Drugs and reagents

Shenma Jingfu Granule (Approval No: HYZZ 205050324) was provided by Yueyang Hospital, Shanghai University of Traditional Chinese Medicine. Anti-VEGF Polyclonal Antibody (K009531P) was purchased from Solarbio (Beijing, China). Anti-HIF-1 Alpha antibody (bs-20399R) was from Bioss (Beijing, China). Anti-vWF antibody (PB9273) was purchased from Boster (Wuhan, China). Standards used for SPR analysis were provided by Standard Technology (Shanghai, China).

The World Flora Online (http://www.worldfloraonline.org) was used to verify the complete plant names on December 8, 2023.

Chemical identification and profiling by UPLC-Q-TOF -MS

The chemical composition of the SMJF Granule was analyzed through UPLC-Q-TOF–MS. In brief, a Waters ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm) was utilized for gradient elution. As the mobile phase, distilled water was mixed with 0.1% formic acid solution and 0.1% acetonitrile solution. Flow rate: 0.3 mL/min. This experiment was conducted with a 30 °C column temperature, and the injection volume was 2 μL. The detection wavelength varied from 190 to 400 nm. Mass spectrometry detection was performed in ESI negative/positive ion mode. Ion Source Gas 1/2: 50 psi; Ion Source Temperature: 500 °C; Ion Spray Voltage Floating: − 4500/5000 V; Declustering Potential: 100 V; Collision Energy: ± 40 eV; TOF mass range: 50 ~ 1700; MS/MS mass range: 50 ~ 1250. Qualitative analysis of SMJF Granule was performed, resulting in total ion flow diagrams in both positive and negative modes under the mentioned conditions.

Five rats were given oral suspensions (1.2 g/kg, seven times the clinical equivalent dose) of SMJF Granule twice daily for five consecutive days to determine the absorption of the ingredients in plasma and brain. On the final day, rats were euthanized, and samples of plasma and brain tissue were obtained and analyzed by UPLC-Q-TOF–MS for the absorbed ingredients of SMJF Granule.

Prediction of SMJF Granule candidate targets in CI

The potential targets of absorbed ingredients in blood and brain were predicted by the PharmMapper (http://www.lilab-ecust.cn/pharmmapper/), Swiss Target Prediction (http://www.swisstargetprediction.ch/) and HERB (http://herb.ac.cn/) databases. Disease targets related to cerebral ischemia were collected by searching DisGeNET (http://www.disgenet.org/web/ DisGeNET/menu/home), Therapeutic Target Database (TTD) (https://db.idrblab.org/ttd/), and OMIM database (https://omim.org/) using the keywords “cerebral ischemia” and “ischemia stroke”. In addition, the Venn diagram was used to screen the intersection targets of the absorbed ingredients of SMJF Granule and cerebral ischemia. To visualize the network of SMJF Granule twelve herbs-ingredients-targets, the Cytoscape software (version 3.8.0) was employed.

Protein–protein interaction (PPI) network and functional enrichment analysis

Understanding the complex relationship between SMJF Granule and cerebral ischemia targets is challenging due to intricate biological processes involving multiple proteins. To address this, the overlapping targets were then uploaded to the STRING database (https://string-db.org/) with “Homo sapiens” as the species, and only those targets with interaction values exceeding 0.4 were considered for further analysis. The resulting PPI network was visualized in Cytoscape. To understand the roles of the targets of SMJF Granule active ingredient in particular signal pathways, GO enrichment analysis and KEGG pathways with modified values of p less than 0.05 for the potential targets of SMJF Granule in the treatment of cerebral ischemic were obtained. The GO biological process, cellular analysis, molecular function, and KEGG pathway dot plot of core targets were plotted by https://www.bioinformatics.com.cn.

Transcriptome analysis

The total RNA from the penumbra tissues of rat brains was extracted by the Trizol reagent kit with the determination of their integrity and concentration. Subsequently, the RNA was randomly fragmented, followed by the synthesis of double-stranded cDNA. The construction of cDNA libraries involved end repair, poly(A) tailing, and sequencing adapter ligation, followed by PCR amplification. After library quantification, high-throughput sequencing of multiple samples was conducted using the Illumina HiSeq sequencing platform in paired-end mode. The FastQC software was used to conduct a quality control examination on preprocessed data. With STAR software, the preprocessed sequences were matched to the sequenced species' reference genome sequence. Subsequently, to determine the quantity of reads mapped to every gene, StringTie software was utilized. Gene length was used to compute each gene’s FPKM (fragments per kilobase per million) values and read counts which were then mapped to the respective genes. Differential expression analysis between different sample groups was conducted using DESeq2 software, with the criteria of |log2FC|≥ 1 and P ≤ 0.05 used to filter differentially expressed genes between the two groups.

Molecular docking

For molecular docking in AutoDock 1.5.6 Vina, we selected the key compounds with the higher degrees in the SMJF Granule twelve herbs-ingredients-targets network and the HIF-1α protein. The 3D structure of the protein was downloaded from the RCSB PDB database (https://www.rcsb.org/), and the 2D structural compounds were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The protein and compounds were preprocessed using PyMol 2.4.0 software. Molecular docking and binding affinity calculations were conducted by AutoDock Vina. The 3D structure of the complexes was visualized with PyMol to observe the interaction between the receptor protein and compounds, including hydrogen bonding.

