Searching for the chemical composition and targets of GZFL
The active ingredients of GZFL were acquired from the TCMSP database (http://lsp.nwu.edu.cn/tcmsp.php). A screening was performed on the active ingredients to identify those with oral bioavailability (OB) exceeding 30% and drug-likeness (DL) surpassing 0.18. The protein targets related to these active ingredients were determined using the TCMSP platform. (Wang et al. 2022).
Prediction of Disease Targets
The keyword "CIRI" was searched in GeneCards (https://www.genecards.org) and OMIM (Online Mendelian Inheritance in Man, https://omim.org) databases to identify targets related to CIRI. These bioinformatics resources combine information on diseases with comprehensive gene targets. Subsequently, the Venn online tool (http://bioinfogp.cnb.csic.es/tools/venny/index.html) was used to intersect these targets with those related to GZFL.
Construction of protein-protein interaction (PPI) network and analysis of core targets
The intersection targets were uploaded to the STRING database (https://string-db.org/cgi/input).The confidence was set at 0.9. The 'free' nodes, which do not interact with other proteins, were utilized to construct the protein-protein interaction (PPI) network. The Cytoscape software was used for further processing of the PPI network to visualize and identify the core targets.
Enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
In this study, the primary targets underwent GO and KEGG pathway enrichment analyses using the clusterProfiler R package. GO annotations for Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) were provided to elucidate the roles of target proteins in the treatment of CIRI with GZFL. Besides, the KEGG pathway enrichment analysis investigated the signaling pathways implicated in GZFL's therapeutic effects on CIRI. Finally, the bioinformatics platform (http://www.bioinformatics.com.cn/) was utilized for the analysis results visualization.
Molecular docking
The topologically important large and small molecules were subjected to molecular docking, employing available crystal structures of proteins obtained from the RCSB PDB database (https://www.rcsb.org/) or alphafold database (https://alphafold.ebi.ac.uk/). The small molecule library for docking was obtained from the TCMSP database (https://old.tcmsp-e.com/tcmsp.php) by searching Chinese medicine and establishing molecular structures. Firstly, the receptor structure preparation was conducted by removing water molecules, adding hydrogens, and carrying out protein conformational sampling using AutodockTools(El-Hachem et al. 2017). Next, the small molecule library was preprocessed using Open Babel and Autodock programs. Docking simulations were performed using Autodock program, followed by visualization of docking results using Pymol software(Baugh et al. 2011).
Cell Culture and Treatment
The PC12 cell line sourced from the Stem Cell Bank in Shanghai, China, was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 mg/mL penicillin/streptomycin under standard cell culture conditions.
An Oxygen Glucose Deprivation/Reoxygenation (OGD/R) cell model was created to mimic CIRI in vitro (Mo et al. 2012). The cells were rinsed with phosphate-buffered saline (PBS) and subsequently incubated for 24 hours. The PC12 cells were primarily cultured at 37℃ under an anaerobic atmosphere composed of 94% nitrogen, 5% carbon dioxide, and 1% oxygen in sugar-free DMEM for 2 hours. The cells were then moved to DMEM that contained 10% FBS and cultured for 24 hours at 37℃ and 5% CO2. This was done to mimic reperfusion injury. The cells in the control (con) group were not subjected to OGD/R treatment.
GZFL incorporates five herbs in equal measure: Poria cocos (Schw.) Wolf, Paeonia lactiflora Pall, Cinnamomum cassia Blume, Prunus persica (L) Batsch, and Paeonia suffruticosa Andr. A total of 200 grams of each herb underwent a two-hour extraction process, twice, using 2 liters of boiling distilled water. Following extraction, the liquids were filtered and freeze-dried. Prior to experimentation, GZFL was freshly prepared using distilled water.
GZFL at different concentrations (2, 4, 8 mg/ml) was used to treat the OGD/R cell model, and the transfection of TNF-α was accomplished using the Lipofectamine 3000 reagent kit (L3000015, Invitrogen, USA).
