Page 115 - 《水产学报》2025年第10期

李森栋，等 水产学报, 2025, 49(10): 109609

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Committee of Southwest University. the 1 , 3 , 5 day and sacrificed for samples collec-

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tion on the 7 day.
1.2 Experimental design and Diets preparation
1.3 Samples collection
WS extract (Batch No. RH11043467; Certific-
ate No. 20200423929988; RuiHerb Bio-Engineering Fish were euthanized using MS-222 (150
Technology Co., Ltd., Shanxi, China) and Withan- mg/kg), weighed, and fish blood were collected
olide A (catalog No. W350943-5mg; Shanghai Alad- from the caudal vein using 1 mL syringes. Blood
din Biochemical Technology Co., Ltd., Shanghai, was settled down at 4 °C for 30 minutes and centri-
China) were purchased from the suppliers. fuged at 500 g for 20 minutes at 4 °C, then the
The experimental diets were prepared by sup- supernatant (serum) was collected and kept at −20
plementing commercial feed pellets with herb °C. Subsequently, the liver, brain and other tissues
extracts at different concentrations, and identical were carefully dissected and collected for experi-
energy values were maintained across all treat- ments. Partial liver tissues were fixed for sections
ments. Briefly, the commercial feed was first and all other tissues samples were immediately
ground, then re-pelleted for the control or herb diet. dropped into liquid nitrogen and kept at −80 °C.

In the preliminary experiments, three concentra-
1.4 RNA-seq Analysis
tions of Withania somnifera (WS) (0.05%, 0.1%,
and 0.2%) were tested, and 0.1% WS extract was Three fish from each group were disected for
found to significantly alleviate the stress response in samples of RNA-seq analysis, resulting in a total of
darkbarbel catfish, unpublished data). The adult fish 15 samples. Total RNAs were extracted and puri-
were fed with 0.1% WS extract dietary for 5 weeks, fied from the brain tissues of female adult darkbar-
the samples were collected, to prevent postprandial bel catfish. The libraries for RNA-seqs were pre-
effects during physiological assessments, all fish pared and sequenced using paired-end reads (150
were fasted for 24 hours prior to be sampled. The cycles + 150 cycles) on an BGI platform. Raw
acute stress response was evaluated under three sequencing data were acquired from a next-genera-
conditions: (1) anesthesia and sampling, (2) air tion sequencing platform and converted into
exposure (90 seconds) before anesthesia and FASTQ format, including sequence information and
sampling, and (3) physical stimulus (fish was injec- quality scores. Data quality control was performed
ted with 0.5 mL of normal saline into the dorsal using the fastp tool (version 0.23.2), including
muscle before anesthesia and sampling. adapter removal, base correction for paired-end
For the tests in the young fish, the intramuscu- overlap, sliding window trimming, and UMI prepro-
lar injections of Withanolide A were carried out cessing. Clean reads were aligned to the reference
three times in 7 days. Briefly, five groups of fish, 10 genome using HISAT2, and gene-level read counts
fish per group were used. One group was set as were generated using featureCounts (Subread pack-
blank without any treatment, one group as the con- age) , with filtering to remove low-quality,
[28]
trol group (injected with equal volume of 0.1% unpaired, and multi-mapped reads. Differential gene
DMSO), and following the previous reports [26-27] , the
expression was analyzed using DESeq2 (version
fish of other three groups were injected with 1.46.0), and pathway enrichment analysis was con-
Withanolide A at different dose (1, 3, 5 mg/kg:
ducted with cluster Profiler (version 4.14.1) and
Withanolide A was dissolved in dimethyl sulfoxide Dr.eg.db.org (version 3.17.0). All analyses were
(DMSO) for the stock solution at 100 mg/mL, then
performed using R version (4.4.2).

diluted by sterile normal saline to prepare a work-
1.5 Real-time quantitative PCR (RT-qPCR)
ing solution for injection). The fish were treated
with Withanolide A by intramuscular injection on The total RNAs were extracted from the brain

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