The preparation of NSTC
NSTC (batch No.: 160510) were provided by Guangdong Huanan Pharmaceutical Co., Ltd. (Dongguan, China). The product (TP, 33.33%; PR, 23.78%; CR, 19.10%; GR, 9.55%; RR, 14.23%) was produced according to the 2015 Chinese Pharmacopoeia (CP) as follows. 1) Ethanol (70%) was added to PR which was heated and extracted twice for 1 h each time. Then the extracts were combined and filtered after ethanol was recovered from the filtrates, and the mixture was concentrated to an appropriate amount, dried, pulverized, and added with an appropriate amount of calcium hydrogen phosphate. They were mixed and dried for later use. CR was added with 80% ethanol and heated twice under reflux for 1 h each time. The mixed extracts were filtered for further use. The dregs of PR and CR, TP (confined in gauze bags), GR, and RR were boiled in water twice, 1 h each time, to obtain decoction which was then filtered, and the filtrate was concentrated to a clear paste with a relative density of 1.04 to 1.10 (40 °C). The ethanol was added to make the alcohol content reach 70%; Afterward, the supernatant was collected and mixed with the alcohol extract of CR. After ethanol was recovered, the mixture was concentrated to an appropriate amount, dried, crushed, and added with an appropriate amount of calcium hydrogen phosphate. After PR dry powders were added, it was granulated with hypromellose ethanol solution, dried, and mixed with talc, silicon dioxide and magnesium stearate to make capsules.
Standardization of NSTC
The constituent analysis of NSTC was carried out by mass spectrometry (MS). NSTC (0.5 g) was put in a container for a 30-min methanol (10 mL) bath at 40 kHz twice. The extract solution (1 mL) was filtered through a 0.22 μm membrane before analysis. The reference standards of typhaneoside, gallic acid, gastrodin, catechin, chlorogenic acid, β-elemene, paeoniflorin, protocatechuate, apigenin, β-ecdysterone, isorhamnetin-3-O-neohespeidoside (National Institute for the Control of Pharmaceutical, China); calycosin, naringenin (Sigma, St. Louis, USA) were dissolved in methanol at the concentration of 0.1 mg/mL for each compound to make the mixed standard solution.
The UPLC-Trip-TOF–MS/MS analysis was performed by the Triple TOF-TM 5600 plus (AB SCIEX, Foster City, CA) hybrid triple quadrupole time-of-flight mass spectrometer equipped with Analyst® TF 1.6 software (AB SCIEX, Foster City, CA). Both negative and positive mode was set for compounds ionization. The conditions of MS detector were as described elsewhere . The chromatographic separation was carried out on a Shimadzu UFLC XR instrument (Shimadzu, Kyoto, Japan) and a Phenomenex Kinetex column (2.1 mm × 100 mm, 2.6 μm, Phenomenex, CA, USA). The mobile phase consisted of 0.1% aqueous formic acid (Sigma, St. Louis, USA, v/v, A) and acetonitrile (Sigma, St. Louis, USA, v/v, B) at a flow rate of 0.3 mL/min. The following gradient elution process were used: isocratic 2% solvent B (0–3 min), linear gradient from 2 to 60% solvent B (3–20 min), 60–100% solvent B (20–32 min), 2% solvent B for 8 min. The injection was 10 μL and the column temperature was 25 ℃.
The Ultimate 3000 DGLG System (Dionex, CA, United States) with DAD was applied for paeoniflorin determination. The mobile phase consisted of 0.1% aqueous formic acid (v/v) (A) and acetonitrile (B) run on a C18 Column (5 μm, 250 mm × 4.6 mm, Dionex CA, United States) at a flow rate of 1 mL/min. The following gradient elution process were used: isocratic 4% solvent B (0–10 min), linear gradient from 4 to 50% solvent B (3–30 min), 50–100% solvent B (30–35 min), 4% solvent B for 5 min. The DAD detector was set at 230 nm while the volume of injection was 10 μL.
