Azadirachta indica attenuates cisplatin-induced nephrotoxicity and oxidative stress

Abstract

We investigated the effects of methanolic leaves extract of Azadirachta indica (MLEN, 500 mg/kg bwt) on cisplatin- (CP-) induced nephrotoxicity and oxidative stress in rats. CP (5 mg/kg bwt) was injected intraperitoneally and MLEN was given by gastric gavage for 5 days before or after CP injection. After 5 days of CP injection, CP-induced injury of the renal tissue was evidenced (i) as histopathological damage of the renal tissue, (ii) as increases in serum uric acid, urea, and creatinine, (iii) as increases in malondialdehyde (MDA) and nitric oxide (NO), (iv) as decreases in the level of glutathione and activities of superoxide dismutase, catalase, glutathione reductase, glutathione-S-transferase, and glutathione peroxidase, and (v) as increase in the expression of nuclear factor kappa B and apoptosis in kidney tissues. However, the oral administration of MLEN to CP-intoxicated rats for 5 days brought back MDA, NO production, and enzymatic and nonenzymatic antioxidants to near normalcy. Moreover, the histological observations evidenced that neem extract effectively rescues the kidney from CP-mediated oxidative damage. Furthermore, PCR results for caspase-3 and caspase-9 and Bax genes showed downregulation in MLEN treated groups. Therefore, Azadirachta indica can be considered a potential candidate for protection of nephrotoxicity induced by cisplatin.

Figure 1

Effects of methanolic leaves extract of neem on body weight and relative kidney weight of rats treated with CP. Values are mean ± SEM (n = 7). ^a P < 0.05, significant change with respect to control; ^b P < 0.05, significant change with respect to CP for Duncan's post hoc test.

Figure 2

Effects of methanolic leaves extract of neem on malondialdehyde, nitric oxide, and glutathione levels in kidney of rats treated with CP. Values are mean ± SEM (n = 7). ^a P < 0.05, significant change with respect to control; ^b P < 0.05, significant change with respect to CP for Duncan's post hoc test.

Figure 3

Effects of methanolic leaves extract of neem on superoxide dismutase and catalase activities in kidney of rats treated with CP. Values are mean ± SEM (n = 7). ^a P < 0.05, significant change with respect to control; ^b P < 0.05, significant change with respect to CP for Duncan's post hoc test.

Figure 4

H&E staining showing histopathological damage induced by CP on the kidney of rats. (a) Control kidney section with normal renal corpuscle and renal tubules. (b) Rats treated with cisplatin with congested shrank and completely degenerated glomeruli, debris in the lumen of some renal tubules, and pyknotic nuclei in renal tubules. ((c) and (d)) Treatment with methanolic leaves extract of neem before or after administration of CP showing normal renal corpuscle and renal tubule more or less like normal structure (X 400). SG: shrank glomeruli, DG: degenerated glomeruli; D: debris; PN: pyknotic nuclei.

Figure 5

NF-κB expression changes in the kidney of rats. (a) Normal kidney showing negative expression for NF-κB. (b) Treated kidney with cisplatin showing positive expression for NF-κB. (c) Kidney of rats treated with methanolic leaves extract of neem after 5 days of cisplatin injection showing mild positive expression for NF-κB. (d) Treated kidney with cisplatin after methanolic leaves extract of neem showing moderate expression for NF-κB (400X). The staining intensity was graded as white star (category 1); blue star (category 2); yellow star (category 3); orange star (category 4); red star (category 5).

Figure 6

Morphological changes visualized under fluorescence microscope with PI staining in the kidney of rats. (a) Normal kidney. (b) Treated kidney with cisplatin. (c) Kidney of rats treated with methanolic leaves extract of neem after 5 days of cisplatin injection. (d) Treated kidney with cisplatin after methanolic leaves extract of neem. The bright cells indicate the cells undergoing apoptosis (400X).

Figure 7

Relative quantification using RT-qPCR of mRNA expression of caspase-3 and caspase-9 and Bax genes in kidney of rats treated with cisplatin and methanolic leaves extract of neem.