Capsaicin ameliorates cisplatin-induced renal injury through induction of heme oxygenase-1

Abstract

Cisplatin is one of the most potent chemotherapy agents. However, its use is limited due to its toxicity in normal tissues, including the kidney and ear. In particular, nephrotoxicity induced by cisplatin is closely associated with oxidative stress and inflammation. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in the heme metabolism, has been implicated in a various cellular processes, such as inflammatory injury and anti-oxidant/oxidant homeostasis. Capsaicin is reported to have therapeutic potential in cisplatin-induced renal failures. However, the mechanisms underlying its protective effects on cisplatin-induced nephrotoxicity remain largely unknown. Herein, we demonstrated that administration of capsaicin ameliorates cisplatin-induced renal dysfunction by assessing the levels of serum creatinine and blood urea nitrogen (BUN) as well as tissue histology. In addition, capsaicin treatment attenuates the expression of inflammatory mediators and oxidative stress markers for renal damage. We also found that capsaicin induces HO-1 expression in kidney tissues and HK-2 cells. Notably, the protective effects of capsaicin were completely abrogated by treatment with either the HO inhibitor ZnPP IX or HO-1 knockdown in HK-2 cells. These results suggest that capsaicin has protective effects against cisplatin-induced renal dysfunction through induction of HO-1 as well as inhibition oxidative stress and inflammation.

Fig. 1.

Effect of capsaicin pretreatment on renal function and histology in cisplatin-induced kidney injury. Capsaicin was administered orally once a day for 5 consecutive days. Cisplatin was injected once at 12 h after capsaicin treatment for 4 consecutive days. The mice were sacrificed on day after the final cisplatin injection, and levels of serum creatinine (A) and BUN (B) were analyzed by an assay kit according to the manufacturer’s instructions (BioVision, USA). *p < 0.05 by one-way ANOVA compared with the cisplatin group (n = 5). Kidney specimens were stained with H&E (C). Damaged areas on tissue sections are marked with black arrows.

Fig. 2.

Effect of capsaicin pretreatment on cisplatin-induced inflammatory mediators. TNF-α (A), IL-1β (B), and IL-6 (C) were analyzed in serum by ELISA. Total kidney lysate was blotted and probed with an anti-TLR4 antibody (upper panel). (D) The blots were reprobed with an anti-actin antibody. Densitometric analyses are presented as the relative ratio of TLR4 to actin (lower panel). *p < 0.05 by oneway ANOVA compared with the cisplatin group (n = 3). Kidney sections were immunohistochemically stained with TLR4 (E), HMGB1 (F), and AGEs (G) antibodies. Cont, PBS (phosphate-buffered saline)-treated group; Cisplatin, cisplatin only treated group; Cisplatin + Cap, cisplatin and 10 mg/kg capsaicin-treated group.

Fig. 3.

Effect of capsaicin pre-treatment on cisplatin-induced oxidative stress markers. Kidney sections were immunohistochemically stained with NOX4 (A) or 4-HNE (C) antibody. Cont, PBS (phosphate-buffered saline)-treated group; Cisplatin, cisplatin only treated group; Cisplatin + Cap, cisplatin and 10 mg/kg capsaicin combined group. (B, D) HK-2 cells were treated with 30 μM cisplatin for 24 h in the presence or absence of 100 μM capsaicin. NOX4 expression was measured by Western blotting (B). To determine ROS level, cells were incubated with 10 μM H2-DCFDA at 37°C for 60 min and then fluorescence intensity was recorded using a fluorometer (D). *p < 0.05 by one-way ANOVA compared with the cisplatin (n = 3).

Fig. 4.

Effect of capsaicin pre-treatment on the expression of HO-1 and cisplatin-induced cytotoxicity. (A) For the quantitative analysis of protein expression level, kidney lysates were analyzed by Western blotting for HO-1 and Actin (upper panel) and signal intensities were quantified (lower panel). *p < 0.05 by one-way ANOVA compared with the control (n = 3). (B) Real-time PCR analysis of HO-1 expression in the kidney. Results were normalized to GAPDH expression and expressed as fold change compared with control. *p < 0.05 by one-way ANOVA compared with the control (n = 3). (C) Western blots and corresponding densitometric analyses of HO-1 expression in HK-2 cells at the indicated times after capsaicin treatment (100 μM). *p < 0.05 by one-way ANOVA compared with the control (n = 3). (D) Real-time PCR analysis of HO-1 expression in HK-2 cells at the indicated times after capsaicin treatment (100 μM). Results were normalized to GAPDH expression and expressed as fold change compared with control. *P < 0.05 by one-way ANOVA compared with the control (n = 3). (E–G) Cell viability was determined by MTT assay. Cell viability was expressed as a percentage of controls. *p < 0.05 by one-way ANOVA compared with the cisplatin (n = 3). N.S, not significant.