Animal and Drugs
Animal experimental protocols were approved by the Gannan Medical University Animal Care and Use Committee. All animal experiments were performed following the Guidelines for the Care and Use of Laboratory Animals of China Medical University. Sprague-Dawley (SD) rats (Male, 250-300g) were purchased from Hunan SJA Laboratory Anima Company (Hunan, China) and were housed at 50-60% relative humidity and a temperature of 22-25℃. The rats drank sterile running water and took SPF grade chow freely. Before performing experiments, all rats were acclimatized to the laboratory environment for at least one week.
GSS was purchased from Shanghai Skychemical Co., Ltd (Shanghai, China). It was dissolved in normal saline (NS) to 1 mg/kg concentration before use. Specific α7nAChR-antagonist α-bungarotoxin (α-BTX, #ab120542) was obtained from Abcam (Cambridge, United Kingdom). It was dissolved in 0.1 M phosphate buffer solution (PBS).
Reagents
Tris, sodium dodecyl sulfate (SDS), 30% acrylamide and protein lysis buffer (#R0010) were obtained from Solarbio Science & Technology Company (Beijing, China). TRzol was purchsed from Ambion company (Texas, United States). Anti-CD11b (#ab1211), anti-IL-1β (#ab9787), anti-CD40 (#ab13545), anti-CD68 (#ab31630), and anti-α7nAChR (#ab10096) were purchased from Abcam (Cambridge, United Kingdom). Anti-IKK (#2682s), anti-phospho-IKK(#2697s), anti-P65-NF-κB (#242s), anti-phospho-P65--NF-κB (#3033s), anti-IκB(#4814s) and anti-phospho-IκB (#9246s) were purchased from Cell Signaling Technology (Massachusetts, United States). Anti-β-tubulin (#MA511732), AlexaFluor488 mouse secondary antibody (#A11029), AlexaFluor488 rabbit secondary antibody (#A11034), enhanced chemiluminescence reagent (ECL) Western blotting detection reagents (#32106), Reverse Transcription Master Mix kit (Invitrogen, #4374966), SYBRÒ Select Master Mix (Life Technologies, #4472908), 0.25% trypsin (Gibco, #25200-056), DMEM medium (Gibco, #31800-014), fetal bovine serum (FBS) (Gibco, #10099-141) and penicillin streptomycin combination (Gibco, #15140-122) were purchased from Thermo Fisher Scientific (Massachusetts, United States). 2-, 3-, 5-triphenyltetrazolium chloride (TTC, #T8877-25G) was purchased from Sigma (United States).
Transient middle cerebral artery occlusion and reperfusion (tMCAO) rat model and drug treatments
Rat tMCAO model was established, as described previously[32, 36]. Briefly, rats were anesthetized with 1% pentobarbital sodium (50 mg/kg, i.p.), and brain ischemia was induced by inserting a 5-cm-long nylon filament (diameter, 0.24-0.28 mm) into the middle cerebral artery for 2 h. Then, the filament was removed to allow reperfusion of the brain for 24 h. Sham rats were performed a comparable surgery as tMCAO rats but without the occlusion of the middle cerebral artery. The rats were randomly divided into five groups: sham group, tMCAO group, GSS group (tMCAO rats treated with GSS), GSS+α-BTX group (tMCAO rats pretreated with α-BTX and subsequently treated with GSS), and α-BTX group (sham rats were administrated with α-BTX). Since α-BTX is not able to pass through the blood-brain barrier, we injected it into the lateral ventricle at a dose of 0.5 µg/kg body weight 30 min before tMCAO surgery. GSS (1.0 mg/Kg) was administrated via sublingual vein injection 10 min after ischemia. Rats without α-BTX and GSS treatments were parallelly given equivalent vehicle (saline). The schematic diagram of animal treatments is shown in Figure S1A and B in supplementary materials. The doses of GSS and α-BTX were chosen based on previous study and our preliminary experiments[32].
lateral ventricular injection
Rats were anesthetized with 1% pentobarbital sodium (50 mg/kg, i.p.) and fixed on a stereotactic apparatus (ZH-1-Lanxing, RWD, China). When bregma was exposed, a burr hole was prepared on the skull in the right hemisphere (1.6 mm lateral and 0.9 mm posterior to bregma) using a power drill (68605, ϕ 1.4 mm, RWD, China). Then an injector (5-μL micro-syringe, Hamilton, USA) connected with micro-pump (KDS310, Harvard Apparatus, USA) was placed its needle into the right lateral ventricle at 3.5 mm depth of sub-dural through the micro-hole. Placement of needle was performed using a stereotactic apparatus. The vehicle or the α-BTX was then administered into the lateral brain ventricle at a speed of 0.25 μL/min. The needle stayed in place for 5 min after injection to prevent backflow.
Neurological test
All rats were received Garcia assessment at 24h after I/R to evaluate neurobiological function. The evaluation includes six tests scoring on a scale from 3 to 18 [37]: spontaneous activity, symmetry in four limb movements, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch.
TTC staining
After the neurological assessment, rats were anesthetized with 1% pentobarbital sodium (50 mg/kg, i.p.) and sacrificed. Rat brain was collected and performed 2,3,5-Triphenyltetrazolium chloride (TTC) staining. In brief, rat brain was consecutively sliced into six coronal sections (2 mm thickness) and then stained with 0.5% TTC solution for 30min at 37℃ in a water bath shaker in the dark. During the staining, the slices were turned over every 5min. After the staining, the slices were washed with PBS and fixed in 4% paraformaldehyde for 6h. The infarcted area and brain area in TTC-stained brain slices were measured using ImageJ 1.37a. The corrected percentage of infarct volume was calculated as the formula: infarct volume (%) = (the area of contralateral hemisphere - the area of non-infarcted ipsilateral hemisphere) /(2 * the area of contralateral hemisphere) *100%[37].
