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. 2021 Oct;9(20):1578.
doi: 10.21037/atm-21-5125.

TRPA1 promotes cisplatin-induced nephrotoxicity through inflammation mediated by the MAPK/NF-κB signaling pathway

Jinyan Yuan #  1   2 , Xiao Liang #  3 , Wei Zhou #  2 , Jing Feng  4 , Zhenyang Wang  1   2 , Shaoxian Shen  5 , Xin Guan  5 , Liangbin Zhao  6 , Fei Deng  1   5
Affiliations

TRPA1 promotes cisplatin-induced nephrotoxicity through inflammation mediated by the MAPK/NF-κB signaling pathway

Jinyan Yuan et al. Ann Transl Med. 2021 Oct.

Abstract

Background: The nephrotoxicity induced by cisplatin (DDP) has been a severe obstacle for its clinical use in anticancer treatment. The apoptosis and inflammation induced by DDP are the main causes of the nephrotoxicity. Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation ligand-gated channel that is involved in the inflammation progress.

Methods: The apoptosis, inflammation, MAPK/NF-κB signaling pathway, and TRPA1 expression were assessed after HEK293 cells had been induced by DDP, and the role of TRPA1 in apoptosis and inflammation of DDP-induced HEK293 cells treated with TRPA1 antagonist HC-030031 was also evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), flow cytometry, and western blot assays.

Results: The cell viability was reduced by DDP in both a time-dependent and dose-dependent manner with a minimal cytotoxic concentration of 10 μM. Moreover, DDP induced an enhancement of the apoptosis and inflammation in a dose-dependent manner, as indicated by the increase of the relative protein level of cleaved-caspase3 (cleaved-cas3), the cleavage product of caspase-3 substrate poly-ADP-ribose polymerase (cleaved-PARP) and inducible nitric oxide synthase (iNOS), and the messenger RNA (mRNA) expression level of interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), and interferon-γ (INF-γ). Additionally, DDP treatment increased the protein phosphorylation expression of IKKβ, JNK, ERK, and p38 in a dose-dependent manner, which was antagonized by the treatment of NF-κB-specific inhibitor BAY 11-7082 and pan-MAPK inhibitor U0126. It was also found that DDP upregulated the expression of TRPA1 at both the mRNA and protein levels in a dose-dependent manner. Besides, block of TRPA1 with HC-030031 relieved the apoptosis, diminished the level of IL-1β, IL-6, TNF-α, and INF-γ, reduced the level of cleaved-cas3, cleaved-PARP, and iNOS, decreased the p-IKKβ, p-JNK, p-ERK, and p-p38 expression, and enhanced the expression of IκBα.

Conclusions: Taken together, these results indicate that TRPA1 regulates DDP-induced nephrotoxicity via inflammation mediated by the MAPK/NF-κB signaling pathway in HEK293 cells.

Keywords: Cisplatin (DDP); MAPK/NF-κB signaling pathway; apoptosis; nephrotoxicity; transient receptor potential ankyrin 1 (TRPA1).

