Teicoplanin Induced HEI-OC1 Cell Loss and Cochlear HC Loss in a Dose-Dependent Manner in vitro

HEI-OC1 cells were subjected to various concentrations of teicoplanin (0, 3, 6, 12, 24 mM) for 24 h to determine the possible cytotoxicity of teicoplanin on auditory cells. Immunostaining results indicated that HEI-OC1 treated with teicoplanin had a decrease in cell quantity and shrunken nuclei. CCK-8 assay and statistical analysis revealed that the HEI-OC1 cell viability was significantly decreased to 65.57 ± 3.15% after 3 mM teicoplanin administration compared to the control group, and it was further reduced to 61.20 ± 1.90%, 51.93 ± 1.69% and 45.23 ± 1.72% after teicoplanin of 6, 12, 24 mM concentrations, respectively. As the concentration of teicoplanin increase, fewer HEI-OC1 cells were able to survive, indicating that the reduction in cell viability was dose-dependent (Fig. 1a, b). According to the IC50 curve, 7.5 mM teicoplanin was about to produce a 50% cell viability in HEI-OC1 cells (Fig. 1b), hence, 7.5 mM teicoplanin treatment for 24 h was selected as the treatment condition for the subsequent HEI-OC1 cell experiments.

Fig. 1

Teicoplanin induced HEI-OC1 cell loss and cochlear HC loss in a dose-dependent manner in vitro. a. Representative immunostaining images of HEI-OC1 cells labeled with HC marker Myosin 7a (green) and DAPI (blue) after different concentrations (0, 3, 6, 12, 24 mM) of teicoplanin administration for 24 h respectively. b. CCK-8 assay showed that the HEI-OC1 cell viability was markedly decreased after teicoplanin administration compared to the control group, and the reduction was in a dose-dependent manner. IC50 curve showed that 7.5 mM teicoplanin was about to cause a 50% cell viability in HEI-OC1 cells. c. Representative immunostaining images of cochlear HCs (phalloidin, green) treated with teicoplanin (0, 3.7, 7.5, 15.0 mM) for 24 h respectively in vitro. d, e. The mean cochleogram showed that teicoplanin caused a basal–apical gradient of HC loss and both IHC and OHC losses were exacerbated with teicoplanin dose increasing. * P < 0.05, ** P < 0.01, *** P < 0.001. Scale bar = 20 μm

Next, based on the dosage response assays of HEI-OC1 cells, cultured cochlea HCs were treated with in vitro teicoplanin at concentration of 3.7 mM, 7.5 mM, and 15 mM for 24 h respectively. Immunostaining and mean cochleograms were utilized to evaluate the location and percentage loss of OHCs and IHCs induced by teicoplanin. As illustrated in Fig. 1c-e, the greatest severe loss of HCs occurred at the base of the cochlea and decreased toward the apex, that is, teicoplanin induced a basal–apical gradient of HC loss. In addition, there was a greater loss of OHCs than IHCs after teicoplanin treatment, and both IHC and OHC losses exacerbated as the teicoplanin dose was increased. Specifically, there were barely any IHCs missing in the apex and middle cochlea turns of the 3.7 mM and 7.5 mM teicoplanin-treated groups, while there were considerable IHC losses in the basal cochlea turns (Fig. 1c, e, Table 3). Treatment with 3.7 mM teicoplanin resulted in modest OHC loss while 7.5 mM teicoplanin led to moderate OHC loss in the middle and basal turns of cochlea, but no significant OHC loss was found in the apical turn in either group (Fig. 1c, e, Table 3). However, 15 mM teicoplanin induced considerable losses of both IHCs and OHCs along the entire length of the cochlea (Fig. 1c-e, Table 3).

Table 3 HC loss (%) in mouse cochlea treated with teicoplaninTeicoplanin Induced Apoptosis in HEI-OC1 Cells and Cochlear HCs

The cellular apoptosis of HEI-OC1 cells and cochlear HCs induced by teicoplanin was assessed using TUNEL assay and cleaved Caspase-3 expression examination. HEI-OC1 cells or cochlea HCs were treated with 7.5 mM teicoplanin for 24 h. TUNEL results revealed that there was no TUNEL-positive HEI-OC1 cells in the control group, but HEI-OC1 cells treated with teicoplanin exhibited nuclei shrinkage and clear TUNEL-positive cells (Fig. 2a). Based on our findings in Fig. 1c that the HCs in the basal turn are the most vulnerable to teicoplanin injury, fluorescence staining results of cochlear basal turn HCs were selected as the representative images. As illustrated in Fig. 2b, the cultured cochlear basal turn HCs remained aligned and no TUNEL-positive HCs were detected in the control group. In contrast, HCs exhibited morphological disorder and significant amount of apoptotic HCs, which were cleaved Caspase 3-postive, were observed in the 7.5 mM teicoplanin-treated group (Fig. 2b). Moreover, the protein levels of cleaved Caspase-3 in HEI-OC1 cells and cochlear HCs were both increased significantly after teicoplanin injury, compared to the control groups (Fig. 2c, d). These results indicated that exposure to teicoplanin induced apoptosis in HEI-OC1 cells and cochlear HCs in vitro.

