Page 117 - 《水产学报》2025年第10期

李森栋，等 水产学报, 2025, 49(10): 109609

expressed in kcal/mol. The Vina score was calcu- lected for proliferation analysis with CCK-8 kit fol-
lated as an indicator of binding affinity, where lowing the manufacturer’s instructions (Dingguo,
lower docking energy values indicated greater bind- Beijing, China) through measuring the absorbance
ing affinity between the protein and the ligand. at 450 nm using a microplate reader (Spectro UV-

VIS Auto, UV-2602, Labomed, Los Angeles) at the
1.7 Biochemical indicators analysis
three time points, at 24, 48, and 72 hours respect-
To confirm the stress and immune responses
ively.

induced by external stimulation in WS and control
groups, COR, HSP70, TNF-α, IL-6, IgM, and ROS 1.10 Western blot analysis
were measured in fish serum using ELISA kits After a 72-hour treatment, the SG3 cells were
(ml104175, ml024582, ml002095, mI098430, harvested and washed once with cold PBS, and
ml106398, ml107146; Shanghai Enzyme-linked lysed with SDS buffer (P0013G, Beyotime) con-
Biotechnology Co., Ltd.), while GLU, SOD, and taining protease inhibitor cocktail (Roche). Equal
MDA were examined using biochemical assay kits amounts of proteins (30 μg) were electrophoresed in
(ADS-W-AJS020, GMS50004.2, GMS50099.1.
10% SDS-polyacrylamide gels and transferred to
Jiangsu Aidi Biomedical Technology Co., Ltd.).
polyvinylidene difluoride membranes (88518,

1.8 Oil Red O staining Thermo Fisher Scientific). Membranes were
blocked with 5% skimmed milk for 2 hour at room
The liver tissue was fixed in 4% paraformalde-
temperature and then incubated with anti-PCNA
hyde for 48 h, then washed twice with phosphate
antibody (11224-1-AP, diluted at 1∶2 000) overnight
buffer saline (PBS). Subsequently, the tissue was
at 4 °C. The protein blots were ultimately detected
immersed in a 30% sucrose solution (prepared with
with ECL reagents (P0018S, Beyotime) and imaged
PBS) at 4 °C for 12 h and then embedded in optimal
cutting temperature compound. Cryosections of 3-5 on a Bio-Rad imaging system. The relative expres-
μm thickness were cutted using a cryotome (Leica sion of PCNA proteins in cells were calculated and
CM1950S, USA) and stained with neutral Oil Red normalized to the level of β-Tubulin protein detec-
O staining (ORO) (Solarbio, G1260, Beijing, ted by anti-β-Tubulin (M20005, Abmart) control
China) to examine the accumulation of lipid using the ImageJ software.

droplets in the liver.
1.11 Fluorescence immunostaining

1.9 Cell culture and Withanolide A treatment
Immunostaining was conducted following the
[31]
To confirming the cell proliferation potency of described in our previous reports . The sections or
Withanolide A, SG3 cells (medaka fish spermatogo- cells were immunostained using the TSA Plus fluor-
nial cell line) were in vitro cultured as the previ- escence kit (NEL74100KT, PerkinElmer, Rodgau,
[30]
ously described , and seeded in 96-well plates at Germany) with the primary antibody against PCNA
~5 000 cells / well. Four experimental treatments (home made and the sequence of peptide antigen
were set and 5 wells in each treatment (n=5): from crucian carp, unpublished data), the second-
Briefly, cells treated by 0.001% DMSO (the con- ary antibody of the horseradish peroxidase (HRP)
trol), and 0.5 μmol/L, 1 μmol/L and 5 μmol/L conjugated anti-mouse IgG (BOSTER, BA1050,
Withanolide A respectively. The cells after a 72- China). The cell nuclei were counter-stained by 10
hour treatment of Withanolide A were collected for µg/mL 4', 6-diamidino-2-phenylindole (DAPI) in
mRNA expression analysis of related target genes PBS. The sections were observed and photo-
via qPCR, or for PCNA protein expression by graphed under a fluorescent microscopy system
Western blotting or the fluorescence immunostain- (Nikon, Y-TV55, Japan; NIS-Elements D5.42.06 64-
ing analysis. Meanwhile, the treated cells were col- bit). The images were analyzed with the software

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