Data preparation and target prediction
Nineteen transition components of NSTC (9 prototype components and 10 metabolites) that had been detected in the brain in the previous study  were used for screening potential targets. Details of their chemical structures and information were shown in Additional file 1: Figure S1 and Table S1 respectively. The chemical structures of the 19 compounds were plotted using Chemical Draw 14.0 and saved in mol and sdf formats. Database retrieval, text information mining, reverse molecular docking and target prediction were performed to identify the putative target proteins of the NSTC comprehensively. We searched in TCMSP (https://lsp.nwu.edu.cn/tcmspsearch.php), TCMID (https://www.megabionet.org/tcmid/), Drugbank (https://www.drugbank.ca/) and other databases with the compound’s name as key words to collect the targets of the compounds. The online reverse molecule docking tool Pharmmapper (https://www.lilab-ecust.cn/pharmmapper/) was used to analyze the possible targets of small molecule compounds by submitting the sdf format file of the compound to the database. Target prediction was performed with the online target prediction tool TargetHunter, and the mol format files of compounds were imported into the online platform to find underlying targets of NSTC.
We searched stroke-related targets in TCMSP, TTD (https://db.idrblab.net/ttd/), DrugBank, OMIM (https://www.ncbi.nlm.nih.gov/omim) for related targets of stroke. Subsequently, text mining was also conducted to construct a database of stroke targets . Wayne analysis was carried out to obtain the common targets of NSTC and stroke using software Venny (Version 2.1). The common targets were presumed to be putative targets that might exert pharmacological effects on stroke, and they would be used for later analysis.
Network construction and analysis
We constructed a network of active components and putative targets of NSTC for the treatment of stroke based on their interactive data. The target proteins were analyzed using the mutiple-proteins model in the STRING database to construct the protein–protein interaction network (PPI). Cytoscape software (Version 3.0) was employed to visualize the PPI. Degree, betweenness centrality and closeness centrality were analyzed by the Network Analyzer plug-in in Cytoscape, which was used to measure the topological importance of nodes in the network. Target protein names were converted into the corresponding gene names by the Uniprot database and imported into the DAVID database for the KEGG pathway enrichment analysis.
One hundred and fifty-eight male C57BL/6 mice, weighed 18–25 g were provided by the Guangdong Medical Laboratory Animal Center (Guangzhou, China). The experimental protocol was approved by the Animal Ethics Committee of the School of Life Sciences at Sun Yat-sen University. All mice were housed in the animal room (25 ± 2 °C, 60 ± 5% relative humidity) with a 12 h dark/light cycle. The mice were assigned to five groups, the Sham group (n = 38), the ischemia stroke group (I/S group, n = 38), the NST 1 group (n = 22), the NST 2 group (n = 22), the NST 4 group (n = 38). Mice in the NST 1, 2, and 4 group were administrated with NSTC suspension at daily dose 0.47 (quivalent to the clinical dose, content powder/body weight), 0.94 (twice the clinical dose), and 1.87 g/kg (quadruple the clinical dose) once a day for 7 days. Mice in the Sham and I/S groups were administrated with pure water once a day for 7 days. The photochemical experiment was performed 30 min after the last administration on the 7th day.
Experimental photothrombotic models
After the last administration on the 7th day, the animals were anesthetized with chloral hydrate (Aladdin Reagent, Shanghai China, 420 mg/kg). An incision was made in the middle of the animals’ scalp to expose and clean the skull. Within a 4 mm diameter region, the left parietal bone was polished to approximately 40–60 μm using a high-speed drill. The irradiation area center was 2 mm posterior to the bregma and 2 mm lateral to the sagittal suture. After the Rose Bengal dye (Bomei Biotechnology, Hefei, China, 100 mg/kg) was intraperitoneally injected, all the mice except for those in the Sham group were put on an electric blanket to maintain body temperature, and the mouse skull was illuminated with a 0.01 mW green laser for 15 min. The scalp was sutured, and the animals were allowed to recover. The mice were kept on administration once a day until sacrificed.
