Preliminary verification of the anti-hypoxia mechanism of Gentiana straminea maxim based on UPLC-triple TOF MS/MS and network pharmacology

Drugs and reagents

The dried root that was naturally air-dried from G.s Maxim was purchased from Qamdo Tibetan Hospital (Tibet, China). Rhodiola capsules were obtained from Rhodiola Research and Development Center, Xizang Military Region. TNF-α, IL-6, and NF-κB enzyme-linked immunosorbent assay kits were obtained from Boster Biological Technology (Pleasanton, USA). Primary antibodies for Bcl-2, Bax, HIF-1, p65, and β-tubulin were purchased from Immunoway (Plano, USA).

Ethyl acetate extraction

A total of 100 g G.s Maxim was added to 500 mL of 95% ethanol for 24 h using a heating reflux device, boiled the material for one hour, and repeated three times. We used a vacuum rotary evaporator (Heidolph, Germany) to evaporate the ethanol and collect the extract. The extraction rate was 10%. Finally, we added double-distilled water to dissolve the resulting drug extract completely.

Extraction was according to the order of polarity of organic solvents; the ethanol extraction of G.s Maxim was extracted with petroleum ether, ethyl acetate, and water-saturated N-butanol solution. After rotary evaporation under reduced pressure at 70 °C, we vacuum freeze-dried the extracts (LGJ-10, China) to obtain powder from each extraction for further use.

Compound identification using UPLC-triple TOF MS/MSSamples preparation

To 0.2 g of the ethyl acetate extract, we added 5 mL 50% methanol-aqueous solution, let it sit for 4 h, then subjected it to ultrasound at 40 °C for 40 min. The supernatants were placed in 1-mL centrifuge tubes, centrifuged at 1300 r/min for 10 min, and passed through 0.22-μm ultrafiltration membranes (Millipore, Bedford, MA, USA). Finally, the material was placed in 1.5-mL automatic sampling tubes. The blank control samples were obtained under the same conditions.

UPLC-triple TOF MS/MS condition

Reverse-phase analysis was performed on a UPLC Nexera system (Shimadzu, Japan) using an ACQUITY UPLC CSH C18 column (2.1 mm × 100 mm, 1.7 μm) (Waters, USA) containing a binary pump, a column oven, and an ESI ionization source. The flow rate was 0.3 mL/min, with mobile phase A composed of 0.1% formic acid and mobile phase B composed of acetonitrile. A gradient elution achieved sample separation: 0.01–15 min, 95–75% A; 15–37.1 min, 75–95% A; 37.1–40 min, 95% A. The mobile phase’s aqueous part pH (0.1% formic acid in H2O) was fixed at 2.47.

The column temperature was set to 40 °C, and the injection volume was 2 μL for each analysis. The samples were filtered through a 0.22-μm ultrafiltration membrane before injection.

Mass spectrometric analysis was performed on a Triple TOF® 5600 System (AB SCIEX, USA) in positive and negative ion modes. The source conditions were as follows: spray voltage of 5500 V in ESI (+) and − 4500 V in ESI (−), nebulizing gas at 50 psi, heating gas at 50 psi, curtain gas™ at 40 psi, and heater temperature at 500 °C. The declustering potential was 100 V. MS, and the scan range was 50–1000 (m/z). The mass spectra results were analyzed using Peakview data processing software.

Network pharmacology analysisIdentification of drug-like compounds (DLCs)

The compounds identified using UPLC-Triple TOF MS/MS were screened using the traditional Chinese medicine systems pharmacology (TCMSP) database (http://tcmspw.com/). Bioactive components with oral bioavailability (OB) ≥ 15% and drug-likeness (DL) index ≥0.18 were selected for subsequent analysis.

Targets related to DLCs and anoxia

The target proteins of bioactive components in the ethyl acetate extraction were retrieved from the TCMSP database. Search for disease-related targets in the Gene Cards database (https://www.genecards.org/) [17] by keyword “anoxia.” The target proteins were standardized in UniProt (http://www.uniprot.org/). We recorded the duplication of drug and disease targets, then designated them as the anti-hypoxia target of the G.s Maxim.

Protein-protein interaction (PPI) analysis

Targets identified in section 2.4.2 were uploaded into the STRING database (https://string-db.org/) [18] to perform PPI analysis, focusing on co-expression and co-localization. Cytoscape (http://www.cytoscape.org/, version 3.8.2) was used to analyze the PPI network, and the core anti-hypoxia targets of the G.s Maxim’s ethyl acetate extraction.