Fig. 2

Teicoplanin induced apoptosis in HEI-OC1 cells and cochlear HCs. a. TUNEL results showed that there was no TUNEL (red) positive HEI-OC1 cells (DAPI, blue) in the control group while nuclei shrinkage and clear TUNEL positive cells were found in HEI-OC1 cells after 7.5 mM teicoplanin treatment for 24 h. b. Representative fluorescence images of cochlear basal turn HCs (phalloidin, green) showed that the cultured cochlear basal turn HCs kept in alignment and no TUNEL (red) positive HCs were detected in the control group, while HCs fell into morphological disorder and many HCs were labelled with TUNEL after exposure to 7.5 mM teicoplanin. c, d. the protein levels of cleaved Caspase-3 in HEI-OC1 cells and cochlear HCs were both increased significantly after teicoplanin injury, compared to the control groups. ** P < 0.01, *** P < 0.001. Scale bar = 20 μm

The Expression of TIGAR was Decreased in HEI-OC1 Cells and Cochlear HCs after Teicoplanin Damage

The expression of TIGAR in postnatal cochlea HCs in the inner ears of C57BL/6 mice of varying ages (P1, 3, 7, 14, 30) was described. Immunostaining results showed that TIGAR was clearly expressed in all three turns of cochlea HCs from P1 to P30. Middle turn HCs were selected as the typical samples, and their images were illustrated in supplementary Fig. 1. Next, to determine whether the TIGAR expression in auditory cells was affected by teicoplanin treatment, HEI-OC1 cells and cochlear HCs were treated with 7.5 mM teicoplanin for 24 h, and then the changes in TIGAR expression were detected. As shown in Fig. 3, immunostaining results revealed that the fluorescence intensity of TIGAR was markedly reduced in survived HEI-OC1 cells (Fig. 3a) and cochlear HCs (Fig. 3b) after teicoplanin treatment in contrast to the undamaged controls. Furthermore, western blot results verified that the expression levels of TIGAR in HEI-OC1 cells and cultured cochlear HCs were considerably lower in the teicoplanin-treated groups compared to the control groups (Fig. 3c, d). Together, these results demonstrated that teicoplanin injury led to a reduction of TIGAR expression in HEI-OC1 cells and HCs, indicating a link between teicoplanin-induced ototoxicity and TIGAR expression.

Fig. 3

The expression of TIGAR was decreased in HEI-OC1 cells and cochlear HCs after teicoplanin damage. a, b. HEI-OC1 cells and cochlear HCs were treated with 7.5 mM teicoplanin for 24 h respectively. Immunostaining results revealed that the fluorescence intensity of TIGAR (red) was markedly reduced in survived HEI-OC1 cells (DAPI, blue) and cochlear HCs (phalloidin, green) after teicoplanin treatment in contrast to the undamaged controls. c, d. Western blot results verified that the expression levels of TIGAR in HEI-OC1 cells and cultured cochlear HCs were both significantly decreased after teicoplanin administration compared to the control groups. ** P < 0.01. Scale bar = 20 μm

Knockdown of TIGAR Expression Decreased HEI-OC1 Cell Viability while the Overexpression of TIGAR Increased it after Teicoplanin Injury

To further investigate the role of TIGAR in teicoplanin-induced damage to auditory cells, HEI-OC1 cells were transfected with shRNA or recombinant adenovirus to downregulate or upregulate TIGAR expression, respectively. The efficiency of shRNA transfection was determined. The control group consisted of untreated HEI-OC1 cells, the shRNA-Control group consisted of HEI-OC1 cells transfected with nonsense shRNA conjugated with GFP (shRNA-GFP), and the shRNA-TIGAR group consist of cells transfected with TIGAR-specific shRNA. Immunofluorescence staining results showed a successful shRNA transfection as indicated by the co-label of GFP and DAPI (Fig. 4a). Western blot showed that protein level of TIGAR was dramatically decreased in shRNA-TIGAR group compared to the normal control group, but remained unchanged in shRNA-Control group (Fig. 4b, c). The efficiency of the virus infection was measured using empty adenovirus vector conjugated with GFP (Ad-GFP), and immunostaining result verified the effective infection via GFP and DAPI co-labelling (Fig. 4d). Western blot showed that the level of TIGAR in Ad-TIGAR-treated cells was significantly upregulated, whereas no change was observed in AD-GFP group compare to the control group (Fig. 4e, f). These results demonstrated that the expression of TIGAR was efficiently knocked down by shRNA-TIGAR while it was overexpressed via Ad-TIGAR in HEI-OC1 cells.

