Lysophosphatidic acid exerts protective effects on HEI-OC1 cells against cytotoxicity of cisplatin by decreasing apoptosis, excessive autophagy, and accumulation of ROS

Abstract

Lysophosphatidic acid (LPA) is an active phospholipid signaling molecule that binds to six specific G protein-coupled receptors (LPA_1-6) on the cell surface and exerts a variety of biological functions, including cell migration and proliferation, morphological changes, and anti-apoptosis. The earliest study from our group demonstrated that LPA treatment could restore cochlear F-actin depolymerization induced by noise exposure, reduce hair cell death, and thus protect hearing. However, whether LPA could protect against cisplatin-induced ototoxicity and which receptors play the major role remain unclear. To this end, we integrated the HEI-OC1 mouse cochlear hair cell line and zebrafish model, and found that cisplatin exposure induced a large amount of reactive oxygen species accumulation in HEI-OC1 cells, accompanied by mitochondrial damage, leading to apoptosis and autophagy. LPA treatment significantly attenuated autophagy and apoptosis in HEI-OC1 cells after cisplatin exposure. Further investigation revealed that all LPA receptors except LPA₃ were expressed in HEI-OC1 cells, and the mRNA expression level of LPA₁ receptor was significantly higher than that of other receptors. When LPA₁ receptor was silenced, the protective effect of LPA was reduced and the proportion of apoptosis cells was increased, indicating that LPA-LPA₁ plays an important role in protecting HEI-OC1 cells from cisplatin-induced apoptosis. In addition, the behavioral trajectory and in vivo fluorescence imaging results showed that cisplatin exposure caused zebrafish to move more actively, and the movement speed and distance were higher than those of the control and LPA groups, while LPA treatment reduced the movement behavior. Cisplatin caused hair cell death and loss in zebrafish lateral line, and LPA treatment significantly protected against hair cell death and loss. LPA has a protective effect on hair cells in vitro and in vivo against the cytotoxicity of cisplatin, and its mechanism may be related to reducing apoptosis, excessive autophagy and ROS accumulation.

Fig. 1. LPA protects HEI-OC1 cells against cytotoxicity of cisplatin.

A HEI-OC1 cells were treated with increasing concentrations of LPA: 0 μM (vehicle control), 1, 3, 5, 10, 15, and 20 µM, and incubated for 0, 4, 12, 24, and 48 h. Cell viability was assessed by cell counting kit-8 (n = 6, Data are represented as means ± SD, *p < 0.05). B HEI-OC1 cells were exposed to LPA (1, 3, 5, 10, 15, and 20 µM) for 4 h and treated with cisplatin (10 µM) for 24 h. (n = 6, Data are represented as means ± SD. *p < 0.05 versus cisplatin group; **p < 0.01; ***p < 0.001; ns, non-significance). C Flow cytometric analysis of apoptotic cells in various treatment groups. Viable cells are shown by the Annexin V-negative and propidium iodide-negative quadrant (Q3, lower left quadrant). The Annexin V-positive and PI-negative quadrant (Q4, lower right quadrant) shows early apoptotic cells. The Annexin V-positive and PI-positive quadrant (Q2, upper right quadrant) shows late apoptotic and necrotic cells. The total number of apoptotic cells was the sum of early (Q4) and late (Q2) apoptotic cells (i.e., Q4 + Q2). n = 3, Data are represented as means ± SD. *p < 0.05 versus control group or LPA group. D Data shown in C are quantified. ****p < 0.0001 and *p < 0.05.

Fig. 2. LPA protects cells by inhibiting cisplatin-induced activation of the apoptotic pathway.

A Immunofluorescence staining with TUNEL and DAPI in the HEI-OC1 cells after different treatments, Scale bar = 100 um, n = 3. B Data shown in A were quantified (**p < 0.01). C Immunoblot detection of caspase 3/cleaved caspase 3 and β-actin expression in different treatment groups cells. D and E data shown in C were quantified (****p < 0.0001; ns, non-significance; n = 3). F Immunoblot detection of Bax and β-actin expression in different groups of HEI-OC1 cells. G Data shown in F were quantified. H WB assay of Bcl-2 and β-actin expression in different groups of HEI-OC1 cells. I Data shown in H were quantified (****p < 0.0001, **p < 0.01 and *p < 0.05, n = 3).

