3.1. Melatonin Ameliorated Cisplatin-Induced Nephrotoxicity:
To induce acute kidney injury, mice were treated with cisplatin intraperitoneally. Administration of melatonin significantly attenuated a serious worsening of kidney function, as determined by reduced serum BUN (Figure 1A) and creatinine (Figure 1B) levels. H&E and PAS staining also showed that melatonin treatment significantly ameliorated cisplatin-induced histological changes like as tubular cast formation, tubular dilatation and tubular cell death (Figure 2A&B).
To further evaluate the effect of melatonin in cisplatin-induced tubular damage, the expression levels of tubular injury markers, including NGAL and Kim-1, were examined. In the cisplatin-treated mice, immunohistochemical staining revealed that expression levels of NGAL and Kim-1 were increased in the damaged tubules. These changes were significantly reduced by melatonin (Figure 3A&B). Consistently, melatonin treatment also significantly dropped the expression of Kim-1 (Figure 3C&D).
3.2. Melatonin Attenuated Cisplatin-Induced Tubular Epithelial Cell Apoptosis:
To investigate the mechanisms underlying the safeguarding effects of melatonin against cisplatin-induced nephrotoxicity, TUNEL staining of kidney sections was conducted. Administration of melatonin remarkably
dropped the increased number of apototic cells in the renal tubules (Figure 4A&B). Moreover, melatonin treatment also significantly dropped the ciplatin-induced elevation of cleaved caspase-3 and cleaved PARP-1 expressions (Figure 4C-E).
3.3. Melatonin Attenuated Cisplatin-Induced Tubular Epithelial Cell Necroptosis:
To investigate the effect of melatonin on cisplatin-induced necroptosis, protein expression of RIPK1 and RIPK3 in the kidneys was examined.
Cisplatin treatment markedly increased their protein levels in the kidneys and these changes were significantly attenuated by melatonin (Figure 5A-C), suggesting that melatonin inhibits cisplatin-induced necroptosis.
3.4. Melatonin Attenuated Inflammatory Responses in the Kidneys of Cisplatin-Treated Mice:
Plasma membrane rupture in necroptosis results in release of intracellular components that stimulate immune system and inflammation (23). Given that cisplatin induces NF-κB activation and subsequently promotes expression of pro-inflammatory mediators, the effect of melatonin on NF-κB phosphorylation in the kidneys was examined.
Administration of melatonin significantly reduced protein level of phospho-NF-κB (Figure 6A&B), in the kidneys of cisplatin-treated mice.
Additionally, pro-inflammatory cytokines’ mRNA expression including TNF-α, IL-6, and MCP-1 were also significantly reduced by melatonin (Figure 6C-E).
3.5. Melatonin Attenuated Cisplatin-Induced Apoptosis and Necroptosis:
Next experiment was whether melatonin could also inhibit cisplatin-induced apoptosis and necroptosis in cultured TCMK-1 cells.
The result was that pretreatment with melatonin dropped the increased number of TUNEL-positive cells after cisplatin treatment (Figure 7A&B). LDH release assay also showed that melatonin significantly reversed cisplatin-induced LDH release (Figure 7C). In addition, elevated cleaved caspase-3 and cleaved PARP-1 in cisplatin-treated cells were significantly decreased by melatonin pretreatment (Figure 7D-F).
Pretreatment with melatonin also significantly reduced the elevated RIPK1 and RIPK3 in cisplatin-treated cells (Figure 7G-I).
Figure 1. Effect of melatonin on renal function in cisplatin-treated mice.
Mice were treated with 20 mg/kg melatonin intraperitoneally.
(A) Serum blood urea nitrogen (BUN) levels. (B) Serum creatinine levels. Con: control; CP: cisplatin; CP+MEL:
cisplatin plus melatonin. n = 10 per group. The values represent the mean ± SEM. *p < 0.05, **p < 0.01 vs. Con. #p
< 0.05, ###p < 0.001 vs. CP.