Fig. 4

Knockdown of TIGAR expression decreased HEI-OC1 cell viability while the overexpression of TIGAR increased it after teicoplanin injury. a. Immunofluorescence staining results showed a successful shRNA transfection in HEI-OC1 cells as indicated by the co-label of GFP and DAPI. b, c. Western blot showed that protein level of TIGAR was significantly decreased in shRNA-TIGAR group of HEI-OC1 cells, but it was remained unchanged in shRNA-Control group compared to the normal control group. d. The efficiency of the virus infection was measured using empty adenovirus vector conjugated with GFP (Ad-GFP), and immunostaining result verified the effective infection via GFP and DAPI co-labelling. e, f. Western blot showed that the level of TIGAR in Ad-TIGAR‐treated cells was significantly upregulated while no change in AD-GFP group was found compared with control group. g. Immunostaining illustrated an obvious cell number reduction and nuclei shrunk in the Teico + shRNA-TIGAR group, whereas an increase of cell number in Teico + Ad-TIGAR group compared to the teicoplanin only group. h. The CCK-8 assay showed that the HEI-OC1 cell viability was further reduced in the Teico + shRNA-TIGAR group, while the cell viability was increased in Teico + Ad-TIGAR group in contrast to the teicoplanin group. *** P < 0.001. Scale bar = 20 μm

We then examined the effect of TIGAR on the viability of HEI-OC1 cells after teicoplanin administration. 24 h after pretreatment with either shRNA-TIGAR or Ad-TIGAR, HEI-OC1 cells were co-treated with 7.5 mM teicoplanin. Immunostaining and CCK-8 assay revealed that the HEI-OC1 cell viability was further reduced in the Teico + shRNA-TIGAR group (38.78 ± 1.36%), whereas it was increased in Teico + Ad-TIGAR group (83.64 ± 2.41%), in contrast to the teicoplanin group (50.92 ± 1.72%), indicating that TIGAR inhibition makes the HEI-OC1 cells more sensitive to teicoplanin-induced cell death and the overexpression of TIGAR protects the survival of HEI-OC1 cells (Fig. 4g, h).

TIGAR Deficiency Aggravated HEI-OC1 Cell Apoptosis but TIGAR Overexpression Protected HEI-OC1 Cells from Apoptosis after Teicoplanin Injury

The TUNEL assay and cleaved-Caspase 3 immunostaining were performed to determine apoptosis in HEI-OC1 following co-treatment with teicoplanin or TIGAR deficiency or overexpression. Apoptotic cells exhibited TUNEL-positive fluorescence (Fig. 5a) or cleaved-caspase3-positive fluorescence (Fig. 5c) after treatment with teicoplanin, and the numbers of TUNEL-positive cells or cleaved-caspase3-positive cells were significantly increased in Teico + shRNA-TIGAR group but decreased in Teico + Ad-TIGAR group (Fig. 5b, d).

Fig. 5

TIGAR deficiency aggravated HEI-OC1 cell apoptosis but TIGAR overexpression protected HEI-OC1 cells from apoptosis after teicoplanin injury. a-d. Apoptotic cells exhibited TUNEL-positive fluorescence (red) or cleaved-caspase3-positive fluorescence (red) after treatment with teicoplanin, and the numbers of TUNEL-positive cells or cleaved-caspase3-positive cells in the Teico + shRNA-TIGAR group were increased significantly whereas were decreased in the Teico + Ad-TIGAR group compared to the Teico group. e, f. Western bolt results showed that teicoplanin increased the protein expression of Bax and cleaved Caspase-3, while it reduced the expression of Bcl-2, compared to the control group. The decreases in the levels of Bcl-2 and increases in the levels of Bax and cleaved Caspase-3 were reversed by TIGAR overexpression, whereas they were exacerbated by TIGAR knockdown, in HEI-OC1 cells after teicoplanin treatment. * P < 0.05, ** P < 0.01, *** P < 0.001. Scale bar = 20 μm

