Page 77 - ARNM-3-3

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

experimental groups received 8 Gy IR before testing, while analysis. To eliminate biological differences, the screening
the control group was directly tested on days 1, 3, 5, and 7. of differential genes was evaluated in terms of fold change
and significance level. The threshold for differential
2.12. Colony forming assay gene screening in this analysis was set as p<0.05 and

The MC38 and IEC6 cells were seeded in 6-well culture |log (FoldChange)| > 1.
2
plates with 800 cells/well in biological triplicates. After
48 h, the cells were treated with 0 UI, 0.25 UI, 0.5 UI, or 1 2.15. Statistical analysis
UI dose of CS@LGG. The assays were terminated at day 14 The experiments were repeated 3 times independently with
by fixing the cells in cold methanol solution for 90 min and similar results. All data were expressed as mean ± standard
staining with 0.1% crystal violet for 90 min. Colonies were deviation (SD). Significance between two groups was
counted using ImageJ. tested by an unpaired two-tailed t-test. Significance among
multiple groups was tested by one-way analysis of variance.
2.13. Alkaline comet assay
Statistical analyses were performed using GraphPad
IEC6 were seeded and adhered before co-cultured with Prism v.9.5 (GraphPad Software). Statistical significance
LGG (1×10 CFUs, 0.1 μL) or CS@LGG (0.1 UI, 1 μL), was indicated as *p<0.05; **p<0.01; ***p<0.001; and not
9
respectively. The cells were then subjected to 8 Gy IR and significant (denoted as “n.s.”).
collected after 2 h, with each group of cell suspensions
adjusted to a density of 1 × 10 cells/mL. The comet assay 3. Results
6
was started with the preparation of the first layer of 1% 3.1. Preparation and characterization of CS@LGG
normal melting point agarose (NMA). NMA was heated to probiotic particles
95℃ until fully melted, then quickly dropped onto a clean
glass slide while still hot. Next, the second layer of 0.7% To further clarify whether LGG biomaterial can alleviate
low-melting-point agarose (LMA) was prepared, melted in radiation-induced damage in gut, CS@LGG particle were
a 65℃ water bath, and mixed with 10 μL of cell suspension. synthesized as illustrated in Figure 1A. SEM images in
The slides were then topped with a third layer of 1% NMA Figure 1B displayed a clear surface morphology of CS,
evenly over the LMA and immersed in pre-chilled lysis LGG, and CS@LGG. The lengths and widths of LGG and
buffer at 4°C for 1 h. After complete lysis, the slides were CS@LGG were 4 ± 0.30 and 4.8 ± 0.23 μm, and 0.8 ± 0.04
washed 3 times with PBS and placed in an electrophoresis and 1.2 ± 0.34 μm, respectively, demonstrating that the
chamber filled with 1.2 L TAE buffer for 30 min for DNA particle sizes of LGG and CS@LGG were stable. Zeta
unwinding. Finally, the slides underwent electrophoresis potential measurements (Figure 1C) showed values of
at 100 V for 15 min in the dark, followed by staining 13.5 ± 10.9, 38.1 ± 10.8, and –19.9 ± 13.7 mV for LGG, CS,
with 10 μL propidium iodide (PI) and the application and CS@LGG, respectively, suggesting that the biological
of coverslips. The slides were then observed under a formation was mainly related to electrostatic interactions.
fluorescence microscope, and the percentage of comet tail Specifically, LGG is enriched in negatively charged teichoic
DNA of at least 50 cells was calculated using CASP 1.2.3 acids, whereas CS carries a positive charge, facilitating
(Krzysztof Konca, Poland). stable binding between them. FTIR analysis (Figure 1D)
further assessed the structure of CS@LGG, showing
2.14. RNA sequencing analysis peaks at 3447.394 and 1636.168 cm , corresponding to
-1
Total RNA was extracted using TRIzol reagent (Invitrogen, amidogen and carbonyl functional groups. In addition,
USA). The concentration and purity of RNA was assessed XPS (Figure 1E) revealed the elemental composition, with
by Nanodrop 2000 and agarose gel electrophoresis, characteristic binding energies and counts at 285.15 (C1s),
respectively. The Majorbio Cloud Platform (SourceForge, 398.38 (N1s), 1070.53 (Na1s), and 531.62 (O1s). Together,
USA) was used to analyze the data. RSEM (version 1.3.1) these results confirmed the successful loading of LGG
was used to evaluate the relationship between samples. into CS, with the final product forming a translucent and
DESeq2, DEGseq, and edgeR were used for differential uniform hydrogel macroscopically (Figure 1F).
gene expression analysis. Gene differential expression
analysis was further applied to gene ontology (GO). 3.2. CS@LGG alleviates damage of acute
GO analysis was performed using Fisher’s exact test and radiation-induced bowel injury in symptom and
the χ -test. The input data for the differential expression pathology
2
analysis were the ReadCount data obtained from the gene In clinical settings, IR of pelvic radiotherapy can lead to
expression level analysis. The edgeR method, which is various abnormalities of the intestinal tract. According
based on negative binomial distributions, was applied for to previous scientific studies and expert consensus,

Volume 3 Issue 3 (2025) 69 doi: 10.36922/ARNM025230026