Neurological function evaluation using mNSS
The mNSS (8 mice each group) was conducted at 24 h and on the 7th day after illumination to appraise neurological recovery of the stroke mice. It consisted of several aspects encompassing motor function, sensory, reflex, and balance tests, with full marks 18 points. The higher the score was, the more serious the nerve damage would be . The detailed process of mNSS methodology and appraisal was shown in Additional file 1: Table S2. 30 min after mNSS test, the mice were sacrificed for western-blot and assay kit test.
TUNEL assay and Nissl staining
Brains (3 mice each group) were collected from mice at 24 h and 7d after stroke which were perfused with 20 ml normal saline followed by 20 ml 4% paraformaldehyde, and were then fixed with 4% paraformaldehyde and dehydrated with 20% and 30% sucrose. The samples were embedded in OTC compound, frozen in liquid nitrogen, and sliced to a thickness of 20 μm using a freezing microtome (Leica CM1950, Leica, German). TUNEL staining and Nissl staining were carefully performed according to the manufacturers’ instructions by one-step TUNEL apoptosis detection kit (Beyontime Biotechnology, Shanghai, China) and Nissl Staining Solution (Beyontime Biotechnology, Shanghai, China) respectively. An anti-fluorescence quenching method was used during tissue sealing. The slides stained with TUNEL were observed under a fluorescence microscope (excitation wavelength ≈ 550 nm, emission wavelength ≈ 570 nm; Leica DM6B, German), and the ischemic area was photographed. The brain slices stained with Nissl were observed under a light microscope. Both Nissl- and TUNEL-positive cells in three randomly selected ischemic areas of the cerebral cortex were counted.
RNA extraction and mRNA library construction
The mice for transcriptomics analysis (8 in each group) were sacrificed at 4 h, 24 h, and 7 d after surgery. Brains were obtained from the mice which were perfused with 20 ml physiological saline. The ischemic tissue of the mouse brain was quickly collected and stored in a centrifuge tube with RNAlater at − 80 °C for subsequent experiments.
Total RNAs were extracted from the samples using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. DNase I was used to digest double- and single-stranded DNAs in total RNAs. The cDNA was reversely transcribed from purified mRNA which was fragmented using buffer at an appropriate temperature. The PCR products were heat-denatured, and circularized by the oligo sequence. The single-strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified and single-end 50 bases reads were generated on the BGISEQ500 platform (BGI, Shenzhen, China). Any unsatisfactory read obtained by sequencing was removed from the raw data to obtain clean reads. HISAT was used to compare the clean reads with the reference genome sequence. To determine the results whether meet the second quality control of alignment, the comparison rate and the distribution of reads on the reference sequence were compared. Quantitative gene analysis and cluster analysis based on gene expression levels were performed, and differentially expressed genes (DEGs) were screened out among the samples. The KEGG enrichment and GO enrichment data analysis was performed on the platform. According to the results of KEGG analysis, DEGs were functionally classified, and the phyper function in the R software was used for an enrichment analysis. The p-values of KEGG pathways and GO enrichment were calculated, and a p of < 0.05 was regarded as statistically significant.
The detection of γ-aminobutyric acid (GABA) and Glutamic acid (Glu) level
GABA (Sigma, St. Louis, USA, 10 mg) and Glu (Sigma, St. Louis, USA, 10 mg) were dissolved by a mixed solution of formic acid, methanol and water (v:v:v = 2:200:800) to prepare a 1 mg/mL mixed standard stock solution. Then it was diluted to concentrations of 10,000, 8000, 6400, 3200, 1600, 800, 400, 200 ng/ml as working solutions respectively. Isoproterenol (25 mg) was dissolved in the same way to prepare a 2.5 mg/ml separate standard stock solution as an IS working solution. The brain tissue (50 mg) was homogenated with 1.89% formic acid solution (500 μL), centrifugated at 14,000 rpm, 4 °C for 10 min, and the supernatant was collected. 5000 μL 1.89% formic acid was added to 100 μL supernatant, and 90 μL mixture was transferred to a new EP tube, and then added with 10 μL working solution, 10 μL IS working solution. Afterward, 200 μL 1% formic acid-acetonitrile was added to obtain a mixture. It was vortexed, mixed, and centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was collected for further analysis.
