Salvianolic acid B inhibits ototoxic drug–induced ototoxicity by suppression of the mitochondrial apoptosis pathway

Abstract It has been claimed that salvianolic acid B (Sal B), a natural bioactive antioxidant, exerts protective effects in various types of cells. This study aims to evaluate the antioxidant and anti‐apoptosis effects of Sal B in a cultured HEI‐OC1 cell line and in transgenic zebrafish (Brn3C: EGFP). A CCK‐8 assay, Annexin V Apoptosis Detection Kit, TUNEL and caspase‐3/7 staining, respectively, examined apoptosis and cell viability. The levels of reactive oxygen species (ROS) were evaluated by CellROX and MitoSOX Red staining. JC‐1 staining was employed to detect the mitochondrial membrane potential (ΔΨm). Western blotting was used to assess expressions of Bax and Bcl‐2. The expression pattern of p‐PI3K and p‐Akt was determined by immunofluorescent staining. We found that Sal B protected against neomycin‐ and cisplatin‐induced apoptotic features, enhanced cell viability and accompanied with decreased caspase‐3 activity in the HEI‐OC1 cells. Supplementary experiments determined that Sal B reduced ROS production (increased ΔΨm), promoted Bcl‐2 expression and down‐regulated the expression of Bax, as well as activated PI3K/AKT signalling pathways in neomycin‐ and cisplatin‐injured HEI‐OC1 cells. Moreover, Sal B markedly decreased the TUNEL signal and protected against neomycin‐ and cisplatin‐induced neuromast HC loss in the transgenic zebrafish. These results unravel a novel role for Sal B as an otoprotective agent against ototoxic drug–induced HC apoptosis, offering a potential use in the treatment of hearing loss.

important issue that has attracted a great deal of attention regarding our understanding of the cellular mechanisms underlying these ototoxic drug-induced ototoxicity, and much valuable effort has been focused on identifying novel otoprotective agents.
The amassing of reactive oxygen species (ROS) is considered one major factor underlying aminoglycosides and cisplatin-induced ototoxicity. 3 Overproduction of ROS in the mitochondria of HCs overwhelms the redox balance, triggering mitochondrial depolarization and cytochrome c release, eventually activating caspase-3 and inducing apoptosis by modulating various intracellular signalling pathways. 1,4 Therefore, the application of antioxidants to attenuate free radicals by using ROS scavengers has been currently proposed as promising otoprotectants for mitochondrial function in ototoxic drug-induced ototoxicity. Among various free radical scavengers, natural herbal compounds have attracted consideration, as they are safer and less expensive.
Salvianolic acid B (Sal B) is a major bioactive compound extracted from Radix Salvia miltiorrhiza (Danshen), a popular therapeutic compound used in traditional Chinese medicine as a ROS scavenger to treat various diseases, including hepatitis, menstrual disorders and cardiovascular diseases. 5,6 Numerous studies have reported that Sal B contains anti-apoptotic, antioxidant, and anti-inflammatory properties in vivo and in vitro. [7][8][9][10] Sal B has been shown to inhibit ischaemia-reperfusion-induced injury in rat brains by scavenging free radicals and enhancing cerebral energy metabolism. 11,12 Sal B also exhibited a protective effect against cerebral ischaemia-reperfusion injury: this may be attributed to the up-regulation of anti-apoptotic protein Bcl-2 expression and maintenance of mitochondrial membrane potential. 13 However, the result and mechanism of Sal B on aminoglycosides and cisplatin-mediated ototoxicity is not clearly expounded upon.
This study's aim was to establish whether Sal B possesses a protective action against aminoglycosides and cisplatin-induced ototoxicity in HEI-OC1 cell line and in transgenic zebrafish and, if so, the possible mechanisms underlying this action.

| HEI-OC1 cell culture and drug treatments
The House Ear Institute-Organ of Corti 1 (HEI-OC1) cell line is a widely used auditory HC line derived from the cochlea of the immortomouse. 14

| Zebrafish larvae and drug treatments
Zebrafish larvae were raised at 28.5°C in embryo medium with a density of 50 larvae to each 100 mm 2 Petri dish. Developmental stages were evaluated as days post-fertilization (dpf). Each zebrafish experiment followed the guidelines of the Institutional Animal Care and Use Committee of Fudan University, Shanghai. For the neomycin studies, 5 dpf larvae were pre-treated with Sal B for 2 hours and then co-treated with Sal B and neomycin (200 µM) for another 1 hour.
For the cisplatin studies, 4 dpf larvae were incubated for 24 hours with a mixture of 50 μM cisplatin and 40 μM Sal B, following 2 hours pre-incubation with Sal B. Control animals were exposed to vehicle (DMSO) and included in each experiment.

