Page 44 - JCTR-11-1

Journal of Clinical and
Translational Research Hesperidin enhances repair of γ-irradiated wounds

weight. Six animals were used for each radiation dose in acid (70 µmoL, 0.1 mL). The mixture was heated for
each concurrent group at each interval, except for the 40 Gy 15 min in a boiling water bath. After centrifugation, the
group, where eight were used for each dose of radiation at absorbance was recorded at 535 nm using a double-beam
each assay time. In total, 240 animals were used for this UV-visible spectrophotometer. The LPx was expressed as
experiment. malondialdehyde in nmol/mg protein.
2.4.3.2. Skin 2.4.3.2.4. Protein estimation
A separate experiment was carried out to study the effect Protein contents were measured using the method
of hesperidin on the status of various antioxidants in the described by Waterborg & Matthews (1994). using bovine
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skin of wounded mice after exposure to 0, 10, 20, or 40 Gy serum albumin as the standard.
of fractionated γ-radiation. The grouping of animals and
production of full-thickness wounds were similar to those 2.4.4. Experiment 4: Histological studies
described in the wound contraction experiment, with skin A separate experiment was conducted to evaluate the
biopsies collected at 0, 1.5, 3, 6, 12, 24, and 48 h after the histological alterations during wound healing after
last exposure. The skin was carefully separated from the exposure to 0, 10, 20, or 40 Gy fractionated γ-radiation.
panniculus carnosus muscle, flash-frozen in liquid nitrogen, The grouping of animals and production of full-thickness
and stored at −70°C until analysis. The skin samples were wounds were similar to those described for the wound
weighed and homogenized in phosphate-buffered saline. contraction experiment. Cross-sectional full-thickness
A minimum of four animals were used for each group biopsies of regenerating wounds from each group were
and each dose of radiation at each post-irradiation time, collected on days 4, 8, and 12 post-irradiation. The
resulting in a total of 280 animals used for this experiment. samples were fixed in 10% buffered formalin, passed
sequentially through different grades of alcohol to ensure
2.4.3.2.1. Glutathione peroxidase (GSHPx) complete dehydration, and then embedded in paraffin
GSHPx activity was estimated using the method described wax. Medial samples were sectioned to a thickness of
by Sazuka et al. Briefly, 100 µL of homogenate was mixed 5 µm perpendicular to the surface, starting from the
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with 200 µL each of EDTA, sodium azide, glutathione center of the wound, and stained with hematoxylin
(GSH), H O and 400 µL of buffer. The reaction mixture and eosin. Sections were assessed in a blinded manner
2,
2
was incubated at 37°C for 10 min, followed by the addition under the light microscope using a planimeter for
of 10% TCA. After centrifugation, the supernatant was fibroblast proliferation, neovascularization, and collagen
collected and mixed with 3 mL disodium hydrogen deposition. For collagen deposition studies, faint
phosphate and 1 mL DTNB. The absorbance of the samples traces of staining reaction, hyalinization, and irregular
was recorded against the blank at 412 nm using a double- arrangement of collagen bundles were considered as
beam UV-visible spectrophotometer. The enzyme activity +, while the most intense reactions with compactly
was expressed as µmol GSH/mg protein. arranged collagen bundles were scored as +++. Two areas
in each section were counted for neovascularization and
2.4.3.2.2. GSH fibroblast proliferation. Elongated spindle-shaped cells
GSH concentration was measured using the method with purple nuclei and pink cytoplasm were identified
described by Moron et al. Briefly, proteins were as fibroblasts and scored accordingly. Blood vessels that
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precipitated using 25% TCA, centrifuged, and the were conspicuous with hematoxylin and eosin stains were
supernatant was collected. The supernatant was mixed scored for vascular repopulation studies. Four animals
with 0.2 moL sodium phosphate buffer of pH 8.0 and 0.06 were used in each concurrent group at each interval for
mmoL DTNB, followed by incubation for 10 min at room each radiation dose, resulting in a total of 120 animals
temperature. The absorbance of the samples was read used for this experiment.
against the blank at 412 nm in a double-beam UV-visible
spectrophotometer and the GSH concentration was 2.5. Data analysis
calculated from the standard curve. Statistical significance between the treatments was
determined using one-way Analysis of variance, while
2.4.3.2.3. Lipid peroxidation (LPx) Student’s t-test was used for biochemical estimations.
LPx was measured using the method described by Buege and Data analysis was conducted using Origin 8 (Origin Lab
Aust. Briefly, the tissue homogenate was mixed with TCA- Corporation, United States). All data are presented as mean
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thiobarbituric acid-HCl solution, butylated hydroxytoluene ± standard error of the mean, and P < 0.05 were considered
(3.5 mmoL, 0.1 mL), and diethylenetriamino-pentaacetic statistically significant.

Volume 11 Issue 1 (2025) 38 doi: 10.36922/jctr.24.00049