We further investigated the potential mechanism underlying the apoptotic response by evaluating the activity of apoptosis-related genes. HEI-OC1 cells treated with 7.5 mM teicoplanin for 24 h exhibited considerably increased expression of pro-apoptotic genes Bax and cleaved Caspase-3, but dramatically decreased expression of anti-apoptotic gene Bcl-2, compared to the control group (Fig. 5e). TIGAR overexpression reversed the declines in Bcl-2 and increases in Bax and cleaved Caspase-3 that were caused by teicoplanin administration in HEI-OC1 cells, whereas TIGAR knockdown exacerbated these changes (Fig. 5e). These results suggested that TIGAR overexpression protects HEI-OC1 cells from apoptosis, while TIGAR knockdown aggravated teicoplanin-induced cell apoptosis.

TIGAR Overexpression Decreased the Generation of ROS, while TIGAR Knockdown Exacerbated the Accumulation of ROS in HEI-OC1 Cells after Teicoplanin Damage

It has been established that ROS a close association with the process of HC damage induced by ototoxic drugs [25,26,27]. To determine the relationship between teicoplanin and oxidative stress in HEI-OC1 cells, the Mito-SOX Red was utilized to evaluate mitochondrial ROS accumulation in HEI-OC1 cells after treatment with teicoplanin and the regulation of TIGAR expression. HEI-OC1 cells were incubated with either shRNA-TIGAR or Ad-TIGAR before the co-treatment with 7.5 mM teicoplanin for 24 h. Immunostaining results demonstrated that the relative fluorescence intensity of Mito-SOX red was significantly upregulated in HEI-OC1 cells treated with teicoplanin compared to control cells (Fig. 6a, b). Pretreatment with shRNA-TIGAR transfection enhanced the fluorescence intensity further, while pretreatment with Ad-TIGAR decreased the fluorescence intensity relative to teicoplanin group (Fig. 6a, b). The accumulation of ROS can induce mitochondria oxidative stress injury. Therefore, we assess the mitochondrial membrane potential (ΔΨm) of HEI-OC1 cells with various pharmacological treatments using the JC-1 mitochondrial staining test. JC-1 aggregates fluorescence (red) accumulated in the mitochondrial membrane emitted a stronger fluorescent signal than monomeric JC-1 (green) in control HEI-OC1 cells, but the monomeric JC-1 signals were increased after teicoplanin injury, indicating the depolarization of the mitochondrial membrane (Fig. 6c). The ratio of JC-1 fluorescence (red/green ratio) was utilized to evaluate the change of the ΔΨm. As shown in Fig. 6d, JC-1 fluorescence was significantly reduced after teicoplanin treatment compared to the control group, and it was further decreased in Teico + shRNA-TIGAR group, whereas it was significantly increased in the Teico + Ad-TIGAR group compare to the Teico group. These results demonstrated that TIGAR was effective at inhibiting the accumulation of ROS in HEI-OC1 cells exposed to teicoplanin, which was directly associated to mitochondrial membrane depolarization.

Fig. 6

TIGAR overexpression decreased the generation of ROS, while TIGAR knockdown exacerbated the accumulation. a, b. The relative fluorescence intensity of Mito-SOX was significantly upregulated after teicoplanin treatment compared to that in control HEI-OC1 cells. The pretreatment with shRNA-TIGAR transfection enhanced the fluorescence intensity further, while pretreatment with adenovirus infection of TIGAR reduced the fluorescence intensity compared to teicoplanin group. c, d. Immunostaining results showed that JC-1 fluorescence (red/green ratio) was reduced significantly after teicoplanin treatment compared to the control group, and the JC-1 fluorescence (red/green ratio) in Teico + shRNA-TIGAR group was further reduced, whereas it was raised significantly in Teico + Ad-TIGAR group in contrast to the Teico group. ** P < 0.01, *** P < 0.001. Scale bar = 20 μm

Antioxidant Treatment with NAC Lowered ROS Level, Rescued HEI-OC1 Cell Loss and Apoptosis as well as Restored p38/p-p38 Expression Levels Induced by TIGAR Deficiency after Teicoplanin Injury

