The first group served as the control group. == 1. Intro == Methotrexate, a folic acid antagonist, is definitely widely used in the treatment of numerous malignancies and inflammatory diseases. However, nephrotoxicity is an important adverse effect of methotrexate therapy [1]. The pathogenesis of methotrexate nephrotoxicity entails multiple pathways, including oxidative stress and swelling [2,3]. Several providers have been used, with various examples of success, to ameliorate or prevent methotrexate nephrotoxicity [24]. Curcumin is an active polyphenolic constituent fromCurcuma longawith notable antioxidant and anti-inflammatory properties [5,6] that render it a good candidate for safety Telotristat against methotrexate nephrotoxicity. Curcumin has shown renal protecting properties against gentamicin- and cisplatin- induced renal toxicities ([7] and [8], resp.) as well mainly because diabetic nephropathy [9]. The present study consequently was designed to assess the possible renoprotective effect of curcumin and to examine the underlying Telotristat mechanism(s) responsible for this effect inside a rat model of methotrexate-induced nephrotoxicity. The mechanism of renoprotection was evaluated Telotristat by assessing the oxidative stress (i.e., malondialdehyde, nitric oxide, glutathione peroxidase, and superoxide dismutase) and inflammatory (i.e., tumor necrosis element-[TNF-] and cyclooxygenase-2 [COX-2]) guidelines. == 2. Materials and Methods == == 2.1. Chemicals == Curcumin was a good gift from DBK Pharma (Cairo, Egypt). Methotrexate was a good gift from Minapharm (Cairo, Egypt). Antibody against COX-2 was purchased from Thermo Fisher Telotristat Scientific Inc./Lab Vision (Fremont, CA, USA). All other chemicals were of analytical grade and were from commercial sources. == 2.2. Animals and Experimental Design == Ten-week-old male Wistar rats weighing 150180 g were used after one week Rabbit Polyclonal to NF-kappaB p65 (phospho-Ser281) for appropriate acclimatization to the animal house conditions (12 h lighting cycle and 25 2C temp) with free access to standard rodent chow and water. All experimental methods were conducted according to the honest standards authorized by the Institutional Animal Ethics Committee recommendations for animal care and use, Minia University or college, Egypt. Animals were randomly divided into four organizations (68 animals each). The 1st group served as the control group. The second group was treated with curcumin (200 mg/kg p.o.) [10] suspended in 1% aqueous remedy of carboxymethyl cellulose daily for three consecutive days and served as positive control. The third group was injected with methotrexate (7 mg/kg, i.p.) [11] daily for three consecutive days to induce nephrotoxicity. The fourth group was given curcumin (200 mg/kg, p.o.) 1 h before methotrexate (7 mg/kg, i.p.) daily for three consecutive days. All organizations received equal quantities of the used vehicles. Rats were sacrificed 24 h after the last methotrexate injection, and blood samples were collected and centrifuged at 3000 g for 10 min to obtain obvious sera. The longitudinal section of the remaining kidney was excised from each animal for histological and immunohistochemical exam. The renal cortex of the rest of the kidneys were snap freezing in liquid nitrogen, stored at 80C, and consequently homogenized in chilly potassium phosphate buffer (0.05 M, pH 7.4) for various biochemical analyses. == 2.3. Biochemical Analysis == Using commercially available kits, serum levels of creatinine and urea (Diamond Diagnostics, Egypt) as well as renal glutathione peroxidase and superoxide dismutase (Biodiagnostic, Egypt) activities were quantified according to the manufacturers’ recommendations. Renal TNF-assay was performed with rat TNF-ELISA kit (RayBiotech, Inc., GA, USA) relating to supplier’s instructions. Renal cortex lipid peroxidation was identified as thiobarbituric acid reacting substance and is indicated as equivalents of malondialdehyde, using 1,1,3,3-tetramethoxypropane as standard [12]. Renal cortex nitric oxide level was measured as total nitrite/nitrate, the stable degradation products of nitric oxide, by reduction of nitrate into nitrite using copperized cadmium, followed by color development with Griess reagent in acidic medium [13]. == 2.4. Histological and Immunohistochemical Exam == Renal cells samples were fixed in 10% neutral buffered formalin, inlayed in paraffin, sectioned, and stained with hematoxylin and eosin for histological exam using light microscopy. Three sections from each animal group were subjected to semiquantitative microscopical analysis using light microscopy (Olympus CX41). Renal changes were graded as slight, moderate, or severe. Scores +, ++, and +++ are slight, moderate, and severe levels, revealing less than 25, 50, and 75% histopathological alterations of total fields examined, respectively. For immunohistochemical.