Samples (50 mg) were added with 180 μL 1.89% formic acid, homogenated, and centrifuged at 14,000 rpm for 10 min at 4 °C. Then the supernatant (2 μL) was mixed with 100 μL 1.89% formic acid, 10 μL IS working solution, and 200 μL 1% formic acid containing acetonitrile, and vortexed. The mixture was centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was obtained for the analysis of GABA and Glu.
The analysis was performed on an Agilent 6410 Triple Quad LC–MS system (Agilent, USA). Chromatographic separation was carried out on an ACE 3 C18-PFP column (150 × 4.6 mm, 2.6 μm, A194318, UK) at 25 ℃. The mobile phase consisted of 0.1% formic acid (v/v) in both acetonitrile (A) and water (B) using a gradient-elution program, linear gradient 95% B at 0–2 min, 10% B at 2–5 min, 10% B at 5–6 min, and 95%B at 6–13 min. The injection volume was 10 μL with the flow rate kept at 0.6 mL/min.
The analytes and IS were ionized by the ESI source in positive ion mode and the ion spray voltage was set at 4000 V. The drying gas flow rate was 12.0 L/min; atomizing gas pressure was 30.0 psi with a source temperature of 325 °C. The Q1 Mass (Da)/Q3 Mass (Da) of Glu, was 148.0/84.0 while GABA was 104.0/45.1. The fragmentation voltage of Glu and GABA was 75 V and 65 V, with collision voltage 13 V and 22 V respectively.
Western blotting analysis
The protein levels of Bax, Bcl-2, and N-methyl-d-aspartate receptor (NMDAR) 1 in brain tissue were quantitated using western blotting analysis. Brain tissues from left hemisphere lysed with 19-fold-volume, ice-cold RIPA lysis buffer were homogenated with on the ice, and centrifuged at 10,000 rpm. The supernatant was obtained for further experiments. Total protein concentration was determined by the enhanced BCA protein assay (Beyondtime Biotechnology, Shanghai China). The protein samples were loaded onto a 10% resolving SDS-PAGE gel and a 10% stacking gel, and fractionated by electrophoresis at 250 V. They were electrotransferred onto PVDF membranes which were subsequently washed three times with Tris-buffered saline containing 0.1% Tween-20 (TBST) for 5 min each time, and blocked in 5% non-fat dry milk in TBST for 2 h. Afterward, they were incubated with primary antibodies, encompassing anti-Bax, anti-Bcl-2 and anti-NMDAR1 (diluted 1:1000, Abam Technology, Cambridge, UK), overnight at 4 °C, and washed three times. The membranes were incubated with the secondary antibody (diluted 1:5000) for 1 h. Protein bands were visualized using a chemiluminescence (ECL) assay kit and photographed using a Syngene Tanon5200 imaging system (Tanon, China). Besides, β-actin (diluted 1:10,000, Santa Cruz Dallas, TX, USA) was used as an internal reference. Expression levels of all proteins were normalized to that of β-actin. The optical density of the bands was determined using Image J software.
SOD, GSH, IL-1β, TNF-α and TGF-β1 assay of Brain tissues biochemistry analysis
After neurological evaluation was completed, the left hemisphere of the brain was completely moved out and stored at – 80 ℃. Brain tissues (30 mg) from left hemisphere were added with pre-chilled cell lysates (600 μL), homogenated on ice, and centrifugated at 3500 rpm. The supernatant was collected. The protein concentration was determined using a BCA assay. The SOD activity, GSH content at were tested by assay kits (Nanjing Jiancheng, Nanjing, China) and expressions The ELISA ssay kits of IL-1β (Meimian, Wuhan, China), TNF-α (Meimian, Wuhan, China) and TGF-β1 (Cusabio, Wuhan, China) were used to quantitate the expression level according to the manufacturers’ instructions.
All data were analyzed using Graphpad prismprogram 5.0 and presented as means ± SEM. One-way ANOVA (nonparametric test) with Dunnett post-hoc test was used to analyzed the histopathological data. The other data was analyzed by one-way ANOVA with Bonferroni post-hoc test. A p value < 0.05 was considered statistically significant.