Page 116 - 《水产学报》2025年第10期

李森栋，等 水产学报, 2025, 49(10): 109609

[11]
tissues of female adult darkbarbel catfish using the database (GSE75614) . The raw data were prepro-
Trizol RNA isolation Kit (Invitrogen 15596-026) cessed using R, and genes with expression levels in
according to the manufacturer’s protocol. The the top 50% were selected for further analysis.
quantity and quality of extracted total RNA were To explore the mechanisms of WS acts in dif-
examined by 1% agarose gel and Nanodrop 2000, ferent tissues, the common genes between RNA-
and cDNAs were synthesized with the RNA reverse seqs and WS targets predicted by Network pharma-
transcript™ RT Reagent Kit (TaKaRa). QRT-PCR cological analysis were identified in brain(this
was performed using Lightcycler-FastStart DNA study) and liver (GSE75614) respectively, followed
Master SYBR Green 1 and Lightcycler480 (Roche), by PPI network construction using STRING
and qPCR reaction was performed in 10 μL, con- (https://cn.string-db.org/) to determine their roles.
taining 1 μmol/L primers, 10 ng cDNA, and 1× To further clarify the mechanism behind WS
SYBR Green qPCR Master Mix (Roche). The function in alleviating the stress response in dark-
2 −∆∆C T method was employed to calculate the barbel catfish, a compound–target interaction net-
expression changes of target genes, following nor- work was constructed. The network of WS–active
malization with the GeNorm-mean of darkbarbel
compounds–targets–stress-related targets were con-
catfish β-actin. PCR was conducted under the condition: structed and visualized using Cytoscape_v3.10.3.
94 °C for 2 min, 33 cycles at 94 °C for 30 s, 57 °C
Additionally, KEGG pathway analysis was conduc-
for 30 s, and 72 °C for 30 s. All samples were
ted using ggplot2 in R to determine the role of WS
examined in triplicate and experiments were
target genes in stress-related pathways.
repeated at least twice. The sequences of the PCR
To evaluate the binding energies and interac-
primer pairs used in this study are listed in Table
tion modes between the candidate compounds and
S2.
their targets, molecular docking was performed

1.6 Network pharmacological analysis and using the CB-DOCK2 online docking server
molecular docking of WS (https://cadd.labshare.cn/cb-dock2/php/index.php)
[29]
based on AutoDock Vina . Pocket detection and
The bioactive compounds of WS were
screened and their potential targets were predicted blind docking were conducted sequentially. The
molecular structures of Withanolide A, Withanolide
by searching the ChEBI (https://www.ebi.ac.uk/
chebi/) and PCIDB (https://www.genome.jp/db/ D, and Withanolide J were obtained from Pub-
Chem (https://pubchem.ncbi.nlm.nih.gov/), and the
pcidb) databases. Relevant active compounds were
protein structures of IARS1 (Uniprot ID: P41252)，
identified with reference to the study by Safar M.
[13]
Alqahtani . SMILES structures were retrieved MAPK10 (PDB ID: 8WGF; resolution: 1.85 Å) and
from PubChem (https://pubchem.ncbi.nlm.nih.gov/), MAPK8 (PDB ID： 8X5M; resolution: 2 Å)were
and compounds meeting the criteria of drug-like- downloaded from the UniProt and PDB databases
ness (DL ≥ 0.18) and oral bioavailability (OB ≥ (http://www.rcsb.org/), respectively. Ligand
30%) were retained. The PubChem IDs (CIDs), molecules were pre-processed by adding hydrogen
molecular formulas (MFs), molecular weights atoms and partial charges, and their initial 3D con-
(MWs), OB, and DL of these compounds were sum- formations were generated using RDKit. Missing
marized for analysis. The potential targets of 13 act- side-chain atoms and hydrogen atoms were added to
ive compounds were predicted using the Swiss Tar- the receptor proteins, and crystallized water was
get Prediction database (http://www.swisstargetpre- removed, all performed automatically by the Open-
diction.ch/), with a probability threshold of >0 for Babel and MGL program tools provided by the
target selection. Transcriptome data of darkbarbel online server. Docking binding energy was calcu-
catfish liver tissue were downloaded from the GEO lated using the AutoDock Vina scoring function and

https://www.china-fishery.cn 中国水产学会主办 sponsored by China Society of Fisheries
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