Reagents
TAK-242 (HY-11109) was obtained from MedChemExpress (Monmouth Junction, NJ, USA). TLR4 (sc-293072), MyD88 (sc-74532), IκBα (sc-1643), and NF-κB p65 (sc-8008) primary antibodies were provided by Santa Cruz Biotechnology (Dallas, TX, USA). COX-2 (12282), GFAP (3670), p38 (8690), JNK (9252), ERK1/2 (9102), p-EEK1/2 (Thr202/Tyr204) (9101), and GAPDH (2118) primary antibodies were provided by Cell Signaling Technology (Danvers, MA, USA). iNOS (ab3523) and TNF receptor-associated factor 6 (TRAF6, ab33915) primary antibodies were provided by ABCAM (Cambridge, MA, USA). Each secondary antibody was provided by Thermo Fisher Scientific (Rockland, IL, USA).
PTH capsules were purchased from Zhangzhou PTH Pharmaceutical Co., LTD. (Zhangzhou, China). In accordance with our reported UPLC–MS/MS method [22], 21 characteristic compounds (taurine, malic acid, citric acid, cholic acid, hyodeoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid, glycodeoxycholic acid, glycocholic acid notoginsenoside R1, ginsenosides Rg1, Rb1, Re, Rf, Rd, Rg2, Rg3, Rh1, and muscone) in the PTH capsules were accurately quantified, and the total content of those compounds was 183.73 mg/g.
Animals
Male Sprague–Dawley rats weighing 230–260 g were provided by the Guangdong Experimental Animal Center (Guangzhou, no. SYXK-2019-0007). Animals were provided food and water ad libitum under SPF conditions at the temperature of 24°C ± 1°C and relative humidity of 60% ± 5%. All animal experiments were conducted with the approval of the Animal Care and Use Committee of Fujian University of Traditional Chinese Medicine.
Preparation of the middle cerebral artery occlusion model
The transient middle cerebral artery occlusion (MCAO) model was established in accordance with our previous protocol [23]. Briefly, male SD rats were anaesthetized with 5% isoflurane (Shenzhen, China) and maintained with 2% isoflurane. The left common, external, and internal carotid arteries were separated. A 0.38 mm nylon suture with a L3800 silicon-coated tip (Guangzhou, China) was carefully inserted into the internal carotid artery from the external carotid artery to occlude the middle cerebral artery. After occlusion for 1.5 h, the nylon suture was withdrawn to induce reperfusion. Sham rats were subjected to the same procedure without inserting the nylon suture into the MCAO.
Drug administration
Experiment 1: After adaption for 7 days, the rats were randomly allocated to three groups (n = 12): (1) Sham group: sham rats intragastrical (i.g.) administered 0.5% CMC-Na (10 mL/Kg); (2) MCAO group, MCAO rats i.g. administered 0.5% CMC-Na (10 mL/Kg); (3) MCAO + PTH group, MCAO rats i.g. administered PTH (10 mL/Kg). PTH was dissolved in 0.5% CMC-Na (18 mg/mL) and administered at the dose of 180 mg/kg once a day for 4 days before MCAO as previously described [18].
Experiment 2: After adaption for 7 days, animals were randomly divided into five groups (n = 6): (1) Sham group: sham rats i.g. administered 0.5% CMC-Na; (2) MCAO group, MCAO rats i.g. administered 0.5% CMC-Na; (3) MCAO + PTH group, MCAO rats i.g. administered PTH (180 mg/Kg); (4) MCAO + TAK-242 group, MCAO rats intraperitoneal (i.p.) administered TAK-242 (3 mg/kg); (5) MCAO + PTH + TAK-242 group, MCAO rats i.g. administered PTH (180 mg/Kg) and i.p. administered TAK-242 (3 mg/kg). TAK-242, an antagonist of TLR4, was dissolved in 10% DMSO and administered at the dose of 3 mg/kg at 1.5 h after MCAO. The other experimental conditions were the same as those described above.
Neurological deficit evaluation
In accordance with the reported procedures and criteria [18], the neurological deficit condition of each rat was evaluated blindly at 24 h after reperfusion.