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/atm-21-5125). The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
DDP inhibits the HEK293 cells viability and increases apoptosis. HEK293 cells were administrated with DDP with the concentration of 0, 5, 10, 20, 40, and 80 μM for 24, 36, and 48 h, respectively. DDP reduced the HEK293 cells viability in a time-dependent (A) and dose-dependent way (B). The cleaved-cas3 and cleaved-PARP protein levels were measured using western blot (C). The means ± SD of three independent samples were present. *P<0.05 and **P<0.01, compared with 0 group. DDP, cisplatin; SD, standard deviation; cleaved-cas3, cleaved-caspase3; cleaved-PARP, cleavage product of caspase-3 substrate poly-ADP-ribose polymerase.
Figure 2
Figure 2
DDP boosted the inflammatory of HEK293 cells. HEK293 cells were administrated with DDP with the concentration of 0, 0.5IC50, IC50 and 2IC50 respectively. The expression level of IL-1β, IL-6, TNF-α, and INF-γ was detected by qRT-PCR (A). Also, the level of iNOS (B) was examined using western blot analysis respectively. The means ± SD of three independent samples were shown. The results were exhibited after being normalized to β-actin. *P<0.05 and **P<0.01, compared with 0 group. DDP, cisplatin; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; INF-γ, interferon-γ; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; iNOS, inducible nitric oxide synthase; SD, standard deviation.
Figure 3
Figure 3
DDP influenced the MAPK/NF-κB signaling pathway. HEK293 cells were treated with DDP at the concentrations of 0, 0.5IC50, IC50, and 2IC50, respectively. The expression level of IKKβ, p-IKKβ, JNK, p- JNK, ERK, p-ERK, p38, and p-p38 (A) were examined by western blot assay. Then, HEK293 cells were treated with IC50 DDP, BAY 11-7082, IC50 DDP + BAY 11-7082 or PBS (control) respectively. The expression of iNOS protein (B) and the expression of IL-1β, IL-6, TNF-α, and INF-γ (C) was examined by western blot and qRT-PCR analysis. After HEK293 cells were treated with IC50 DDP, U0126, IC50 DDP + U0126 or PBS (control) respectively, the expression of iNOS protein (D), the expression level of IL-1β, IL-6, TNF-α, and INF-γ mRNA (E), and (F) the expression of IκBα, p-IκBα, IKKβ, and p-IKKβ protein was determined using western blot analysis and qRT-PCR analysis. The means ± SD of three independent samples were exhibited. The data were analyzed after being normalized to β-actin. *P<0.05 and **P<0.01, compared with 0/control group; #P<0.05 and ##P<0.01, compared with IC50 DDP group. DDP, cisplatin; PBS, phosphate-buffered saline; iNOS, inducible nitric oxide synthase; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; INF-γ, interferon-γ; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; mRNA, messenger RNA; SD, standard deviation.
Figure 4
Figure 4
The TRPA1 expression was increased both at mRNA and protein levels in a dose-dependent manner. HEK293 cells were administrated with DDP with the concentration of 0, 0.5IC50, IC50, and 2IC50 (A), or IC50 DDP, BAY 11-7082, IC50 DDP + BAY 11-7082 or PBS (control) (B) respectively. The TRPA1 expression was examined using western blot analysis. The means ± SD of three independent samples were exhibited. The results were analyzed after being normalized to β-actin. *P<0.05 and **P<0.01, compared with 0/control group; #P<0.05 and ##P<0.01, compared with IC50 DDP group. DDP, cisplatin; PBS, phosphate-buffered saline; SD, standard deviation.
Figure 5
Figure 5
HC-030031 relieved the apoptosis and the expression level of apoptosis-related proteins. HEK293 cells were treated with IC50 DDP, HC-030031, IC50 DDP + HC-030031, or PBS (control), respectively. The apoptotic of the HEK293 cells was determined via flow cytometry after PE and APC co-staining (A), and the expression level of cleaved-cas3 and cleaved-PARP was examined by western blot (B). The means ± SD of three independent samples were shown. The results were shown after being normalized to β-actin. β-actin. *P<0.05 and **P<0.01, compared with control group; #P<0.05 and ##P<0.01, compared with IC50 DDP group. DDP, cisplatin; PBS, phosphate-buffered saline; PE, phycoerythrin; APC, allophycocyanin; cleaved-cas3, cleaved-caspase3; cleaved-PARP, cleavage product of caspase-3 substrate poly-ADP-ribose polymerase; SD, standard deviation.
Figure 6
Figure 6
HC-030031 alleviated the inflammation. HEK293 cells were treated with IC50 DDP, HC-030031, IC50 DDP + HC-03003, or PBS (control), respectively. The expression level of IL-1β, IL-6, TNF-α, and INF-γ of the HEK293 cells was determined by qRT-PCR (A), and the expression of iNOS was determined via western blot (B). The means ± SD of three independent samples were present. The results were shown after being normalized to β-actin. *P<0.05 and **P<0.01, compared with control group; #P<0.05 and ##P<0.01, compared with IC50 DDP group. DDP, cisplatin; PBS, phosphate-buffered saline; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; INF-γ, interferon-γ; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; iNOS, inducible nitric oxide synthase; SD, standard deviation.
Figure 7
Figure 7
HC-030031 functioned on the MAPK/NF-κB signaling pathway. HEK293 cells were dealt with IC50 DDP, HC-030031, IC50 DDP + HC-030031, or PBS (control) respectively. The expression of IκBα, p-IκBα, IKKβ, p-IKKβ, JNK, p-JNK, ERK, p-ERK, p38 and p-p38 was examined using western blot assay. The means ± SD of three independent samples were shown. The data were exhibited after being normalized to β-actin. *P<0.05 and **P<0.01, compared with control group; #P<0.05 and ##P<0.01, compared with IC50 DDP group. DDP, cisplatin; PBS, phosphate-buffered saline; mRNA, messenger RNA; SD, standard deviation.
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