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Global Translational Medicine Hesperetin alleviates pulmonary injury

was laid in ventrodorsal position, was thumped with a Deparaffinized pulmonary tissue sections were dehydrated
cylindrical object weighing 0.4 kg dropped from 0.5 m with ethanol, washed in PBS, placed in a citrate buffer
above. Thus, the chest cage of the rat received an energy to reacquire antigens, and then heated in a microwave
amounting to 1.96 J, measured according to the formula: (Arcelik, MD550) for 5 min. After cooling down, the
E = m × g × h (I) sections were treated with 3% hydrogen peroxide to
suppress endogenous peroxides. Tissue incubation with
where E = Energy, m = Mass of cylindrical object (0.4 kg), primary antibody was performed in a humidified chamber
g = Gravity (9.8 m/s ), and h = Height (0.5 m). Animals in for over 1 h at 37°C. Each tissue section was incubated with
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the PC + hesperetin group were administered hesperetin streptavidin-biotin-labeled antibodies for 30 min, and
for 7 days with the aid of intragastric gavage at a dose of subsequently, each of them was labeled with AEC. Contrast
100 mg/kg/day dissolved in 1 mL physiological saline. At staining was completed using Mayer’s hematoxylin.
the end of the 7 day, the rats were opened on the midline The iNOS-positive cells in the pulmonary tissues were
th
under anesthesia (ketamine-xylazine; 90 – 15 mg/kg, ip), enumerated per squared millimeter (mm ).
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and blood samples were taken by cardiac puncture and
pulmonary tissue was removed for future analyses. 2.8. ELISA analysis
2.5. Determination of lung wet/dry weight ratio Pulmonary MDA levels (nmol/mg protein) were
determined using a commercial kit according to the
The left superior lobe of the lungs was used to assess manufacturer’s instructions. Commercial ELISA kits
pulmonary edema. The fresh tissue was washed with were employed to measure the pulmonary levels of MDA
phosphate-buffered saline (PBS), dried, and weighed. The (catalog no: CK-E10376; Eastbiopharm, Hangzhou,
wet/dry weight ratio was calculated according to a previous China) and TNF-α (KRC3011, Thermo Scientific), as well
study by weighing the tissues again after being kept at 80°C as the serum levels of IL-1β (BMS630, Thermo Scientific)
for 24 h. 17 and IL-6 (BMS625, Thermo Scientific), according to the
2.6. Histopathological analysis manufacturers’ instructions. The absorbance of the tested
samples was measured at 450nm using Multiscan Go
The left middle lobe was immersed in a 10% buffered Spectrophotometer (Thermo Fisher Scientific, MA, USA).
neutral formalin solution for fixation for 48 h before The levels of MDA and proinflammatory cytokines were
hematoxylin-eosin (H&E) and Masson trichrome determined with the help of standard curves.
staining. After fixation, tissue samples were soaked in
tap water overnight and then processed in a series of 2.9. Statistical analysis
routine procedures before being submerged in paraffin. Data are expressed as mean ± standard deviation in this
Paraffin blocks were cut to sections with 5 µm thickness paper. The Kruskal–Wallis test was used to evaluate the
using a rotary microtome (Slee, MPS, Germany) and then data, and Mann–Whitney U test was employed to analyze
subjected to H&E staining, Masson trichrome staining, the differences among groups. All statistical analyses
and immunohistochemical iNOS labeling. H&E and were performed using the PASW statistic 18.0 (SPSS Inc,
Masson trichrome staining were conducted according to Chicago, IL, USA). Differences were considered statistically
steps described elsewhere. H&E-stained slides were rated significant at P < 0.05.
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by two histopathologists, who were blinded to the study
groups, for histopathological changes, interalveolar edema, 3. Results
interalveolar hemorrhage, and neutrophil infiltration using
a range of grades from 0 to 4 (0: none; 1: mild; 2: moderate; 3.1. Histopathological findings and lung wet/dry
3: severe; 4: overwhelming) with total maximum score of weight ratio
12. The distribution of connective tissue in pulmonary Histopathological investigation of lung tissue was
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tissue and alveolar wall thickening was confirmed with performed using H&E and Masson trichrome staining
Masson trichrome staining. Microscopic examination (Figure 1). The H&E staining revealed that the lung
was performed with an Olympus CX41 (Olympus, Tokyo, tissue structure in the control group was normal. In the
Japan) and image analysis system (Kameram Gen III, PC group, alveolar wall thickening, alveolar hemorrhage,
Argenit, Istanbul, Turkey) software. alveolar edema, and increased leukocyte infiltration
were observed. In the PC + hesperetin group, these
2.7. Immunohistochemical iNOS labeling histopathological changes were also present but at a milder
Immunohistochemical iNOS labeling was conducted degree. In addition, milder alveolar thickening and edema
using the avidin-biotin complex peroxidase technique. in the PC + hesperetin group were confirmed with Masson

Volume 3 Issue 2 (2024) 3 doi: 10.36922/gtm.2568