Infarct volume measurement
Magnetic resonance imaging (MRI) test was used to determine the infarct volume as follows: A rat was anesthetized with 5% isoflurane and fixed in the animal cradle with its head inside the horizontal magnet bore of a BioSpec 70/20 USR 7.0 T MRI scanner (Bruker BioSpin, Ettlingen, Germany). Then, the coronal plane image was prescribed beginning at 3 mm behind the olfactory bulb. T2WI scans were performed with a turbo-rapid acquisition relaxation enhancement sequence. All images were obtained with the repetition time of 4200 ms, echo time of 55 ms, field of view of 32 × 32, matrix size of 256 × 256, slice thickness of 1 mm, and number of slices of 21. Image J software was used to calculate the infarct volume.
Hematoxylin–eosin staining
Each rat was anesthetized with 5% isoflurane, and its heart was perfused successively with saline and 4% paraformaldehyde. Then, its brain was removed, fixed, dehydrated, and embedded. The brain was cut into 5 µm thick sections and stained with hematoxylin and eosin (Beyotime, Shanghai, China). Finally, the sections were photographed under a DMi8 light microscope (Leica, Wetzlar, Germany).
qRT-PCR analysis
Ischemic brains were removed from deeply anesthetized rats at 24 h after reperfusion. Total RNA was extracted by using Trizol (Invirtogen) and reverse transcribed into cDNA with a Superscript First-Strand Synthesis System (Life Technologies, Grand Island, NY). Quantitative analysis was performed by using SYBR Green real-time PCR master mix (Life Technologies). GAPDH was used as the internal control in this study. The PCR primer sequences are listed as follows:
IL-1β Forward: 5'-GTGTTTTCCTCCTTGCCTCTGAT-3'
Reverse: 5'-GCTGCCTAATGTCCCCTTGAAT-3'
IL-6 Forward: 5'-CTGTCTGACCCATGTGAGCTG-3'
Reverse: 5'-TTTGTCGTTGCTTGTCTCTCCTT-3'
TNF-α Forward: 5'-ATGGGCTCCCTCTCATCAGT-3'
Reverse: 5'-GCTTGGTGGTTTGCTACGAC-3'
MCP-1 Forward: 5’-CAGGTCTCTGTCACGCTTCT-3’
Reverse: 5’-GTAGTTCTCCAGCCGACTCA-3’
GAPDH Forward: 5'-CAACGGGAAACCCATCACCA-3'
Reverse: 5'-ACGCCAGTAGACTCCACGACAT-3'
Immunofluorescent staining
Each brain was removed and embedded in paraffin as described above. After dewaxing and rehydration, each brain section (5 µm) was subjected to antigen retrieval solution for 15 min in a microwave oven and then blocked with 5% BSA containing 10% goat serum for 2 h. It was incubated overnight at 4°C with mouse monoclonal anti-NF-κB p65 antibody (1:100) or anti-Iba-1 antibody (1:300) diluted in the blocking solution. Subsequently, it was washed with PBST and incubated with FITC-conjugated goat antimouse IgG (1:200) for 1 h at 25°C. The nuclei were counterstained with DAPI (Beyotime) and cover-slipped. Finally, the immunofluorescent images were captured under a DMi8 microscope (Leica) and magnification of 200⋅. The number of NF-κB p65 or Iba-1-positive cells in five similar fields of the ipsilateral cortex from three sections per rat was counted by an investigator who was blinded to the treatment.
Western blot analysis
Proteins were extracted from brain tissues by using RIPA lysis buffer. BCA Protein Assay kit (Beyotime, Shanghai, China) was used to measure the protein concentration of each sample. Western blot analysis was conducted in accordance with our previous study [18]. In brief, each protein sample (30 µg) was separated through SDS-PAGE then transferred and blocked. Primary antibodies against TLR4 (1:500), MyD88 (1:500), TRAF6 (1:5000), IκBα (1:500), NF-κB p65 (1:500), p38 (1:1000), p-p38 (1:1000), JNK (1:1000), p-JNK (1:1000), ERK1/2 (1:1000), p-ERK1/2 (1:1000), COX-2 (1:1000), iNOS (1:500), GFAP (1:1000), and GAPDH (1:1000) were used. GAPDH was used as a control. Images were visualized by using ChemiDoc XRS + imaging system (Bio-Rad, Hercules, CA), and the target bands were scanned and analyzed quantitatively by using Image J software.
Statistical analysis
Data were expressed as means ± SEM and analyzed with SPSS software (version 20.0). One-way AVONA was used when the data conformed to normal distribution, whereas Kruskal–Wallis test or Mann–Whitney test was used when the data conformed to non-normal distribution. p < 0.05 was defined as statistically significant.