| Cell viability
Cell Counting Kit-8 (CCK-8) was employed to gauge cell viability according to the manufacturer's instructions. In brief, HEI-OC1 cells were seeded at a density of 5000 cells/well in 96-well plates in three replicates and incubated overnight in acceptable conditions. After drug treatment in 100 μL culture medium, CCK-8 (Sigma, 96992) was added to each well for 4 hours. The optical density (OD) values were measured at 450 nm using a plate reader (Bio-Rad). The positive control was subject to the same procedure without cell seeding, while the negative control was left drug-free.

| Flow cytometry analysis of apoptosis
Apoptosis was detected from flow cytometry using an Annexin V-FITC and propidium iodide (PI) kit (BD Biosciences, 556547) according to the manufacturer's instructions. Cells were collected by centrifugation at 3000 × g for 5 minutes, washed twice with cold PBS and gently resuspended in 1 × binding buffer at a concentration of 1 × 10 6 cells/mL. Annexin V-FITC (5 μL) and PI (5 μL) were introduced, gently mixed with cell suspension and incubated for 15 minutes at illuminated room temperature. The cells were immediately analysed using flow cytometry.

| Caspase-mediated apoptosis assay
HEI-OC1 cells were cultured in 6-well plates at a density of 2 × 10 5 cells per well. Cells after treatment, they were washed in pre-warmed PBS and stained using 5 μM caspase-3/7 reagent (Molecular Probes, Life Technologies, United States, C10723) in serum-free DMEM at 37°C for 30 minutes. Fluorescence microscopy and flow cytometry were used to analyse the fluorescent signal intensity of the cells.

| Mitochondrial transmembrane potential measurement
Mitochondrial transmembrane potential (ΔΨm) was estimated by

| TUNEL assay
Apoptosis was determined by TUNEL assay using an in situ cell detection kit (Roche) according to the manufacturer's instructions.
Samples were stained with TUNEL reaction mixture at 37°C for Inc, Silver Spring), and the number was normalized to the total viable cells to determine TUNEL-positive rate.

| ROS assay
The levels of ROS were detected using CellROX green reagent
After being quickly rinsed three times with fresh water, the larvae were anaesthetized and fixed with 4% PFA.

| Statistical analysis
All values were shown as mean ± SEM and established through oneway analysis of variance (ANOVA) or two-tailed, unpaired Student's t test. Statistical analyses were conducted using GraphPad Prism 6 software, with P < 0.05 considered statistically significant.

| Sal B protected viability of HEI-OC1 cells upon neomycin and cisplatin damage
To ascertain the optimal in vitro neomycin ototoxicity model, HEI-OC1 cells were treated with increasingly concentrated neomycin in the medium (0, 1, 2, 5, 10, 20 or 30 mM) for 24 hours, and their viability was analysed by CCK-8 assay. As shown in Figure 1A, neomycin treatment significantly reduced the cell viability in a dosedependent manner, compared to the non-treated control group ( Figure 1A). Neomycin at a concentration greater than 10 mM markedly reduced cell viability; thus, a neomycin concentration of 10 mM was used for the in vitro study. To determine whether Sal B could protect HEI-OC1 cells from neomycin-induced damage, cells were pre-treated with Sal B concentrations of 0, 10,20,30,40 and 50 μM for 2 hours, and then co-treated with 10 mM neomycin for 24 hours. We observed a significant protective effect of Sal B at 40 and 50 μM, and a maximal protective effect at a concentration of 40 μM, compared with culture treated with neomycin alone (****P < .0001, **P < 0.01, respectively) ( Figure 1B). Notably, Sal B alone (20, 40 and 60 μM) for 24 hours had no significant influence on the viability of HEI-OC1 cells ( Figure S1). These results showed Sal B can protect viability of HEI-OC1 cells upon neomycin ototoxicity.
To evaluate the protective effect of Sal B against cisplatin-induced ototoxicity, HEI-OC1 cells were exposed to increasing concentrations of cisplatin in the culture medium (0, 10, 20, 30, 40, 50 and 60 μM) for 24 hours. As shown in Figure 1C, cisplatin from 30 to 60 μM exposure decreased cell viability from 67.34% ± 4.64% to 33.01% ± 1.18%, compared with the non-treated control group.
Based on the cell viability data, we selected 30 μM cisplatin treatment for 24 hours as the optimal condition for HEI-OC1 cell injury to the study of cisplatin ototoxicity, as the viability was significantly decreased in comparison with the non-treated control group. In order to test whether Sal B was able to protect HEI-OC1 cells from this cisplatin-induced ototoxicity, the cells were pre-treated with various Sal B concentrations (0, 10, 20, 30, 40 and 50 μM) for 2 hours, after which they were co-cultured with 30 μM cisplatin for 24 hours.
Using CCK-8 assay, we confirmed that cell viability in the presence of Sal B was indeed significantly higher than in its absence of Sal B ( Figure 1D). Based on these data, we selected 40 μM Sal B, which was associated with maximal cell viability, as the optimal concentration for subsequent experiments. These findings suggested Sal B was able to ameliorate cisplatin-induced damage to HEI-OC1 cells.