To further investigate the effect of TIGAR on suppressing ROS accumulation in HEI-OC1 cells induced by teicoplanin, a rescue experiment was performed using the ROS scavenger NAC in TIGAR-deficient HEI-OC1 cells following teicoplanin-induced injury. The dose of NAC was chosen according to our published studies [21, 28], and the result of NAC dose responses which showed that 2 mM NAC pre-treatment successfully rescued the HEI-OC1 cell loss from teicoplanin damage (Supplementary Fig. 2). Then the HEI-OC1 cells were pre-treated with 2 mM of NAC for 2 h before the co-treatment of 7.5 mM teicoplanin and shRNA-TIGAR. The accumulation of ROS was detected by the intensified immunostaining signals of Mito-SOX Red in HEI-OC1 cells. The relative fluorescence intensity of Mito-SOX was reduced significantly in the Teico + NAC group compared to the teicoplanin group, and in the Teico + shRNA-TIGAR + NAC group compare to the Teico + shRNA-TIGAR group (Fig. 7a, b). Moreover, the analysis of JC-1 fluorescence staining demonstrated that JC-1 fluorescence (red/green ratio) in HEI-OC1 cells treated with teicoplanin was significantly increased in NAC-treated groups compared to control groups (Fig. 7c, d).

Fig. 7

Antioxidant treatment with NAC lowered ROS level, rescued HEI-OC1 cell loss and apoptosis as well as restored p38/p-p38 expression levels induced by TIGAR deficiency after teicoplanin injury. a, b. The relative fluorescence intensity of Mito-SOX was reduced significantly in Teico + NAC group compared to the teicoplanin group, and in Teico + shRNA-TIGAR + NAC group in contrast to Teico + shRNA-TIGAR group after NAC treatment. c, d. Immunostaining results showed that JC-1 fluorescence (red/green ratio) in groups with NAC treatments had a significant increase compared to their control groups in HEI-OC1 cells after teicoplanin treatment. e, f. Representative images showed that apoptotic cells exhibited TUNEL-positive fluorescence (red) after treatment with NAC were reduced in Teico + NAC group compared to the teicoplanin group, and they were also decreased in Teico + shRNA-TIGAR + NAC group compared to Teico + shRNA-TIGAR group. g. The CCK-8 assay showed that the number of surviving HEI-OC1 cells in Teico + NAC group was significantly increased compared to the teicoplanin only group, as well as in Teico + shRNA-TIGAR + NAC group versus Teico + shRNA-TIGAR group. h, i. The western bolt results demonstrated P38 expression level was reduced while p-P38 expression level was increased in HEI-OC1 cells after teicoplanin injury. After treatment with NAC, P38 protein level was increased and the p-P38 expression was decreased in Teico + NAC group compared to Teico group, as well as in Teico + shRNA-TIGAR + NAC group in contrast to Teico + shRNA-TIGAR group. β-actin served as control. **p < 0.01, ***p < 0.001. Scale bar = 20 μm

We then detected the survival and apoptosis of HEI-OC1 cells with or without NAC treatment. TUNEL assay revealed that the TUNEL-positive cells were reduced in the Teico + NAC group compared to the teicoplanin group, and they were also reduced in the Teico + shRNA-TIGAR + NAC group compared to the Teico + shRNA-TIGAR group (Fig. 7e, f). The NAC treatment significantly increased the number of surviving HEI-OC1 cells in the Teico + NAC group (87.44 ± 8.10%) compared to the teicoplanin-only group (50.50 ± 3.41%), as well as in the Teico + shRNA-TIGAR + NAC group (59.55 ± 3.17%) versus the Teico + shRNA-TIGAR group (39.62 ± 3.56%) (Fig. 7g).

To further explore the potential underlying mechanisms of regulating ROS accumulation in HEI-OC1 cells, we examined the P38 MAPK signaling pathway, which is involved in oxidative stress-induced cell apoptosis [29]. The expression and phosphorylation of p38 were identified in HEI-OC1 cells treated with teicoplanin and TIGAR knockdown. As illustrated in Fig. 7h, TIGAR knockdown aggravated the teicoplanin-induced decrease in p38 expression and rise in p-p38 in HEI-OC1 cells. In contrast, a significant increase of p38 protein level and a decrease in p38 phosphorylation level were observed in Teico + NAC group compared to the Teico group, as well as in Teico + shRNA-TIGAR + NAC group compare to Teico + shRNA-TIGAR group (Fig. 7h).

Collectively, our observations revealed that the antioxidant treatment with NAC successfully reduced the ROS accumulation, reversed the increased apoptosis and cell loss in HEI-OC1 cells induced by TIGAR knockdown following teicoplanin injury, and prevented the formation of ROS. In addition, the data suggested that teicoplanin exposure might activate the ROS/P38 signaling pathway in HEI-OC1 cells, and that NAC treatment contributes to restoring the p38/p-p38 expression levels induced by teicoplanin injury and TIGAR deficiency.