Cell Counting Kit-8(CCK8) assay
Logarithmic phase PC12 cells were inoculated at 5×103 cells per well into 96-well plates and cultured for 24 hours. Subsequently each well was exposed to 10 µL of CCK-8 reagent (C0037, Beyotime Biotechnology, Shanghai, China), and incubated continuously for 1.5 hours. The optical density at 450 nm was measured with an ELISA plate reader (BioRad, Hercules, USA).
Terminal deoxynucleotidyl-transferase mediated dUTP‐biotin nick end‐labeling (TUNEL) staining
Cells were suspended in 1× Binding Buffer at a concentration of 1×10^6 cells/mL. Subsequently, a 100 µl aliquot of the cell suspension was used for cell apoptosis detection following the TUNEL kit (MK-1013-100, Boster, China) instructions. Initially, the cells were washed with PBS, followed by fixation with 4% paraformaldehyde for 30 min. Afterward, the cells were rinsed twice with PBS, each time for 2 min. Diluted Proteinase K solution was added to digest the cells for 10 min. Subsequently, TdT and BIO-d-UTP were added, and the labeling was performed at 37 ℃ for 2 h. The cell nuclei were stained with DAPI staining solution, and the slides were mounted with an anti-fade reagent. Staining patterns were examined using a fluorescence microscope, and the percentage of TUNEL-positive cells was determined. The apoptosis rate was determined as the ratio of TUNEL-positive cells to total cells, expressed as a percentage.
Western blot
Following OGD/R and GZFL treatment, the total cellular protein of PC12 cells was extracted through RIPA lysis buffer (P0013, Beyotime Biotechnology, Shanghai, China) which included phenylmethanesulfonyl fluoride (PMSF). The protein level was quantified using the bicinchoninic acid method (P0012, Beyotime Biotechnology, Shanghai, China). Equivalent protein quantities were resolved on 10% SDS-PAGE and subsequently transferred onto PVDF membranes (Bio-Rad Laboratories, USA).After blocking with 5% skim milk for 1 hour, membranes were incubated with primary antibodies against TNF-α (ab183218, 1:1000, Abcam) and GAPDH (ab9485, 1:2500, Abcam) overnight, at 4°C. The secondary antibody goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) (ab6721, 1:2000, Abcam) was added for 2 hours at room temperature. The membranes went through three PBST washes, and the protein bands were visualized by ECL (P0018S, Beyotime Biotechnology, Shanghai, China) using GAPDH as an internal reference.
Enzyme linked immunosorbent assay (ELISA)
The PC12 cell culture medium was harvested and centrifuged at 1000 g for 20 minutes at 4°C to remove impurities and cellular debris. Subsequently, the supernatant was obtained by centrifuging the lysed brain tissue from the infarct area. ELISA assay kits (ab229440 for IL-1β, ab222503 for IL-6, and ab208979 for MCP-1 from Abcam) were utilized to measure the concentrations of the mentioned cytokines according to the manufacturer’s instructions.
Oxidative stress detection
The supernatants from the cell culture medium and brain tissue homogenate were collected and the levels of Malondialdehyde (MDA), Superoxide Dismutase (SOD) activity, and Glutathione Peroxidase (GSH-Px) activity were measured using commercially available kits (MDA: A003-1-2, SOD: A001-3-2, GSH-Px: A005-1-1, Nanjing Jiancheng Bioengineering, Nanjing, China), following the manufacturer's instructions.