| Sal B protected HEI-OC1 cells from neomycin-and cisplatin-induced apoptosis
To investigate whether the effects of Sal B on HEI-OC1 cells were due to the reduction of apoptosis, an Annexin V-FITC and Values were represented as the mean ± SEM from three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns. not significant vs the non-treated control group; ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs the group treated with 10 mM neomycin or 30 μM cisplatin only Conversely, Sal B pre-treatment significantly inhibited neomycininduced apoptosis ( Figure 2B). The results showed that neomycin promoted HEI-OC1 cell apoptosis, which was significantly inhibited by Sal B pre-treatment. To verify these findings, we performed TUNEL staining to detect the apoptotic HEI-OC1 cells after 10 mM neomycin treatment for 24 hours ( Figure 2C). We found a significantly higher percentage of TUNEL-positive cells in the neomycin alone group, compared with the non-treated control group. The Sal B pre-treated group had significantly lower percentages of TUNELpositive cells after neomycin damage than the neomycin alone group ( Figure 2C,D).
We next performed caspase-3/7 staining to investigate the underlying molecular mechanism ( Figure 3A). Cells in the neomycin alone group showed apoptotic properties, whereas fewer cells displayed these properties in the Sal B pre-treatment group ( Figure 3A).
Flow cytometry results showed that Sal B pre-treated cells had sig-  and Bax in the control, Cis and Sal B-Cis groups, and the columns (E and F) showed the quantification of the fold changes of expression. The data expression for protein was normalized by GAPDH. Data were presented as the mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001

| Sal B reduced neomycin-and cisplatininduced ROS generation and increased mitochondrial membrane potential in HEI-OC1 cells
Neomycin was reported to cause the overproduction of radical oxygen species (ROS), which is integral to apoptosis as it activates multiple apoptotic pathways. 15 We investigated whether Sal B was able to inhibit this process. Cells from different groups were collected and stained with ROS indicator dye CellROX green. As expected, neomycin resulted in a noticeable elevation of green fluorescence of CellROX in HEI-OC1 cells, compared with the non-treated control group. Conversely, pre-treatment with Sal B significantly inhibited ROS production induced by neomycin exposure (Figure 5A-C).
We next monitored mitochondrial ROS using the probe MitoSOX which is considered one of the earliest hallmarks of mitochondrial dysfunction, which ultimately leads to apoptosis. 16 ΔΨm was assessed using the JC-1 staining. As shown in Figure 5G, HEI-OC1  (Figure 6G,H). Collectively, these results suggested ototoxic drug-induced mitochondrial dysfunction in HEI-OC1 cells, and Sal B was able to protect mitochondrial function.

| Sal B attenuated neomycin-and cisplatininduced cytotoxicity through activation of the PI3K/ AKT pathway
Accumulating evidence indicates that many signalling pathways

