Page 100 - Read Online

Page 4 of 14 Chen et al. Microbiome Res Rep 2025;4:6 https://dx.doi.org/10.20517/mrr.2024.38

group, the CCFM1376 group, and the QJSWX160M2 group (n = 8). Throughout their 8-week rearing phase,
the mice in the control and model groups received 200 μL of physiological saline daily through oral gavage,
whereas mice in the L. johnsonii-supplemented groups were daily given oral gavage of 200 μL of L. johnsonii
10
at a concentration of 1 × 10 colony-forming units (CFU)/mL. The control group was given a standard
reference feed, while the other three groups were given the high-cholesterol feed. The standard reference
feed (TP23302) and a high-cholesterol feed (TP28600), characterized by 15% fat, 1.25% cholesterol, and 0.5%
bile salts, were both sourced from Trophic Animal Feed High-Tech Co., Ltd in China .
[14]

Determination of biochemical parameters in mouse serum and liver
Biochemical parameters in mice serum, such as total cholesterol (TC), triglyceride (TG), LDL-C, and high
density lipoproteins cholesterol (HDL-C), were assessed using a Mindray fully automated biochemical
analyzer (Shenzhen Mindray Animal Medical Technology Co., LTD). The levels of TC, TG, and LDL-C in
mouse liver were determined according to the instructions of the assay kits provided by Nanjing Jiancheng
[15]
Bioengineering Institute, Nanjing, China .
RNA extraction and gene expression analysis in tissues
For 50 mg liver or ileum tissue samples, TRIzol reagent, 500 μL in volume, was introduced to the mixture,
which was then thoroughly homogenized using a high-throughput grinder at 4 °C to fully disrupt the cells.
Subsequently, the mixture was treated with 200 μL of DNase- and RNase-free water, followed by
centrifugation at 12,000 × g and 4 °C for 15 min to retrieve the supernatant. The supernatant was mixed
with an equal volume of isopropanol, followed by centrifugation at 12,000 × g and 4 °C for 10 min to pellet
the precipitate. The supernatant was aspirated off, and the pellet was washed twice with 75% ethanol and
air-dried. The pellet was resuspended in DNase- and RNase-free water, and complementary DNA (cDNA)
was prepared through a reverse transcription reaction according to the instructions of the reverse
transcription kit (R333-01 Vazyme Biotech Co., Ltd., Nanjing, China). Gene expression levels within the
mouse liver and ileum tissues were quantified through quantitative real-time polymerase chain reaction,
conducted on a BioRad thermocycler with the quantitative real-time PCR (qPCR) Master Mix from Vazyme
[16]
Biotech Co., Ltd, Nanjing, China .

Determination of bile acid content in tissue and feces
Twenty mg freeze-dried feces, 20 mg ileal contents, and 20 mg liver were weighed into a 1.5 mL centrifuge
tube. One mL of chromatography-grade methanol was added for homogenization; 50 μL of serum was
added into a 1.5 mL centrifuge tube, and 1 mL of chromatography-grade acetonitrile was added for
homogenization. After incubation at room temperature for an hour, the sample was centrifuged under cold
conditions (4 °C, 12,000 × g, 15 min) to isolate the supernatant for subsequent use. The pellet was then
treated with 1 mL of methanol, thoroughly mixed, incubated for 15 min, and centrifuged again under the
same cold conditions. The supernatants were pooled, and the process was reiterated. Subsequently, the
pooled supernatant was freeze-dried to remove all liquid content, followed by the addition of 1 mL of
chromatographic-grade methanol for dissolution, and then centrifuged once more under the same
conditions. The supernatant was filtered through a 0.22-micron filter, and an aliquot of the sample was
transferred into an injection vial for analysis.

For the determination of bile acid content, liquid chromatograph mass spectrometer (LC-MS) was
employed with a Thermo U3000 system equipped with an ACQUITY UPLC® HSS T3 1.8 µm column (2.1 ×
100 mm). The auto-sampler was maintained at 15 °C, with a flow rate set to 0.30 mL/min, and the column
temperature regulated at 35 °C. An injection volume of 2 µL was utilized for the gradient elution, which
involved a mobile phase consisting of 1 mM ammonium acetate in water (A) and in methanol (B). The
gradient elution program is as follows: 0 min, 20% B; 0-6 min, 20%-60% B; 6-26 min, 100% B; 26-28 min,
100%-50% B; 28-30 min, 50%-20% B; 30-32 min, 20% B .
[17]