Page 76 - ARNM-3-3

Advances in Radiotherapy
& Nuclear Medicine CS@LGG for acute radiation-induced bowel injury alleviation

2.7. Biosafety assessment PBS and the sections were further subjected to HE staining

Each group of mice contained five individuals and was for 5 min. Finally, the sections were dehydrated and
dosed orally with CS (10 mg/mL, 200μL), LGG (1 × 10 9 mounted with neutral balsam. Histological assessment was
CFUs, 200 μL), or CS@ LGG (1 UI, 200 μL). On day 30, performed using a general optical microscope and ImageJ
after air drying. The percentage of positive-stained cells,
the mice were sacrificed, and their major organs, such staining intensity score, and final score were calculated
as heart, liver, spleen, lung, and kidney, were harvested. and subjected to statistical analysis. The ratio of positive
Blood samples were centrifuged (1000 × g, 5 min, cells was divided into four levels: ≤25% (1 point), 26–50%
room temperature) and aliquoted for routine blood (2 points), 51–75% (3 points), and >75% (4 points). The
examination, hepatotoxicity, and nephrotoxicity analysis. staining intensity score was also divided into four levels: No
These tests were performed at the Animal Testing Center, positive coloration (0 point), light yellow (1 point), brown
Nanfang Hospital. Organ samples were immersed in PFA yellow (2 points), and tan (3 points). Finally, the IHC score
(4%, 1 mL/sample) for 24 h. The tissue samples were was obtained by multiplying the staining intensity score by
then embedded and subjected to longitudinal sectioning the percentage of positive cells score.
for further pathological staining, while blood and serum
samples were prepared for biochemical and routine tests. 2.9. In vivo intestinal permeability assay

2.8. Histopathology analysis Intestinal permeability assays were performed with 4 kDa
FITC-dextran. After 4 h of food and water deprivation, the
2.8.1. Hematoxylin-eosin (HE) staining mice in four groups were given 0.5 mg/kg of FITC-dextran
The mice intestine, colon, and other organs were collected orally. The mice were then sacrificed, and serum samples
and rinsed in pre-cooled PBS. They were then immersed were collected to measure the fluorescence intensity of
in PFA and fixed for more than 24 h. Next, the tissues were FITC using a Varioskan LUX microplate reader (Thermo
dehydrated and paraffin-embedded for sectioning at 5 μm Fisher, USA). The mice serum from the control group
thickness. The prepared sections were dewaxed and stained served as the blank sample.
with hematoxylin first and eosin last. Finally, the sections 2.10. In vitro and in vivo ELISA
were dehydrated and mounted with neutral balsam. After
air drying for more than 24 h, they were placed under an To determine the levels of cytokines (IL-6, IL-1β, and TNFα)
optical microscope (Leica Microsystems, Shanghai, China) in colon tissue and serum, colon tissue homogenates and
for observation. ImageJ software (National Institutes of serum samples were prepared and analyzed using ELISA
3
Health, USA) was applied to analyze the length of villi, the kits. First, mice colon segments (1 mm ) were harvested,
depth of crypts, and the ratio of the former to the latter. homogenized in PBS at 4°C (1:10 w/v), centrifuged (12,000
First, the intestinal villi were measured for length, counts, ×g, 10 min, 4°C), and the supernatant was collected for
as well as area, and underwent manual correction and testing. Second, serum was separated from blood samples
statistical analysis. Second, the numbers, depth, and area by centrifugation (1,000 ×g, 5 min, room temperature) and
of intestinal crypts were calculated as described above. stored at 4°C for further analysis.
Finally, statistical analysis of the key data for villi, crypts, 2.11. In vitro cell assay
and their ratio was performed using GraphPad Prism v.9.0
(Dotmatics, USA). 2.11.1. Cell counting kit-8 (CCK-8) cell viability assay
2.8.2. Immunohistochemistry (IHC) staining The MC38 and IEC6 cells were seeded into 96-well
plates with 2,000 cells/well for the proliferation assay and
The paraffin tissue sections of mice intestine and colon 5,000 cells/well for cytotoxicity experiments, with three
underwent antigen retrieval with EDTA-Na (1×, high- replicates in each group. After incubation at 37°C, 5%
pressure heating, 8 min), blocking with goat serum (5%, CO humidified atmosphere for adherence, each group
2
Biosharp) for 20 min, and were then probed with Ki67 underwent different treatments and was prepared for
antibody (1:800), OCCLDN antibody (1:800), CLDN3 measurement. As specified by the CCK-8 kit, the working
antibody (1:800), γH2A.X antibody (1:600). After overnight solution was prepared with CCK-8 and DMEM at 1:100
incubation, the sections were washed with PBS for 5 min, in dark. Then, the cell culture medium was discarded in
3 times, and incubated with horseradish peroxidase- the tested wells, and 100 μL/well of the pre-made solution
conjugated anti-rabbit immunoglobulin G (1:500, MXB) was added, followed by an 1-h incubation. Finally, OD
for 1 h on the next day. The sections were then washed values were measured at 450 nm using a microplate reader
with PBS and incubated with DAB substrate. Once the (Multiskan, Thermo Fisher, USA), and the OD value was
samples turned brown, the reactions were stopped with positively correlated with the cell viability. In this study, the

Volume 3 Issue 3 (2025) 68 doi: 10.36922/ARNM025230026