A- Animals: 36 mature male Wistar albino rats, weighing 180 ± 20 g, were utilized in this investigation. They were purchased from the faculty of pharmacy- Assuit University, Assuit, Egypt. The institutional animal care and use committee of Beni-Suef University (BSU-IACUC) was in charge of overseeing the handling, nourishment, housing, acclimation, and experimental animal protocols. Their permission number is 022–360.
B-Drug used:
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Cyclosporine: Sigma-Aldrich (St. Louis, MO) provided the cyclosporine, which was administered to the animals by IP injection at a dose of 20 mg/kg each day for the previous seven days.
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Sitagliptin: The animals received sitagliptin orally for 14 days at a dose of 10 mg/kg/day from Sigma-Aldrich (St. Louis, MO).
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Hesperidin: The animals were fed hesperidin orally at a rate of 200 mg/kg/day for 14 days straight, courtesy of Sigma-Aldrich (St. Louis, MO).
C-experimental Design And Grouping Of Rats: -
o Group І: Rats were only given the vehicle for 14 days. The standard control group was this one.
o Group ІІ: Rats given a single 20 mg/kg/day i.p. injection of CsA every day for the previous seven days.
o Group ІІІ: Sitagliptin was administered orally to rats once daily for 14 days at a dose of 10 mg/kg.
o Group ІV: Rats received both CsA and sitagliptin treatments as previously indicated. Sitagliptin administered 7 days before and 7days after CsA that was administrated last 7 days.
o Group V: For 14 days straight, rats were given a single oral dose of 200 mg/kg/day of hesperdin prepared in distal water by oral gavage.
o Group VІ: Rats received both CsA and hesperdin treatments as previously indicated. CsA was administered 7th days after of hesperdin (that’s was administrated 7days before CsA and 7 days after CsA).
Before scarification, blood samples were drawn from the retro-orbital plexus into clean, dry tubes. Following that, blood samples were centrifuged for 5 minutes at 4000 RPM. For the purpose of measuring the serum's parameters, the serum was separated and transferred to sterile screw-capped vials.
Animals were killed by cervical dislocation after blood assembly, and the kidneys were promptly removed and three times cleansed in ice-cooled normal saline [20]. To prepare 20% kidney homogenates, a portion of each kidney was homogenized (1/5 w/v) in ice-cold Tris-HCl buffer (pH 7.4, 0.1 M), and the homogenates were then stored at -20°C for biochemical examination [20].
D-biochemical Analysis:
1- Determination of serum Albumin (g/dl)
determined utilizing Dumas' techniques [21].
2. Determination of serum creatinine (mg/dl): That determined according to the method of Toora and Rajagopal [22].
3-Determination of myeloperoxidase (MPO)
According to the manufacturer's recommendations, serum MPO was calculated using the Quantikine ELIZA kit from R&D Systems, catalogue number DMYE00B, for the quantitative assessment of MPO concentration in serum.
4-Determination of Cystatin-C (CYS-C)
that was established using the technique of Finney et al. [23] and Erlandsen et al. [24]
5-Determination of serum glucose
that was established using the technique of Trinder [25].
6. Determination of tissue malondialdehyde (MDA): that was established using the technique of Satoh et al. [26]
7. Determination of catalase (CAT) activity: that was established using the technique of Fossati et al. [27]
8. Determination of glutathione reduced (GSH): that was established using the technique of Beutler et al. [28]
E- Histopathological Examination
All of the animals' kidneys underwent paraffin embedding and 10% formalin fixation. After that, specimens were embedded in blocks in order to slice them at five micron intervals. Following this, slices were prepared for hematoxylin and eosin (H&E) staining. The kidney was examined, and then photographs were taken.
F- Determination Of Tnf-α And Western Plot Analysis:
Measurement of TNF-α in sera
TNF-α was measured in sera using an ELISA kit according to Aderka et al. [29], employing ELISA kits according to Montero-Julian et al. [30]
Western Blot Analysis:
To achieve clear supernatant, tissue samples were centrifuged after being homogenized in RIPA buffer. The total protein content was determined using the Bradford reagent. SDS-PAGE was used to isolate 30 µg of protein per gel lane, and the protein was then transferred to a PVDF membrane. The membranes were then treated with primary antibodies against TNF-alpha Antibody after being blocked in Tris-buffered saline with Tween 20 (TBST) containing 5% non-fat milk powder (Novus Biologicals USA). By ensuring that protein loading is uniform throughout the gel, the housekeeping protein β-actin was utilized as a loading control to normalize the quantities of protein observed. The membranes were TBST-washed before being incubated for an hour with horseradish peroxidase-conjugated secondary antibodies from Novus Biologicals in Littleton, Colorado, USA. An improved chemi-luminescence kit was used to find immuno-labeling (BioRad, Hercules, CA). Finally, utilizing ImageJ to scan the acquired blots and quantify band intensities (NIH, Bethesda, Maryland, USA) [31].
G- Immunohistochemical determination of Bax and NrF-2.
Rehydrate and deparaffinize the tissue portion. Slide should be incubated for ten to fifteen minutes in hydrogen peroxide to decrease endogenous peroxidase's nonspecific background staining. Wash in the buffer twice. Incubate tissue in digesting enzyme if necessary.
To prevent nonspecific background staining, wash four times in buffer, apply block, and then incubate for 5–10 minutes at room temperature. Note: Don't wait longer than 10 minutes otherwise the intended stain may not be as strong. Once in the buffer, wash. Apply the primary antibody and incubate as directed by the manufacturer.
4 times in the buffer, wash. Apply the biotinylated link antibody, then let it sit at room temperature for 15 to 20 minutes. 4 times in the buffer, wash. Apply Streptavidin/HRP, and then let it sit at room temperature for 20 minutes. 4 times in the buffer, rinse. One 5ml vial of DAB Substrate should contain 8 drops of DAB chromogen. Mix thoroughly, then apply to tissue for five minutes. Rinse once in distilled water. Apply the DAB Chromogen/Substrate mixture, and then wait a further five minutes before continuing. Rinse the coverslip, counterstain, and DI water three times using a permanent mounting medium [32].
Statistical analysis:
ANOVA (Analysis of Variance) - One Way Analysis of Variance and Duncan Multiple Range Test (DMRT) were used in the statistical analysis to compare the effects of the various treatment groups on the various variables under investigation. The statistical analysis was made using SPSSPC + version 28 –Computer program.