Bee Venom Mitigates Cisplatin-Induced Nephrotoxicity by Regulating CD4(+)CD25(+)Foxp3(+) Regulatory T Cells in Mice

Abstract

Cisplatin is used as a potent anticancer drug, but it often causes nephrotoxicity. Bee venom (BV) has been used for the treatment of various inflammatory diseases, and its renoprotective action was shown in NZB/W mice. However, little is known about whether BV has beneficial effects on cisplatin-induced nephrotoxicity and how such effects might be mediated. In the present study, the BV-injected group showed a significant increase in the population of Tregs in spleen. Although there was no significant difference in the numbers of Tregs 3 days after cisplatin injection between the BV- and PBS-injected groups, more migration of Tregs into the kidney was observed 6 hours after cisplatin administration in BV group than in PBS group. In addition, BV-injected mice showed reduced levels of serum creatinine, blood urea nitrogen, renal tissue damage, proinflammatory cytokines, and macrophage infiltration into the kidney 3 days after cisplatin administration. These renoprotective effects were abolished by the depletion of Tregs. The anticancer effect of repeated administrations of cisplatin was not affected by BV injection. These results suggest that BV has protective effects on cisplatin-induced nephrotoxicity in mice, at least in part, through the regulation of Tregs without a big influence on the antitumor effects of cisplatin.

Figure 1

Increased CD4⁺CD25⁺Foxp3⁺ Tregs in vitro and in vivo. BV (1 μg/mL) was treated in splenocytes separated from Foxp3^EGFP mice for 3 days. There was no significant difference in the proportion of dead cells between BV- (20.7%) and PBS-treated (19.3%) groups. This in vitro BV treatment significantly increased the number of CD25⁺Foxp3⁺ T cells among the CD4⁺T cells (a, b). And Foxp3^EGFP mice were injected with BV (1 mg/kg) or same volume PBS once a day for 5 days. This in vivo BV injection also significantly increased the number of CD25⁺Foxp3⁺ T cells among the CD4⁺T cells (c). Tregs were analyzed by flow cytometry gated for CD4-positive cells (a). The values shown indicate the mean ± SEM. ***P < 0.001 versus control; n = 5 (b), *P < 0.05 versus control; n = 3 in vivo (c).

Figure 2

Survival in cisplatin-treated mice. Survival curves of mice treated with 25 mg/kg of cisplatin that were followed up for up to 240 h. BV-treated mice had a 43.75% survival rate, whereas all of the control mice expired. The values shown indicate the mean ± SEM. *P < 0.05 versus control; n = 16.

Figure 3

The protective effects of BV on cisplatin-induced nephrotoxicity in mice. Mice received BV (1 mg/kg) once a day for 5 days. The control group received the same volume of PBS. After the fifth administration of BV or PBS, all mice received a single injection of cisplatin (25 mg/kg). Blood samples were obtained at 24 h and 48 h after cisplatin administration, and mice were sacrificed under ether anesthesia at 72 h after cisplatin administration. Blood and kidney samples were extracted for analysis. Blood was obtained at 24, 48, and 72 h after cisplatin injection (n = 18). Renal dysfunction was measured as creatinine (a) and BUN (b). Kidney sections were stained with H&E 72 h after cisplatin injection. PBS treatment alone (cont); PBS and cisplatin treatments (Cis); BV and cisplatin (Cis + BV) (400x, bar = 50 um) (c). Tubular injury damage was scored from 0 to 4 (0, none; 1, <10%; 2, 10–25%; 3, 25–75%; 4, >75%) (d). The values shown indicate the mean ± SEM. NS: no significance (P > 0.05). *P < 0.05, ***P < 0.001 versus Cis.

Figure 4

Survival test in CD25-depleted mice. After CD25 depletion, mice were treated with 25 mg of cisplatin/kg intraperitoneally and followed up for up to 114 h to produce survival curves. At 114 h, all of the control mice and BV-treated mice had expired (NS, P > 0.05 versus cont; n = 8).

Figure 5

The inefficacy of bee venom in CD25-depleted mice. All mice were treated with anti-CD25 antibody (0.1 mg/mouse, i.p.) twice, before and after BV injection. Nephrotoxicity was induced by cisplatin (25 mg/kg i.p.). Blood was obtained at 24, 48, and 72 h after cisplatin injection (n = 12). Renal dysfunction was reflected by the levels of creatinine at 48 h, 72 h (a) and BUN at 48 h, 72 h (b). Kidney sections from CD25-depleted mice were stained with H&E at 72 h after cisplatin injection. PBS treatment alone (cont); PBS and cisplatin treatments (Cis); BV and cisplatin (Cis + BV) (400x, bar = 50 μm) (c). Tubular injury damage was scored from 0–4 (0, none; 1, <10%; 2, 10–25%; 3, 25–75%; 4, >75%) (d). The values shown indicate the mean ± SEM. NS, P > 0.05 versus Cis.

Figure 6

Proinflammatory cytokines in the kidney. Proinflammatory cytokines were measured in kidney tissue from both of normal mice and CD25-depleted mice by ELISA. Renal IL-6 and TNFα were decreased in the BV-treated group (Cis + BV) compared with the PBS-treated group (Cis). However, there was no difference between the BV-treated group and PBS-treated group in CD25-depleted mice. The values shown indicate the mean ± SEM. *P < 0.05 versus Cis.

Figure 7

The number of Foxp3-positive cells in the kidney. Tregs in the kidney were detected by EGFP signal under the confocal microscope imaging. Foxp3-positive cells were indicated by arrows (a). There was no difference in the number of Foxp3 positive cells between PBS-treated group and BV-treated group before cisplatin administration. However, Foxp3-positive cells in the kidney increased 6 hours after cisplatin administration. In BV-pretreated group, after cisplatin administration, the number of Foxp3-positive cells in the kidney was significantly higher than in PBS-pretreated group (b). All data are presented as the mean ± SEM. NS, P > 0.05, *P < 0.05 versus cont, and ^# P < 0.05 versus Cis, as determined by one-way ANOVA followed by Tukey's multiple comparison test.

Figure 8

Macrophage infiltration into kidney. Macrophage accumulation in the kidney was detected by immunostaining for F/480-positive cells at 72 h after cisplatin administration. The number of F4/80-positive cells in each section was counted in 10 fields per slide at an original magnification of 200x. The infiltration in BV-treated mice was compared with those of PBS-treated mice and CD25-depleted mice. The values shown indicate the mean ± SEM. ***P < 0.001 versus Cis.

Figure 9

Influence of BV on the antitumor effect of cisplatin. All mice received injections of EL4 lymphoma cells (2 × 10⁶/0.1 mL) s.c. into the right flank. BV (1 mg/kg) was injected once per day for 5 days from day 5 of tumor inoculation (n = 8). Cisplatin was injected 3 times (on days 11, 14, and 17) at a concentration of 5 mg/kg. All mice were sacrificed at day 20, and the tumors were photographed (a). The tumor size was measured every three days after BV injection for 16 days. After sacrifice, the tumors were fully separated from the mice, and the size (b) and weight (c) of the tumors were measured. All data are presented as the mean ± SEM. ***P < 0.001 versus cont; NS, P > 0.05 versus Cis, as determined by one-way ANOVA followed by Tukey's test.