Cochlear Marginal Cell Pyroptosis Is Induced by Cisplatin via NLRP3 Inflammasome Activation

Abstract

Better understanding the mechanism of cisplatin-induced ototoxicity is of great significance for clinical prevention and treatment of cisplatin-related hearing loss. However, the mechanism of cisplatin-induced inflammatory response in cochlear stria vascularis and the mechanism of marginal cell (MC) damage have not been fully clarified. In this study, a stable model of cisplatin-induced MC damage was established in vitro, and the results of PCR and Western blotting showed increased expressions of NLRP3, Caspase-1, IL-1β, and GSDMD in MCs. Incomplete cell membranes including many small pores appearing on the membrane were also observed under transmission electron microscopy and scanning electron microscopy. In addition, downregulation of NLRP3 by small interfering RNA can alleviate cisplatin-induced MC pyroptosis, and reducing the expression level of TXNIP possesses the inhibition effect on NLRP3 inflammasome activation and its mediated pyroptosis. Taken together, our results suggest that NLRP3 inflammasome activation may mediate cisplatin-induced MC pyroptosis in cochlear stria vascularis, and TXNIP is a possible upstream regulator, which may be a promising therapeutic target for alleviating cisplatin-induced hearing loss.

Keywords: NLRP3; TXNIP; cisplatin; marginal cells; pyroptosis.

Figure 1

Cisplatin induced MC damage in vitro in a time- and concentration-dependent pattern. (A) MCs grow in clumps and express CK-18 characteristically. Scale bar: 200 µm. (B) Cell viability of MCs under cisplatin treatment at gradient concentrations of 0, 0.1, 0.5, 1, 5, 10, 50, 100, 500, and 1,000 (μmol/L) for 24, 48, and 72 h, respectively. (C) Representative images that cisplatin induced the variation of the proportion of MCs with membrane rupture (PI+, the percentage of cells in Q1 and Q2 quadrant) with the time prolongation detected by flow cytometry. The control group was not treated with cisplatin, and the other groups were treated with 5 μmol/l cisplatin for 24, 48, and 72 h, respectively. The above experiment was repeated at least 3 times. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Cisplatin induced an increased expression of NLRP3 in MCs at both the mRNA level and the protein level. (A) The expression difference of NLRP3 mRNA in MCs between the control and the cisplatin group (5 μmol/l cisplatin for 24 h). (B, C) The increased expression of NLRP3 protein in the cisplatin group compared with the control detected by immunofluorescence (B) and Western blotting (C). Green fluorescence indicates FITC-labeled NLRP3 protein, and blue fluorescence indicates DAPI-labeled nuclei. Scale bars: 50 µm. Representative results of at least three repeated experiments are shown. *p < 0.05, ***p < 0.001.

Figure 3

Downregulation of NLRP3 decreased the number of MCs with cell membrane rupture (A) or DNA rupture (B). (A) The differences in the percentage of MCs with cell membrane rupture (the cells in the Q1 and Q2 quadrants) between the groups. (B) The differences in the percentage of MCs with DNA fracture (TUNEL positive) between the groups. Blue fluorescence indicates DAPI-labeled cell nuclei, and red fluorescence indicates DNA fragmentation labeled with Alexa Fluor 640. The red fluorescence overlapping with the nucleus was judged to be TUNEL-positive cells (white arrows). Scale bar: 100 µm. Representative results of at least three repeated experiments are shown. **p < 0.01, ***p < 0.001.

Figure 4

Downregulation of NLRP3 reversed the cisplatin-induced ultramicroscopic changes in MCs. (A) The morphologic changes of MCs after different treatments under TEM. The red arrows indicate the ruptured cell membrane and local lighter-stained cytoplasm. Scale bars: 2 µm. (B) The morphological changes of MCs observed by SEM. The red arrows indicate the withered tree-like cell membrane at the edge of the cell. Scale bars: 50 µm. Representative results of at least three repeated experiments are shown.

Figure 5

The expression differences of key molecules in NLRP3 inflammasome-mediated pyrolysis (NLRP3, ASC, caspase-1, IL-1β, and GSDMD) in mRNA level (A) and in protein level (B) in the control, cisplatin, cisplatin+siNC, and cisplatin+siNLRP3 groups. (C) The difference in the IL-1β concentrations in the supernatant of each group. Representative results of at least three repeated experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001. ns, no significance.

Figure 6

The expression differences of NLRP3, ASC, caspase-1, and GSDMD in the control, cisplatin, cisplatin+siNC, and cisplatin+siNLRP3 groups. Blue fluorescence indicates DAPI-labeled cell nuclei, and green fluorescence indicates FITC-labeled target proteins. Representative results of at least three repeated experiments are shown. Scale bar: 100 µm.

Figure 7

The effect of downregulation of TXNIP on cisplatin-induced NLRP3 inflammasome-mediated pyrolysis-related protein expression in MCs at mRNA level (A) and protein level (B). (C) The difference in the concentrations of IL-1β in the supernatant in each group. (D) The variation of NLRP3 expression before and after TXNIP-siRNA transfection. Scale bars: 100 µm. Representative results of at least three repeated experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8

Downregulation of TXNIP decreased the number of MCs with cell membrane rupture (A) or DNA rupture (B). (A) The differences in the percentage of MCs with cell membrane rupture (the cells in the Q1 and Q2 quadrants) between the groups. (B) The differences in the percentage of MCs with DNA fracture (TUNEL positive) between the groups. Blue fluorescence indicates DAPI-labeled cell nuclei, and red fluorescence indicates DNA fragmentation labeled with Alexa Fluor 640. The red fluorescence overlapping with the nucleus was judged to be TUNEL-positive cells (white arrows). Scale bars: 100 µm. Representative results of at least three repeated experiments are shown. *p < 0.05, **p < 0.01.

Figure 9

Downregulation of TXNIP reversed the cisplatin-induced ultramicroscopic changes in MCs, similar to that after downregulating NLRP3. (A) The morphologic changes of MCs after different treatments were evaluated under TEM. The red arrows indicate the ruptured cell membrane and local lighter-stained cytoplasm. Scale bars: 2 µm. (B) The morphological changes of MCs were observed by SEM. The red arrows indicate the withered tree-like cell membrane at the edge of the cell. Scale bars: 15 µm. Representative results of at least three repeated experiments are shown.

Figure 10

The schematic diagram of cisplatin-induced NLRP3 inflammasome-mediated MC pyroptosis.