Fig 1.
Effect of EGCG on cisplatin-induced renal dysfunction in mice.
Cisplatin caused significant renal dysfunction as measured by the levels of creatinine (Panel A) and BUN at 72 hours (Panel B). Cisplatin administration resulted in severe kidney injury which was attenuated by EGCG treatment (at dose 100mg/kg). Results are mean ± S.E.M. n = 4/group.* p<0.05 versus vehicle; and # p<0.05 versus cisplatin.
Fig 2.
Effect of EGCG on cisplatin-induced kidney tubular damage in mice.
Cisplatin administration resulted in severe tubular damage as shown by histological examination using PAS staining. The damage was attenuated by EGCG treatment at dose 100mg/kg (designated as EGCG 100). Results are mean ± S.E.M., n = 4/group.* p<0.05 versus vehicle; and # p<0.05 versus cisplatin.
Fig 3.
Effect of EGCG on cisplatin-induced oxidative/nitrative stress in mitochondria.
Panel A. Quantitative measurement of protein nitration from mitochondrial fraction by ELISA demonstrated cisplatin induced content of protein niration. EGCG attenuated cisplatin induced mitochondrial protein nitration. Panel B. Quantitative measurement of HNE adducts from mitochondrial fraction by ELISA. A trend similar to protein nitration was also observed. Results are mean ± S.E.M. n = 4/group.* p<0.05 versus vehicle; and # p<0.05 versus cisplatin.
Fig 4.
Effect of EGCG on cisplatin-induced changes in mitochondrial enzyme complex activities in mice.
Cisplatin reduced the enzyme activities of mitochondrial electron transport chain namely (Panel A) NADH dehydrogenase Activity (Complex I), (Panel B) Succinate Dehydrogenase Activity (Complex II) and (Panel C) Cytochrome Oxidase Activity (Complex IV). EGCG administration protected mitochondrial enzyme against cisplatin induced damages. Results are mean ± S.E.M. n = 4/group.* p<0.05 versus vehicle; and # p<0.05 versus cisplatin.
Fig 5.
Effect of EGCG on cisplatin-induced nitrative stress in mice.
Panel A. Histological examination revealed significant protein nitration in the renal tubules of the cisplatin-treated group. EGCG treatment reduced the level of protein nitration similar to vehicle level. Panel B. Quantitative measurement of 3-nitrotyrosine adducts in protein also demonstrated similar trend. Results are mean ± S.E.M. n = 4/group.* p<0.05 versus vehicle; and # p<0.05 versus cisplatin.
Fig 6.
Effect of EGCG on cisplatin-induced pro-inflammatory cytokines in mice.
Real-time PCR based analyses of pro-inflammatory cytokines TNFα and IL1β indicated profound increase in cisplatin treated mice. EGCG treatment attenuated cisplatin induced TNFα and IL1β production. Results are mean ± S.E.M. n = 4/group.* p<0.05 versus vehicle; and # p<0.05 versus cisplatin.
Fig 7.
Effect of EGCG on cisplatin-induced nuclear translocation of NFκB, induction of p53 and apoptosis.
Panel A. Immuno-blot analyses of nuclear fraction from kidney demonstrated increase in p65 in cisplatin treated mice. The level is reduced in EGCG and cisplatin treated group. Panel B. Immuno-blot analyses of total lysate from kidney demonstrated increase in p53 in cisplatin treated mice. Panel C. Quantitative measurement of Caspase 3 activity and DNA fragmentation. Results are mean ± S.E.M. n = 4/group.* p<0.05 versus vehicle; and # p<0.05 versus cisplatin
Fig 8.
Effect of EGCG on cisplatin-induced apoptosis and mitochondrial ROS generation in HK-2 cells.
Panel A: Representative dot plot data showing early apoptosis (AnnexinV-APC staining) and late apoptosis (with Sytox Green) as measured by flow cytometry. Cisplatin was used at 60μM and EGCG was added at 10μM 2h prior to cisplatin addition. Panel B. Quantitative determinations of total cell death including early and late apoptosis were presented. Results are mean ± S.E.M. n = 4/group.* p<0.05 versus vehicle; and # p<0.05 versus cisplatin. Panel C: Representative histogram analyses of MitoSOX Red intensities from the same samples were presented. Panel D: Quantitative determinations of MitoSOX Red mean intensities. Results are mean ± S.E.M. n = 4/group.* p<0.05 versus vehicle; and # p<0.05 versus cisplatin.
Fig 9.
Schematic diagram of protection mechanism of EGCG in cisplatin induced kidney injury.
EGCG inhibit cisplatin induced mitochondrial ROS (Reactive Oxygen Species) in the renal tubular cells which caused cell death. Cell death in the tubular cells leads to pro-inflammatory response with cytokines (TNFα and IL1β). These process leads to leukocytes infiltration with additional burst of oxidative stress. EGCG also neutralize these pro-inflammatory cytokines. All combinatorial effects leads to reduced inflammation and cell death, thus protecting against cisplatin induced kidney injury.