Gene ontology and pathway enrichment analysis

Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG)-pathway enrichment was built using the DAVID Bioinformatics Resources (https://david.ncifcrf.gov/summary.jsp) [19]. Its targets with the involved pathways were obtained using enrichment analysis and explored the potential biological effects for the G.s Maxim’s ethyl acetate extraction targets.

Animals and treatments

Male specific pathogen-free Sprague Dawley rats were obtained from the Xian Jiaotong University Animal Center (SCXK (shaan) 2018–003, Xian, China). Rats were housed in the Xizang Minzu University Laboratory Animal Center with a 12 h–12 h light-dark cycle. They were fed regular chow and purified water ad libitum. The animal experiment was conducted following the internationally accepted laboratory animal use and care principles. It is reviewed by the Ethics Committee of Xizang Minzu University (Ethics Approval No. 20200–7). Effective parts of G.s Maxim were extracted as described in section 2.2. We added water to achieve 6.66 g/kg, 3.33 g/kg, and 1.67 g/kg (calculated by raw drug quantity). Rhodiola capsules were used as the positive control.

The rats were randomly divided into six groups (n = 8): (1) Control; (2) Hypoxia; (3) 228 mg/kg body weight Rhodiola capsules + Hypoxia; (4) 6.66 g/kg body weight the G.s Maxim’s ethyl acetate extraction + Hypoxia; (5) 3.33 g/kg body weight the G.s Maxim’s ethyl acetate extraction + Hypoxia; and (6) 1.67 g/kg body weight the G.s Maxim’s ethyl acetate extraction + Hypoxia. Rats in control groups were maintained in normal conditions; rats in medication groups were intragastric ally administered compounds for 15 consecutive days.

After the final administration, all rats except those in the control group were placed in a hypobaric oxygen chamber (7000 m, 24 h). At the end of modelling, rats were anesthetized by intraperitoneal injection of urethane; then, brain tissues were removed for pathological examination. We measured serum levels of TNF-α, IL-6, and NF-κB and brain expression of HIF-1α, p65, Bax, and Bcl-2.

The brain tissue was fixed in 10% formaldehyde solution for 12 h, then dehydrated, made transparent, and embedded in paraffin. After sectioning, the specimens were stained with hematoxylin and eosin (HE) and observed under a light microscope. The levels of TNF-α, IL-6, and NF-κB in serum were measured using ELISA according to the manufacturer’s instructions. Western blotting was performed as follows: The total protein of cerebrum samples was extracted in RIPA lysis buffer. According to molecular weight, proteins in brain tissue were separated by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes, blocked with 5% skim milk for 3 h, and incubated with the corresponding primary antibodies overnight at 4 °C. After washing in buffer, the membranes were incubated with a conjugated secondary antibody for 1 h at room temperature. Finally, the membranes were exposed to ECL reagent, and bands were detected using the Image Lab detection system. The intensity of each band was analyzed using Image J software.

Molecular docking

The crystal structure of core targets (JUN, TNF, TP53, AKT1, HIF-1α, NF-κB) was obtained from RCSB Protein Data Bank (http://www.rcsb.org/), and their corresponding PDB IDs were as follows: 6NOA, 1TNR, 6IUA, 5AAR, 4H6J and 1RAM [20,21,22,23,24,25]. MOL2 format of active compounds was obtained from the TCMSP database, and their corresponding MOL IDs were as Table 2. Auto Dock Tools Version 1.5.6 (http://mgltools.scripps.edu) and Pymol (https://pymol.org/2/) were applied for molecular docking.

Auto Dock Tools generated and optimized all 3D structures of ligands and proteins. These crystal structures were imported into Auto Dock Tools software for dehydration, hydrogenation, and isolation of original ligands. The optimized targets were constructed in a docking grid box, and the active site of molecular docking was determined using the ligand coordinate in the target protein complex [26]. Finally, molecular docking experiments selected the best affinity conformation as the final docking conformation.

Statistical analysis

Results were expressed as the mean ± SD. Analysis of variance was performed using GraphPad Prism 8.01. Significant differences between groups were defined as p < 0.05. Density analysis of the western blotting bands was performed using Image J software.

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