In Vitro and In Vivo Nephroprotective Effects of Nelumbo nucifera Seedpod Extract against Cisplatin-Induced Renal Injury

Figures

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  Figure 1. Effect of LSE on the survival of NRK−52E cells and effect of cisplatin with/without LSE on NRK-52E cell viability. (A) NRK−52E cells were treated with 0, 5, 10, 25, 50, and 100 μg/mL of LSE for 24 h. Cellular survival was measured by Guava® Muse® cell analyzer. (B) NRK−52E cells were treated with 0, 2, 4, 6, 8, and 10 μM of cisplatin for 24 h. Cell survival was measured by Guava® Muse® cell analyzer. (C) NRK−52E cells were treated with or without LSE (5, 10 μg/mL) for 24 h in the presence or absence of cisplatin (8 μM). Quantification data were presented as mean ± SD of three independent experiments by using Sigma plot 10.0 software. ## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01 compared with the cisplatin group.

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  Figure 2. Effect of LSE on cisplatin-induced ROS production in NRK−52E cells. NRK−52E cells were treated with or without LSE (5, 10 μg/mL) for 24 h in the presence or absence of cisplatin (8 μM). (A) The ROS production was assayed with flow cytometry. (B) The DCF-positive cells divided by the total number of cells were calculated. Quantification data were presented as mean ± SD of three independent experiments by using Sigma plot 10.0 software. ## p < 0.01 compared with the control group; * p < 0.05 compared with the cisplatin group.

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  Figure 3. Effect of LSE on cisplatin-induced apoptosis in NRK−52E cells. NRK−52E cells were treated with or without LSE (5, 10 μg/mL) for 24 h in the presence or absence of cisplatin (8 μM). (A) The depolarization was assayed by JC-1 staining with flow cytometry. (B) The apoptotic cells were assayed by DAPI staining and the DAPI-positive cells were presented as a percentage of the total number of cells. (C) The apoptotic cells were analyzed by flow cytometry and apoptotic values were the percentage of apoptotic cells divided by the total number of cells. Quantification data were presented as mean ± SD of three independent experiments by using Sigma plot 10.0 software. ## p < 0.01 compared with the control group; * p < 0.05 compared with the cisplatin group, ** p < 0.01 compared with the cisplatin group.

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  Figure 4. Effect of LSE on cisplatin-induced apoptotic pathway in NRK−52E cells. NRK−52E cells were treated with or without LSE (5, 10 μg/mL) for 24 h in the presence or absence of cisplatin (8 μM). (A) The protein levels of p53 and phospho−p53. (B) The protein levels of Bcl2, Bax, p−Bad, Bad, and t−Bid. (C) The protein levels of cytosol and mitochondrial cytochrome c. (D) The protein levels of pro−caspase 9, caspase 9, pro−caspase 3, caspase 3, and CAD were analyzed by Western blotting. Quantification data were presented as mean ± SD of three independent experiments by using Sigma plot 10.0 software. # p < 0.05 compared with the control group, ## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01 compared with the cisplatin group.

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  Figure 5. Effects of LSE on the kidney biochemical parameters, lipid peroxidation, and antioxidant capacity in nude mice induced by cisplatin treatment. (A) BALB/c nude mice model and workflow (B) Serum BUN (Blood urea nitrogen), Cre (creatinine), UA (urea acid) and urine ACR (albumin to creatinine ratio) levels were analyzed. (C) The lipid peroxidation of the kidney was assessed by measuring the TBARS assay and the antioxidant capacity of the kidney was assessed by measuring the TEAC assay. Quantification data were presented as mean ± SD of three independent experiments by using Sigma plot 10.0 software. # p < 0.05, ## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01 compared with the cisplatin group.

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  Figure 6. Effects of LSE on kidney histopathology and apoptosis in nude mice induced by cisplatin treatment. (A) Representative 200X and 400X images of kidney sections stained with hematoxylin-eosin to display renal structure. (B) Representative 200X and 400X images of kidney sections stained with TUNEL to display renal apoptosis degree. (C) Quantification data of kidney apoptosis were presented as mean ± SD of three independent experiments by using Sigma plot 10.0 software. ## p < 0.01 compared with the control group; ** p < 0.01 compared with the cisplatin group.

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  Figure 7. Effect of LSE on kidney expression of apoptosis-related proteins in nude mice induced by cisplatin treatment. (A) The protein levels of p53, p-p53, Bax, Bcl2, and CAD were analyzed by Western blotting. (B) Quantification data were presented as mean ± SD of three independent experiments by using Sigma plot 10.0 software. ## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01 compared with the cisplatin group.