| Sal B protected against neomycin-and cisplatin-induced HC death in zebrafish lateral line
To investigate the protective effect of Sal B in vivo, a transgenic zebrafish line tg (Brn3C: EGFP), which express membrane-bound green fluorescent protein in hair cells under the control of the brn3c promoter, was used. We first examined Sal B's potential effect on neomycin toxicity. Larvae treated with neomycin alone showed a significant HC loss compared to that of control animals ( Figure 8A,B). However, pre-treatment of zebrafish larvae with 40 μM Sal B for 2 hours, followed by co-treatment with 200 μM neomycin for 1 hour, showed protection of neuromast HCs compared to that of the neomycin alone ( Figure 8A,B). Next, we studied Sal B's potential otoprotective effect on cisplatin by pre-treating larval zebrafish at 4 dpf with Sal B concentration of 40 µM for 2 hours followed by co-treatment with 50 µM cisplatin for 24 hours. As shown in Figure 8A 20 was used in control larvae and larvae exposed to Sal B 40 μM for 2 hours. Figure 8C shows that rapid dye entry into the HCs was comparable between the control and Sal B treatment groups. Quantification of the FM1-43FX-positive cells did not show any significant differences ( Figure 8D), indicating that Sal B did not affect the MET activity.
The TUNEL assay was conducted to ascertain whether ototoxin induced the death of neuromast cells by apoptosis, and whether that apoptosis could be prevented by Sal B treatment. As seen in Figure 8E, while the ototoxin alone treatment group showed more incidents of cell apoptosis compared with that of the undamaged control group, the Sal B pre-treatment group showed significantly reduced incidents of apoptosis compared those treatments with ototoxin alone. As aminoglycosides' and cisplatin's ototoxic effects are associated with ROS accumulation, MitoSOX Red staining was performed to evaluate ROS generation ( Figure 8F). Increased staining was detected in the neuromast of ototoxin alone treated larvae; however, little staining was ob- It has been documented that increased production of intracellular ROS level is a major mechanism underlying ototoxic insult-induced HC death. 26,27 Excessive ROS violates proper action of the HCs, reducing antioxidant defence, triggering release of apoptosis-related factor cytochrome c from mitochondria and caspase-3 pathway activation and leading to apoptosis. 28 To determine whether Sal It is well known that HC apoptosis is the major mechanism underlying the aminoglycoside-and cisplatin-induced ototoxicity and ROS-associated mitochondrial damage is involved in the hair cell apoptosis. Currently, antioxidants and anti-apoptotic agents have been shown to interfere with ROS pathways and to prevent HC loss in mammals. 29 In this study, we demonstrated that administration of Sal B dramatically decreases the mitochondrial ROS and prevents mitochondrial dysfunction and apoptosis of HEI-OC-1 cells after ototoxic drug injury. Note that although Sal B treatment significantly attenuates neomycin-or cisplatin-induced injury, there is still a loss of HCs compared to undamaged controls. Thus, exploring more effective mitochondria-targeted antioxidants that can be coadministrated with Sal B will be critical to maximize the protective effect of Sal B to rescue HC damaged by ototoxic drugs. Apart from the inhibition of mitochondrial apoptotic pathway, hair cell survival is also dependent upon multiple mechanisms. For example, recent work has shown that autophagy activation is an important contributor to attenuate the neomycin-or cisplatin-induced ototoxicity by suppressing ROS accumulation. [30][31][32] Additionally, our previous study showed that G9a inhibitor and some LSD1 inhibitors could protect against ototoxic drug-induced HC death, suggesting that epigenetic mechanisms are involved in ototoxic drug-induced hearing loss. 33,34 The present study focuses on the mitochondrial apoptotic pathway F I G U R E 9 Schematic illustrating the possible mechanisms of protective effect of Sal B against ototoxic drug-induced apoptosis. As illustrated, neomycin or cisplatin treatment markedly induces the production of ROS and triggers mitochondrial apoptotic pathway. Sal B treatment could reduce the generation of ROS; decrease the expression levels of apoptosis proteins; increase the expression levels of anti-apoptotic proteins; induce the activation of anti-apoptotic PI3K/AKT pathway; and eventually ameliorate the oxidative stress-induced apoptosis in neomycin-or cisplatin-induced HC injury; no other mechanisms or their crosstalk was analysed. Thus, more complete picture of molecular and epigenetic pathways should be elucidated in future work by use of appropriate pharmacologic or genetic approaches.
Increasing evidence has suggested that overproduction of ROS can stimulate numerous intracellular signalling pathways, such as PI3K/AKT and MEK/ERK pathways. 17,35 PI3K/AKT is known to have potential anti-apoptotic functions through several downstream targets including phosphorylation of Bad (serine-136) and has recently been shown to be involved in promoting hair cell survival. 17 Our present findings demonstrated that Sal B did not notably affect the phosphorylation of ERK; it seems possible that ERK activation might not be the direct targets of Sal B. Additionally, our data demonstrated that exposure to Sal B could lead to an increased phosphorylation of PI3K and AKT after ototoxic drug treatment. These findings indicate that the anti-apoptotic effect of Sal B on ototoxic drug-induced ototoxicity might be partly attributed to the restoration of activation of PI3K/AKT signalling. Future research to investigate the precise mechanisms that underlie the otoprotective effect of Sal B in HCs will shed more light on our understanding of HC survival molecular mechanisms. Hence, these results might be important in future clinical settings. Even with consideration of these studies, application to auditory HCs of mammals still requires investigation. Therefore, additional studies should occur to determine the protective effect of Sal B in the mammalian inner ear.
In summation, we demonstrated that Sal B exhibited significant effect on protection against aminoglycoside antibiotics and cisplatin-induced ototoxicity by suppressing the production of ROS and mitochondrial apoptotic pathway (as summarized in Figure 9). Our findings have yielded an initial instance indicating Sal B as a preventive or therapeutic agent in the treatment of ototoxic insult-induced hearing loss.