Cell culture and treatments
BV2 microglial cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). BV2 cells were grown in Dulbecco’s modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in a cell incubator with 5.0% CO2 at 37°C. Cells were seeded in culture dishes at a density of 1×106 cells/mL and treated with LPS (1.0 μg/mL) for 24 h to induce M1 polarization. A schematic diagram for in vitro experiments is shown in Figure S1C and D in supplementary materials.
To determine the effects of GSS on M1 polarization, cells were randomly allocated into four groups: control group (treated with vehicle), LPS group (treated with LPS), LPS+GSS group (treated with LPS plus GSS treatment, 10 μM), and GSS group (treated with GSS alone). Cells were treated with GSS for 24 h.
In order to determine whether α7nAChR-mediated NF-κB signaling was involved in the effects of GSS on microglial M1 polarization, cells were randomly divided into six groups: control group (treated with vehicle), LPS group (treated with LPS), LPS+GSS group (treated with LPS plus GSS treatment, 10μM), GSS group (treated with GSS alone), α-BTX+LPS+GSS group (pretreated with α-BTX followed by LPS and GSS treatment), and α-BTX+LPS group (pretreated with α-BTX followed by LPS incubation). Cells were pretreated α-BTX (10nM) 30min before given LPS incubation.
Western blot
Ischemic penumbra brain tissues were collected for western blotting. Brain tissues were added protein lysis buffer and homogenized on ice for 1h. BV2 cells were also lysed with protein lysis buffer on ice for 1h. Then these samples were centrifuged at 12 000 g for 15 min to obtain the supernatants. The protein concentration in samples was measured by BCA kit according to the manufacturer’s instructions. Equivalent total protein (30 μg) in each sample was separated in SDS/PAGE gel by electrophoresis and transferred to PVDF membrane. After blocking with 5% non-fat milk, the membrane was incubated with following primary antibodies overnight at 4℃: CD11b (1:1000), IL-1β (1: 500), CD40 (1:500), CD68 (1:500), α7nAChR (1:500), IKK (1:1000), phospho-IKK (1:500), P65-NF-κB (1:1000), phospho-p65--NF-κB (1:500), IκB (1:1000), phospho-IκB (1:500) and β-tubulin (1:2000). Then the membrane was incubated with respective secondary antibodies (1:5000) at room temperature for 1h and then incubated with an enhanced chemiluminescence reagent for 1 min. Finally, protein band images were captured, and the gray-scale of each band was analyzed using Chemiluminescence Imaging System (Amersham™ Imager 600, United States).
Quantitative PCR (qPCR)
Total RNA was isolated from tissues or cells using TRIzol reagent according to the manufacturer’s instructions. 4 μg total RNA was used to synthesize the first strand cDNA using Reverse Transcription Master Mix kit (SYBRÒ Select Master Mix). Then PCR reaction was performed as following reaction system: cDNA 2 μL, 1x SYBRÒ 10 μL,10 μM primer 2 μl,adding ddH2O to a total volume of 20 μL. Parameters for PCR reaction were as bellows: pre-denatured at 95℃ for 20 min follow by 40 cycles (desaturated at 95℃ for 10s, annealed at 61℃ for 20s, and extended at 72 ℃ for 25s). PCR results were normalized to GAPDH and expressed as folds of sham or control group. The sequence of primers used in this study was shown in Table 1.
ELISA assay
The concentration of IL-1β in the culture supernatant was determined using enzyme-linked immunosorbent assay (ELISA) kit (R&D systems, Minneapolis, MN) according to the manufacturer’s instructions.
Immunofluorescence
Rats were anesthetized and fixed the whole animal with 4% paraformaldehyde through transcardial perfusion. The brain was then taken out, immersed in 4% paraformaldehyde, dehydrated with 10% sucrose, and cut into brain slices (30 μm thickness) by freezing microtome. Dried brain slices were hydrated with PBS at room temperature (RT) for 20min, and then the slices were incubated in 0.3 % Triton X-100/PBS solution at RT for 10-15 min. After washed with PBS for 5 min×3 times, the slices were blocked with 3% BSA at RT for 30 min and incubated with anti-CD11b antibody (1:200) at 4℃ overnight. Then the slices were washed with PBS and incubated with AlexaFluor488 mouse secondary antibody (1:2000). Finally, the slices were stained with DAPI and sealed with 50% glycerin. The immunofluorescent images were captured under a fluorescence microscope (Carl Zeiss Lsm880, Germany).
Immunofluorescence for BV2 cells was performed as bellows: cells were fixed with cold methanol at -20℃ for 5 min, washed with PBS for 5 min×3 times. The subsequent staining procedure was the same as above brain slice immunofluorescence, except for incubating with anti-IL-1β (1:100) or anti-P65-NF-κB (1:100), and then incubated with AlexaFluor488 rabbit secondary antibody (1:2000).
Statistical analysis
Data were presented with mean ± standard deviation (SD). Statistical analysis was performed using SPSS 20.0 software. One-way ANOVA with Newman-Keuls test was used to compare the differences between the means of more than two groups. The value of P<0.05 was considered statistically significant.