Fig. 3. HEI-OC1 pretreatment with LPA inhibits ROS production and apoptosis induced by cisplatin.

A DCFH-DA assay to detect intracellular ROS. HEI-OC1 cells were exposed to 0 µM cisplatin (vehicle control), 15 µM LPA, 15 µM LPA + 10 µM cisplatin, and 10 µM cisplatin. After 4-h LPA pretreatment, cisplatin was used to treat the cells for 24 h. B Data shown in A were quantified. ***p < 0.001, Scale bar = 100 μm.

Fig. 4. Pretreatment with LPA reduces cisplatin-induced ultrastructural changes in cells.

A Morphological and structural changes in the different treatment groups. Scale bar: 50 mm. B Transmission electron microscopy study for the presence of HEI-OC-1 cells that underwent apoptosis. Cells were exposed to 0 µM cisplatin (vehicle control), 15 µM LPA, 15 µM LPA + 10 µM cisplatin, and 10 µM cisplatin. After 4-h LPA pretreatment, cells were subjected to cisplatin treatment for 24 h. B′ is a locally enlarged picture of the site shown by the red dashed box in each treatment group of Fig. B, mark the mitochondria with red arrows.

Fig. 5. LPA treatment inhibited cisplatin-induced autophagy.

A Immunofluorescence staining images of LC3B in HEI-OC1 cells exposed to cisplatin (10 µM) for 24 h and with or without 15 µM LPA. B Immunoblot detection of LC3B-II and LC3B-I expression in cisplatin- and/or LPA-exposed cells. C, D LC3B expression quantification in B ****p < 0.0001, *p < 0.05, Scale bar = 50 mm.

Fig. 6. Tracking paths for the tap and light/dark photoperiodic variation model.

A, D Heatmap of the tracked path of tap and light/dark modes for each experimental group containing six zebrafish individuals. Blue marks show the area of the passage of zebrafish, and the color intensity represents the tracked path’s cumulative frequency [77]. B Quantification of the running distance of zebrafish in each treatment group. C Quantification of the movement velocity of zebrafish in each treatment group. E, F Running distance and movement velocity analysis of zebrafish in each treatment group under light/dark conditions, respectively (light and dark cycles were run for 10 min each). G Quantification of the movement velocity of zebrafish in each treatment group. H Quantification of the running distance of zebrafish in each treatment group. Six zebrafish were studied in each group. p < 0.0001, ***p < 0.001, **p < 0.01 and *p < 0.05, n = 6.

Fig. 7. LPA protects against cisplatin-induced HCs loss in zebrafish lateral lines in vivo.

A Schematic diagram of the distribution of hair cells in the lateral line neuromast throughout the body of zebrafish and the observation of the dorsal and lateral view of hair cells. B Survival of neuromast hair cells in the lateral line of zebrafish in different treatment groups. C A partial enlarged view of the box in Figure B. Zebrafish strains: [Et(krt4: EGFP)]; control group, do not process; Cisplatin group, 10 μM Cisplatin ; LPA group, 15 μM LPA; LPA+Cis group, adding 15 μM LPA for 4 h, then adding 10 μM Cisplatin for 24 h; The above groups were treated for the same time.

Fig. 8. Silencing of the LPA₁ receptor prevents LPA’s protective effects on cisplatin-induced apoptosis.

A qRT-PCR for the relative expression of the LPA receptors _1–6 (n = 3). B Effective silencing of the LPA₁ receptor using siRNA. siRNA_(LPA1) expression was significantly decreased in HEI-OC1 cells as compared to that in cells transfected with siRNA_(control) ****p < 0.0001, n = 3, as estimated by one-way ANOVA. C FCM of apoptotic cells after transfection with siRNA_(LPA1). D Quantization of flow detection data in C ****p < 0.0001, n = 3, as determined by one-way ANOVA.