Middle cerebral artery occlusion reperfusion (MCAO/R) model
Male Sprague-Dawley (SD) rats (8–10 weeks old, weighing 220-280g) were procured from the Hubei Province Animal Experiment Center and housed in a specific-pathogen-free environment. After a week of acclimatization to a 12:12 hour light/dark cycle, the rats were provided with ad libitum access to food and water. The rats were randomly assigned to groups of six each. The rats were randomly assigned into four groups, each consisting of six rats: Sham group, MCAO/R group, MCAO/R + GZFL group (2 g/kg or 4 g/kg). The MCAO/R model was established following the previously described method (Zhao et al. 2021). Rats were intraperitoneally injected with 1% pentobarbital (50 mg/kg) to induce anesthesia and placed supine and immobilized on a surgical table. Following the removal of neck fur and disinfecting the skin, the right carotid artery (CCA), the external carotid artery (ECA), and the internal carotid artery (ICA) were exposed. The CCA and ECA were separated, and a 0.285 mm nylon suture was used to ligate and block the opening of the MCA. Brain blood flow was monitored with a Laser Doppler Flowmeter (Periflux 5000, Sweden), and MCAO was considered successful when the blood flow decreased by 80%. After two hours of surgery, the suture was withdrawn from the MCA to allow reperfusion. The sham-operated rats underwent the same procedures and vascular separation, but MCAO and reperfusion were not conducted. Throughout the study, all rats were kept on a heating pad maintained at 37°C. After confirming the success of the model, the rats in the MCAO/R group were randomly allocated to subgroups and orally administered GZFL (2 g/kg or 4 g/kg) once a day for seven consecutive days based on earlier reports(Chen et al. 2016). The sham group received an equal volume of normal saline. Following confirmation of neurological function scores, the rats were euthanized, and brain tissue samples were collected and frozen in liquid nitrogen for further investigations. All animal experiments were approved by the Animal Ethics Committee of our institution and conducted in compliance with the NIH Guide for the Care and Use of Laboratory Animals.
Neurological function score
After the induction of MCAO/R, the neurological impairment of the rats was assessed 24 hours after the final administration. The neurological deficit was scored according to Longa's five-point method(Longa et al. 1989), which was used to evaluate the degree of neurological dysfunction. This study employed the following specific scoring criteria: A score of 0 indicated no symptoms of neurological deficits and normal activity. A score of 1 indicated that the contralateral forelimb of the lesion was incapable of complete extension. A score of 2 indicated that when crawling, the rats would turn to the opposite side. A score of 3 indicated that the rats would walk their body to the opposite side. Finally, a score of 4 indicated that the rats were unable to walk independently and experienced a loss of consciousness. The severity of neurological impairment escalates with higher scores.
Hematoxylin–eosin (H&E) staining
The rat brain tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned into slices that were 5 µm thick. Paraffin sections were then de-waxed with xylene and rehydrated in a series of alcohol solutions resulting in various concentrations. The samples were then stained with Hematoxylin and Eosin (HE) to detect any pathological changes. An optical microscope (Olympus, Tokyo, Japan) was utilized to visualize the changes in brain tissue.
Nissl Staining
The Nissl staining kit (Solarbio, Nanjing, China) was employed to detect morphological changes in neurons. Rat brain tissue paraffin sections were deparaffinized with xylene and dehydrated via ethanol gradient concentrations. The sections underwent immersion in Nissl staining solution, followed by a 30-minute staining process in a constant temperature chamber at 50–60°C, post-distilled water wash. The sections were washed once more with distilled water and differentiated utilizing 95% ethanol. They were also dehydrated using absolute ethanol, made transparent utilizing xylene, and eventually sealed with neutral gum for observation purposes. Observation of the staining outcomes was performed with a microscope, and images were captured.
TUNEL assay
Subsequently, the detection was performed following the instructions of the TUNEL kit (MK1011-100, Boster, China). Firstly, the sections were rinsed with distilled water and then digested with Proteinase K solution for 10 min. Subsequently, TdT and BIO-d-UTP were added, and the labeling was performed at 37 ℃ for 2 h. After rinsing with TBS, 50 µl of blocking solution was added, and the sections were incubated at room temperature for 30 min. Following DAB color development and hematoxylin counterstaining, the sections underwent dehydration, clearing, and mounting. The staining results were observed under a microscope, where apoptotic cells were stained brownish yellow.
Statistical analysis
Data analysis and graphing were performed using GraphPad Prism software, presenting results as mean ± standard deviation. One-way ANOVA was conducted for comparisons among multiple groups, while the t-test was used for comparisons between two groups. Statistical significance was set at a P value of < 0.05.