either probe was incubated at a concentration of 20 µM for 30 min. The samples were then fixed with 4% PFA, and fluorescent signals from the probes were detected using the Carl Zeiss LSM 880 confocal microscope.
Cell culture and treatment
To investigate the effect of AGNHW on peroxynitrite scavenging in vitro, we used brain microvascular endothelial b.End3 cells from the American Type Culture Collection (ATCC) and cultured them in a humidified atmosphere with 5% CO2 and 95% air at 37 °C. Cell culture medium was high glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) with 10% fetal bovine serum (Gibco, USA) and 1% penicillin–streptomycin (Life Technologies).
b.End3 cells were subjected to oxygen and glucose deprivation (OGD) followed by reoxygenation to simulate oxidative stress injury. During OGD treatment, the cell culture medium was replaced with the same medium but without glucose and placed at 37 °C in a humidified airtight chamber saturated with 95% N2/5% CO2. We wanted the oxygen concentration to be less than 1%, as measured by an oxygen analyzer (Sable Systems, Las Vegas, NV, USA). Cells in the control group were incubated in normal DMEM medium at 37 °C in a humidified incubator with 5% CO2 and 95% room air. After 5 h of OGD treatment, cells were reoxygenated and incubated for another 5 h in normal DMEM medium. During reoxygenation, cells were treated with t-PA (20 µg/ml) or the same volume of vehicles. b.End3 cells were also treated with AGNHW extract (50 µg/ml, 100 µg/ml) or vehicle.
Peroxynitrite and superoxide detection in vitro
We used hydroethidine (HEt) and HKYellow-AM fluorescent probes to detect superoxide and peroxynitrite in b.End3 cells, similar to in vivo detection. The b.End3 cells were incubated with HEt and HKYellow-AM (10 µM) separately at 37 °C for 20 min after 5 h of OGD plus 5 h of reoxygenation, with or without t-PA treatment or AGNHW treatment. A fluorescence microscope (Carl Zeiss) with an Axio Vision digital imaging system was used to detect the fluorescent signals.
Immunostaining was used to look at the expression of 3-NT, matrix metalloproteinase-9, tight junction protein claudin-5, and extracellular matrix collagen IV in brain tissues 24 h after a stroke. Frozen brain sections were blocked with 5% goat serum (Thermo Fisher Scientific) for 1 h before being incubated with primary antibodies overnight at 4 °C: anti-3-NT (Abcam, 1:50), MMP-9 (Santa Cruz, 1:100), Claudin-5 (Thermo Scientific, 1:200), and Collagen IV (Thermo Fisher Scientific) (Abcam, 1:800). Secondary antibodies were incubated at room temperature for 2 h, including Alexa Fluor 568 Goat anti-mouse (Invitrogen), Alexa Flor 488 Goat anti-rabbit (Invitrogen), Alexa Flor 647 Goat anti-mouse (Invitrogen). The nucleus was stained with DAPI. Fluorescence signals were detected and captured using a Carl Zeiss LSM 780 confocal microscope system.
Western blot analysis
Protein was extracted from brain hemispheres on ice using RIPA buffer containing 1% protease and a phosphorylate inhibitor cocktail. We loaded the same amount of total protein for electrophoresis after quantifying the protein concentration, and then transferred the protein to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% bovine serum albumin (BSA) for 1 h, the membranes were incubated overnight at 4 °C with the following primary antibodies: β-actin antibody (Cell signaling, 1:4000), p47 phox (Santa Cruz, 1:200), p67 phox (Santa Cruz, 1:200), iNOS (Abcam, 1:500), 3-NT (Abcam, 1:500). HRP-conjugated secondary antibody (Cell Signaling Technology) was incubated with the membrane at room temperature for 2 h. Protein bands were detected using ECL Advance (GE Healthcare Bio-Sciences, USA) with the Bio-Rad system. Image Lab software was used to calculate the intensities of all bands.
The MMP-9 band activity on gelatin gels was investigated using gelatin zymography . Brain native protein samples were loaded in 10% acrylamide gel containing 1 mg/ml gelatin (Sigma) for electrophoresis. Following electrophoresis, gels were washed 2.5% Triton-100 to remove SDS before being incubated with developing buffer at 37 °C for 48 h. After Coomassie blue staining, active MMP-9 was visible as a transparent band. Wash the gels with destain buffer (40% methanol, 10% acetic acid, and 50% ddH2O) until the MMP-9 bands became sharp. The MMP-9 was photographed digitally, and the intensity of the bands was analyzed using ImageJ software (NIH, Bethesda, MD, USA).
Measurement of t-PA activity
We examined whether AGNHW affects the t-PA activity by using a commerical assay kit (Abcam), with the manufacture provided protocol. The kit's standard t-PA can convert plasminogen to plasmin, which can then be quantified to reflect its activity. We tested whether AGNHW extracts affect t-PA activity by adding water extract or ethanol extract to the reaction system at a final concentration of 100 µg/ml. As a control, the same volume of blank solution was used. At 24 h after the reaction, the OD value was measured with a microplate reader at a wavelength of 405 nm (BioRad).
Using GraphPad Prism, we performed a one-way ANOVA followed by a post-hoc Tukey test on the data. The statistical significance level was set at P 0.05. In graphs, all data were expressed as Mean ± SEM.