An extract of Rhodobacter sphaeroides reduces cisplatin-induced nephrotoxicity in mice

Abstract

Cisplatin is used as a treatment for various types of solid tumors. Renal injury severely limits the use of cisplatin. Renal cell apoptosis, oxidative stress, and inflammation contribute to cisplatin-induced nephrotoxicity. Previously, we found that an extract of Rhodobacter sphaeroides (Lycogen™) inhibited proinflammatory cytokines and the production of nitric oxide in activated macrophages in a dextran sodium sulfate (DSS)-induced colitis model. Here, we evaluated the effect of Lycogen™, a potent anti-inflammatory agent, in mice with cisplatin-induced renal injury. We found that attenuated renal injury correlated with decreased apoptosis due to a reduction in caspase-3 expression in renal cells. Oral administration of Lycogen™ significantly reduced the expression of tumor necrosis factor-α and interleukin-1β in mice with renal injury. Lycogen™ reduces renal dysfunction in mice with cisplatin-induced renal injury. The protective effects of the treatment included blockage of the cisplatin-induced elevation in serum urea nitrogen and creatinine. Meanwhile, Lycogen™ attenuated body weight loss and significantly prolonged the survival of mice with renal injury. We propose that Lycogen™ exerts anti-inflammatory activities that represent a promising strategy for the treatment of cisplatin-induced renal injury.

Figure 1

The effects of Lycogen™ and cisplatin on the viability of mesangial cells (MES-13). MES-13 cells were treated with the indicated concentrations of Lycogen™ or cisplatin for 48 h. Cell viability was measured after (A) Lycogen™ treatment or (B) cisplatin treatment using a WST-1 assay. *** p < 0.001 (mean ± SD, n = 6). Each experiment was repeated three times with similar results.

Figure 2

Lycogen™ reduced cisplatin-induced cell apoptosis. MES-13 cells were pretreated with Lycogen™ at concentration of 0, 2, 4, 8 or 16 μM for 48 h. Next, cisplatin (5 μg/mL) was added to the cells for 48 h. (A) Cell viability was measured using a WST-1 assay; (B) The expression of nuclear p53 and caspase 3 was measured by Western blot analysis. Proliferating cell nuclear antigen (PCNA) and β-actin expression served as loading controls for nuclear proteins and total proteins, respectively. Inserted values indicate the relative protein expression when compared with pro-caspase 3. * p < 0.05, ** p < 0.01, *** p < 0.001 (mean ± SD, n = 6); (C) TNF-α secreted by MES-13 cells is mediated by TLR4 signaling. The cells were preincubated with anti-TLR4 or with control IgG, and then treated with or without Lycogen™ for 48 h. TNF-α was measured using an enzyme-linked immunosorbent assay. Each experiment was repeated three times with similar results.

Figure 3

Lycogen™ ameliorated cisplatin-induced renal dysfunction. Mice were treated with Lycogen™ (1 mg/kg) for three consecutive days, starting on day 0. Mice were given an intraperitoneal injection (i.p.) of cisplatin (30 mg/kg) on day 3. Control mice received PBS. The effect of Lycogen™ on (A) creatinine and (B) blood urine nitrogen (BUN) levels 72 h after cisplatin administration. * p < 0.05 (mean ± SD, n = 4). (C) Sections of kidney were stained with PAS or TUNEL 72 h after the cisplatin injection. The arrows indicate the location of positive TUNEL staining in the kidney. Each experiment was repeated three times with similar results.

Figure 4

Lycogen™ ameliorated cisplatin-induced renal inflammation. Mice were treated with Lycogen™ (1 mg/kg) for three consecutive days starting on day 0. Next, mice were injected i.p. with cisplatin (30 mg/kg) on day 3. Control mice received PBS. (A) TNF-α and (B) IL-1β levels in the sera as measured by ELISA 72 h after cisplatin treatment. * p < 0.05 (mean ± SD, n = 4). (C) Lycogen™ reduced cytokine expression in the kidney. A portion of the mice received Lycogen™ treatment. After three days, mice were killed, kidneys were collected, and the renal lysates were analyzed for TNF-α and IL-1β expression using immunoblot analysis. Each experiment was repeated three times with similar results.

Figure 5

The effect of Lycogen™ on cisplatin-induced renal injury in mice. (A) The mice were orally administered Lycogen™ (1 mg/kg) for three consecutive days after an i.p. injection of cisplatin (30 mg/kg) and the body weight of mice was recorded daily (mean ± SD, n = 8, * p < 0.01 for cisplatin-induced renal injury mice pretreated with Lycogen™ versus cisplatin-induced renal injury mice pretreated with PBS); (B) Kaplan-Meier survival curves up to day 14 are shown (n = 8). Significant differences between continuous variables was assessed using Student’s t-test. Mice survival analysis was performed using the Kaplan-Meier survival curve and log-rank test. * p < 0.05, ** p < 0.01; (C) The mice were injected i.p. with cisplatin (30 mg/kg) and then orally administered Lycogen™ (1 mg/kg) for three consecutive days beginning 16 h after the cisplatin administration. The body weights of mice were recorded (mean ± SD, n = 8); (D) Kaplan-Meier survival curves up to day 14 are shown (n = 8).