Figure 2. Effect of melatonin on tubular damage in cisplatin-treated mice. (A) Renal hematoxylin and eosin (H&E) and periodic acid-schiff (PAS) stainings. Scale bar, 50 μm. (B) Tubular injury score. Con: control; CP: cisplatin; CP+MEL: cisplatin plus melatonin. n = 10 per group. The values represent the mean ± SEM. ***p < 0.001 vs. Con. #p < 0.05 vs. CP.
Figure 3. Effects of melatonin on expression of tubular injury markers in cisplatin-treated mice. Renal immunohistochemical staining using anti-neutrophil gelatinase-associated lipocalin (anti-NGAL) antibody (A) and anti-kidney injury molecule-1 (anti-Kim-1) antibody (B). Scale bar, 25 μm. (C) Renal expression level of Kim-1 protein. (D) Relative expressions of Kim-1. Con: control; CP: cisplatin; CP+MEL: cisplatin plus melatonin. n = 10 per group. The values represent the mean
± SEM. **p < 0.01 vs. Con. ##p < 0.01 vs. CP.
Figure 4. Effect of melatonin on cisplatin-induced tubular apoptosis. (A) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining on kidney sections.
Scale bar, 50 μm. (B) Number of TUNEL-positive cells. (C) Renal expression levels of cleaved caspase-3 and cleaved poly ADP-ribose polymerase 1 (PARP-1). Relative expressions of cleaved caspase-3 (D) and cleaved PARP-1 (E). Con: control;
CP: cisplatin; CP+MEL: cisplatin plus melatonin. n = 10 per group. The values represent the mean ± SEM. **p < 0.01, ***p
< 0.001 vs. Con. ##p < 0.01, ###p < 0.001 vs. CP.
Figure 5. Effect of melatonin on cisplatin-induced necroptosis in the kidneys. (A) Renal expression levels of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Relative expressions of RIPK1 (B) and RIPK3 (C). Con: control; CP:
cisplatin; CP+MEL: cisplatin plus melatonin. n = 10 per group.
The values represent the mean ± SEM. ***p < 0.001 vs. Con.
##p < 0.01, ###p < 0.001 vs. CP.
Figure 6. Effect of melatonin on cisplatin-induced inflammation in the kidneys. (A) Renal expression level of phospho-nuclear factor kappa-light-chain-enhancer of activated B (phospho-NF-κB) p65. (B) Relative expressions of phospho-NF-κB p65. Relative mRNA expressions of renal tumor necrosis factor-alpha (TNF-α) (C), interleukin-6 (IL-6) (D), and monocyte chemoattractant protein-1 (MCP-1) (E). Con: control; CP: cisplatin; CP+MEL:
cisplatin plus melatonin. n = 10 per group. The values represent the mean ± SEM. **p < 0.01, ***p < 0.001 vs. Con. #p < 0.05, ##p
< 0.01 vs. CP.
Figure 7. Effects of melatonin on cisplatin-induced apoptosis and necroptosis in cultured mouse renal tubular epithelial cells. The cells were pretreated with melatonin (1 mM) for 1 h and after that treated with cisplatin (2 μg/mL) for 24 h. (A) Renal TUNEL stainings. Scale bar, 200 μm. (B) TUNEL-positive cells. (C) Lactate dehydrogenase (LDH) release assay. (D) Cellular expression levels of cleaved caspase-3 and cleaved PARP-1. Relative expressions of cleaved caspase-3 (E) and cleaved PARP-1 (F). (G) Cellular expression levels of RIPK1 and RIPK3. Relative expressions of RIPK1 (H) and RIPK3 (I).
Con: control; CP: cisplatin; CP+MEL: cisplatin plus melatonin. n
= 10 per group. The values represent the mean ± SEM. *p <
0.05, ***p < 0.001 vs. Con. #p < 0.05, ##p < 0.01, ###p < 0.001 vs.
CP.