Herbal data collection
The active ingredients of Erigeron breviscapus, Radix salviae, Lycii fructus, Croci stigma and Ginkgo folium were acquired from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) Database (http://tcmspw.com), which focuses on TCM [15], under the screening conditions of oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. We also used TCMSP to obtain drug-related targets (DRTs) and then acquired their gene symbols from the UniProt Database (https://www.uniprot.org).
Acquisition of glaucoma-related targets
Disease target information was collected from 3 databases: the Online Mendelian Inheritance in Man Database (OMIM, https://omim.org), GeneCards Database (https://www.genecards.org) and GEO Datasets of NCBI (https://www.ncbi.nlm.nih.gov/gds) [16]. We searched the key term “glaucoma” in OMIM and GeneCards to obtain targets and in GEO to obtain the gene chip data of GSE9944 and the platform files of GPL571 and GPL8300. The screening of differential gene expression was performed using the limma package for R software (version 3.6.0) with the filters of P > 0.05 and |log2 fold change (FC)|> 0.5. Finally, we summarized the targets and differentially expressed genes above into known glaucoma-related targets (GRTs).
Construction of glaucoma-targets-drug and protein–protein interaction (PPI) networks
The drug-disease common targets were collected via R software (version 3.6.0) and are shown as a Venn diagram. Then, we used Cytoscape software (version 3.7.2) to construct and visualize the Glaucoma-Targets-Drug network. Under the screening conditions of “Organism = Homo sapiens” and “High Confidence (> 0.9)”, the common targets were also used to build the PPI network using the STRING Platform (version 11.0). The nodes and edges between the nodes consisted of the network above, in which the degree of the node is the number of edges connected to the node, and a higher degree indicates greater importance of the node.
Gene ontology (GO) biological processes enrichment analysis
To explore the functional processes of common drug-disease targets, we performed GO biological process enrichment analysis on these targets in the R/Bioconductor environment (http://www.bioconductor.org/). Based on the gene ratio, we show the top 20 processes (P < 0.05) as a histogram and bubble chart.
Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment analysis and construction of a functional pathway network
To clarify the potential molecular mechanisms, KEGG pathway enrichment analysis was performed by Metascape Platform software (https://metascape.org/), and we also created a heatmap of the top 20 KEGG pathways. Then, 8 pathways highly related to glaucoma were selected to construct the functional pathway network, which also involved related targets and active ingredients. The functional pathway network was constructed using Cytoscape software.
Molecular docking study
We performed molecular docking to verify the connective validity of active ingredients and core targets using AutoDock Vina software (version 1.1.2) [17]. The 3D structures of active ingredients and target proteins were obtained from the TCMSP database and the RCSB PDB database (http://www.rcsb.org/), respectively. The proteins were dehydrated and hydrogenated, and a grid box was set via AutoDockTools software (version 1.5.6). To determine exhaustive docking patterns, the docking of ingredients and targets was performed in a flexible and unrestrained manner, allowing the ligand to move through the entire volume of the grid box. After the steps above, the docking results were visualized using PyMOL software (https://www.pymol.org).
Animal and environmental conditions
Male Sprague–Dawley (SD) rats (200 ± 20 g) from the Laboratory Animal Centre, Chengdu University of Traditional Chinese Medicine, were used, and the experiment was approved by the Committee of Scientific Research and the Committee of Animal Care of Chengdu University of Traditional Chinese Medicine (permission number: 2020–12). Animals were adaptively fed for one week at a cozy temperature of 23 ± 2 ℃ and temperate humidity of 40–60%. We abided by guidelines about animal use from the Association for Research in Vision and Ophthalmology (ARVO) throughout the study.
Chemicals and antibodies
Baicalein (S25956), sodium carboxymethyl cellulose (CMC-Na, S14017) and balanced salt solution were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). A TdT-mediated dUTP nick-end labeling (TUNEL) staining kit (11684795910) was purchased from Roche Group (Basel, Switzerland). BCL-2 (bs-20351R) was procured from Bioss Co., Ltd. (Beijing, China). FITC-conjugated goat anti-rabbit IgG (GB22303) and FAS eyeball fixative (G1109) were provided by Servicebio (Wuhan, China). DAPI (ZLI-9557) and citrate repair solution (ZLI-9065) were obtained from Zsbio Co., Ltd. (Beijing, China). Newborn calf serum (22012-8612) was obtained from Tianhang Co., Ltd. (Hangzhou, China).
Animal modeling, grouping and treatment
A total of 20 SD rats were anesthetized by intraperitoneal injection of 2% pentobarbital sodium solution at a dose of 50 mg/kg weight and then underwent episcleral vein cauterization (EVC) surgery in the right eye by the same skillful surgeon. EVC surgery complied with the procedures designed by Shareef [18]: three small incisions of the conjunctiva in the right eye were created to expose the episcleral veins, and then three vein stems were cauterized using a cautery pen. The left eyes of the sham-operated group rats received a sham procedure that only opened the conjunctiva to expose the episcleral veins and did not cauterize them. Random grouping after the modeling was performed as follows: 10 rats underwent sham operations on the left eyes as a control group (CON) as with as right eye modeling as COH (intact left eyes were used as self-contrast), and 10 rats in the BAL (right-eye modeling). The intragastric administration of drugs began one day after modeling surgery, until the end of the experiment 28 days later: the groups above received suspensions of baicalein (200 mg/kg weight) and 0.5% CMC-Na solution. Gavage was performed in the morning from 10:00–12:00 to avoid possible differences between days.