SPR analysis

In this study, the HIF-1α protein was chosen for SPR analysis based on network pharmacology. Standards were dissolved in DMSO to 20 mM. SPR assays were performed using a Biacore T200. Briefly, the protein was immobilized on a CM5 chip via EDC/NHS cross-linking. Standard compounds were diluted (0.01–128 µM) in 5% DMSO-phosphate buffered saline. Sensor surface stability was assessed by repeating the average concentration. Analytes were injected at 30 μL/min with 60 s binding and 120 s dissociation times. Biacore T200 evaluation software was used for affinity curve fitting, employing a 1:1 ratio steady-state affinity model. Meanwhile, kinetic parameters were calculated.

Animal grouping and treatment

Male SPF-grade SD rats (Shanghai SLAC Laboratory Animal Co., Ltd; license number SCXK(Hu) 2022–0004) were kept in an observation room with a 12-h cycle of light and darkness, maintaining temperature (22–24 °C) and humidity (40–70%). During the period of adaptive feeding, the rats were provided with unrestricted access to sterile water and conventional laboratory chow for one week. Animal experiments were approved by Yueyang Laboratory Animal Center Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (YYLAC-2022-169).

Following a prior study, we induced cerebral ischemia with an MCAO/R model in rats. Anesthesia was administered via a 2% intraperitoneal sodium pentobarbital injection at 40 mg/kg. After the neck incision, we exposed and separated the left common carotid artery and vagus nerve. An appropriately sized nylon monofilament was placed into the internal carotid artery through the common carotid artery, followed by ligation to block the left middle cerebral artery's origin. After 2 h, we removed the monofilament for reperfusion.

Three groups of rats were assigned at random: sham, MCAO/R, and MCAO/R + SMJF Granule (1.2 g/kg) groups. Sham control rats underwent a similar surgical procedure without MCAO ligation. SMJF Granule was administered 24 h post-MCAO/R, twice daily for 7 days. Rats in the sham and MCAO/R group received an equivalent volume of saline.

Neurobehavioral score

Neurological deficits in rats were evaluated in a single-blind fashion after reperfusion by Longa et al. [13]. The scoring criteria were as follows: 0 points: no nerve damage symptoms; 1 point: incomplete extension of the right forepaw; 2 points: rightward turn; 3 points: rightward collapse; 4 points: impaired walking and unconsciousness. Rats with scores of 1 to 3 were included in the results.

Triphenyl tetrazolium chloride (TTC) staining

After the brain tissue was removed, it was immediately frozen at − 20 °C for 15 min. Coronal sections were then taken with an even thickness of 2 mm, resulting in 6 layers of sections. These sections were then incubated in a water bath protected from light with a configured 2% TTC staining solution at 37 °C for 30 min. To ensure uniform staining on both sides, the sections were turned every 5 min. After staining, 4% paraformaldehyde was added for fixation. The percentage of brain infarct volume was calculated by the Image J software. Percentage of infarct volume (%) = total infarct volume/whole brain volume*100%.

HE and Nissl staining

To examine the pathological alterations, the brain tissue specimen was underwent fixation using 4% paraformaldehyde. Then the sample was sliced to a 3 μm thick paraffin section for HE and Nissl staining. Microscopic observation of stained cerebral sections was then performed, facilitating the study of morphological transformations within the cerebral cortex and the hippocampal CA1 region.

Real-time quantitative PCR analysis

RNA was extracted and purified from the ischemic penumbra using the kit according to the Beyotime manufacturer's instructions. The mRNA expression of candidate targets was assessed by real-time quantitative PCR with fluorescence detection. The primer sequences used in this study are shown in Table 2. The relative expression levels of target genes were normalized to that of β-actin using the 2−ΔΔCt method.

Table 2 Sequences of the primers used for RT-qPCRWestern blotting

The total proteins of ischemic penumbra tissue were extracted, and measured through a BCA protein assay. Afterward, equal amounts of protein were electrophoretically separated by SDS-PAGE and immobilized on nitrocellulose membranes (Millipore Corporation, Bedford, USA), which were subsequently immersed in 5% skimmed milk at room temperature for 2 h. Next, the primary antibodies were incubated with the membranes overnight at 4 °C. Subsequently, the secondary antibodies were added and incubated at room temperature for 1 h. The Odyssey system (LICOR, Lincoln, Nebraska) was utilized for visualization. The quantification of protein band densities was performed using Image J software.

Immunohistochemical staining

The infarcted cerebral hemispheres were fixed in 4% paraformaldehyde for 48 h, paraffin-embedded, and cut into 3 μm sections. Sections were deparaffinized, hydrated, blocked with goat serum for 1 h, and incubated overnight at 4 °C with diluted anti-HIF-1α, vWF, or VEGF antibodies (1:100). After washing with PBST, secondary antibodies were added for 30 min at room temperature, followed by DAB chromogenic staining. Nuclei were stained with hematoxylin for 2 min, and positive DAB expression was observed as brownish-yellow. Slides were mounted with neutral resins after drying. Eventually, sections were viewed and photographed under an inverted optical microscope (Olympus).

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 8.0 software. The mean ± SEM was used to express the data. Differences between groups were assessed using one-way ANOVA, with a p-value of less than 0.05 considered statistically significant.