IOP measurement
The IOP assay was performed preoperatively (day surgery), the first day after surgery (before drug administration), the third day of surgery, the seventh day after surgery, the fourteenth day after surgery, and the twenty-first day after surgery by a TONOLAB tonometer TV02 (Icare, Finland). The measurements of IOP were performed at least three times on each eye, and the integer nearest to the mean value of the measurement results above was recorded as the final IOP of the eye. During the measurements, the animal was in a fully conscious state. According to the IOP results of the first day after surgery (before drug administration), we excluded with IOP (operated eye) < 22 mmHg from the subsequent experiments.
Hematoxylin and eosin stain
H&E staining was performed to quantify the thickness of the ganglion cell complex (GCC) and changes in retinal structure. After 28 days, rats were sacrificed by anesthesia, and their eyeballs were removed. Ocular tissues were fixed by formaldehyde, acetic acid, and saline (FAS) eyeball fixative, dehydrated with gradient concentrations of ethanol, and then sliced into 7 μm-thick slides with a Rotary Slicer-RM2016 (Leica, Germany). Slides stained with H&E were sealed with neutral balsam, with 3 samples per group and 3 slices on average. The overall GCC thicknesses (from the ganglion cell layer (GCL), to the nerve fiber layer (NFL)) were measured. All measurements were carried out at distances of 1.0–1.5 mm (central) retina from optic disc with ImageJ software (Bethesda, MD, USA).
Terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay
Rats were sacrificed after 28 days, and three eyes were removed from each group. TUNEL assay was performed to quantify RGC apoptosis in the retina. TUNEL staining was processed by an In Situ Cell Death Detection Kit, Fluorescein (Roche Group, Switzerland). After dewaxing, the slides were repaired with citric acid microwave for 8 min, washed with phosphate buffer saline (PBS) three times, 5 min each time. Prepared with fluorescent TUNEL incubation solution in the dark (A:B = 1:30) and incubated for 1 h at the temperature of 37 °C, washed three times with PBS, and stained with DAPI for 15 min, followed by washing with PBS. The slides of the TUNEL stain were sealed by gelatin glycerin. The images of the slides above were collected and analyzed by Pannoramic 250 Flash (Danjier, Jinan, China).
Immunofluorescence staining of the retina
After the experiment, the rats were sacrificed, and the eyeballs were removed. Ocular tissues were fixed by formaldehyde, acetic acid, and saline (FAS) eyeball fixative, dehydrated in sucrose, and then sliced into 7 μm-thick slides with a Rotary Slicer-RM2016 (Leica, Germany). After dewaxing, the slides were immersed in 0.01 M citrate buffer (pH 6.0), washed three times with PBS for 5 min each time, and blocked at room temperature for 30 min in newborn calf serum. Primary antibody was added (BCL-2 1:100) dropwise, followed by refrigeration overnight at 4 °C, washing three times with PBS for 5 min each time, addition of the secondary antibody dropwise 37 °C for 30 min, washing with PBS the same as before, addition of DAPI and incubation at room temperature for 10 min, washing with PBS and sealing with anti-fluorescence attenuation reagent. The images of the slides above were collected and analyzed by a Pannoramic 250 Flash (Danjier, Jinan, China).
Transmission electron microscopy for optic nerve
Rats were sacrificed after 28 days, and three optic nerves were removed from each group. The optic nerve samples were fixed in 3% glutaraldehyde and postfixed in 1% osmium tetroxide (Leica Company, Germany). After dehydration by gradient acetone dehydration, the optic nerve samples were permeated in Epon 812 epoxy resin (Beijing Keyi, Beijing, China). Then, the permeated samples were embedded in flat molds and heating polymerized for embedded blocks. Ultrathin sections (50 nm) were created with an ultramicrotome and double-stained with Reynolds’s lead citrate and 0.5% aqueous uranyl acetate (Beijing Keyi, Beijing, China). The microscopic examination was performed with a JEM-1400PLUS TEM (JEOL, Tokyo, Japan), and the images were analyzed with OlyVIA software (version 2.8).
Statistical analyses
SPSS software (Version 26.0) was used for statistical analysis, and GraphPad Prism software (Version 7) was used for plotting. All of the images were measured by ImageJ software (Bethesda, MD, USA). Data are expressed as the mean ± standard deviation (SD). Measurement data conformed to a normal distribution and were compared by the independent sample t test analysis for two groups. One-way ANOVA was used for multigroup analysis; the measurement data did not conform to a normal distribution, and the nonparametric rank sum test was used. P < 0.05 was considered statistically significant.