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Effect of baicalein from Scutellaria baicalensis on prevention of noise-induced hearing loss
Neuroscience Letters 469 (2010) 298–302
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Effect of baicalein from Scutellaria baicalensis on prevention of noise-induced hearing loss Tong Ho Kang a , Bin Na Hong b , Channy Park b , Se Young Kim a , Raekil Park c,∗ a b c
College of Life Sciences, Kyung Hee University, Gyeonggi 446-701, Republic of Korea Department of Audiology, Nambu University, Gwangju 506-824, Republic of Korea Vestibulocochlear Research Center & Department of Microbiology, College of Medicine Wonkwang University, 344-2 shinyong-dong, Iksan, Jeonbuk 570-749, Republic of Korea
a r t i c l e
i n f o
Article history: Received 14 October 2009 Received in revised form 2 December 2009 Accepted 3 December 2009 Keywords: Noise-induced hearing loss Scutellaria baicalensis Baicalein Mouse Auditory
a b s t r a c t Noise-induced hearing loss (NIHL) has been thought to primarily involve damage to the sensory hair cells of the cochlea via mechanical and metabolic mechanisms. This study examined the effects of baicalin, baicalein, and Scutellaria baicalensis (SB) extract against NIHL in a mouse model. Mice received oral treatment with SB, baicalin, baicalein beginning 30 min prior to noise exposure and continuing once daily throughout the study. Hearing threshold shift was assessed by auditory brain stem responses for 35 days following noise exposure. Central auditory function was evaluated by auditory middle latency responses. Cochlear function was determined based on transient evoked otoacoustic emissions. SB significantly reduced threshold shift, central auditory function damage, and cochlear function deficits, suggesting that SB may protect auditory function in NIHL and that the active constituent may be a flavonoid, baicalein. © 2009 Elsevier Ireland Ltd. All rights reserved.
Noise-induced hearing loss (NIHL) is a major cause of deafness worldwide and has been thought to primarily involve damage to the sensory hair cells of the cochlea through mechanical injury and/or metabolic deficits [21]. Over the past decade, it has become clear that oxidative stress, induced by noise overexposure, can lead to cell injury, sensory cell death, and permanent NIHL [7]. Hair cell death, induced by noise exposure, arises through the overproduction of free radicals such as reactive oxygen species (ROS), reactive nitrogen species (RNS), and other free radicals through the action of oxidative stress generators [20,23]. Pharmacological protection against NIHL by chemicals has been well reviewed by Lynch and Kil [14]. Successful approaches have included a wide variety of antioxidants [13], glutamate antagonists [1], nitric oxide synthase (NOS) inhibitors [15]. In response to acoustic overexposure, cochlear GSH levels initially increase and then decline steeply. GSH-related enzymes such as gamma glutamyl cysteine synthase, glutathione reductase, and glutathione peroxidase are modulated by loud noise exposure [10,16]. The replenishment of GSH with a glutathione prodrug such as NAC, Dmethionine, or an ester of GSH could reduce hearing loss from loud noise [8,12]. Treatment with ebselen after noise exposure reduced both OHC loss and the swelling of the stria vascularis. This might be due to the preservation of endogenous glutathione peroxidase from ROS/RNS [11].
∗ Corresponding author. Tel.: +82 63 850 6777; fax: +82 63 852 0220. E-mail address: [email protected] (R. Park). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.12.009
Scutellaria biacalensis Georgi (SB), commonly known as Skullcap, is rich in polyphenols and flavonoids; baicalin, baicalein, oroxylin A, and wogonin. Baicalin and baicalein, major flavonoids from SB, have attracted considerable attention because it has a variety of interesting activities such as antitumor, anti-inflammatory, and antioxidant effects [2]. However, no protective effect of a natural product in NIHL in a mouse model has been previously reported. In this study, we evaluated the otoprotective effects of SB, at different orally administered doses, in a NIHL mouse model. We also determined the most effective component from SB (i.e., baicalein or baicalin) in the NIHL mouse model. Seven-week-old male ICR mice (Jung-Ang Lab Animal, Seoul, Korea) were used. They were housed under a 12/12-h light/dark cycle, with food and water provided ad libitum. All experimental procedures were performed in accordance with the Principles of Laboratory Animal Care (NIH publication, #80-23, revised in 1996) and the Animal Care and Use Guidelines of Nambu University, Korea. Experimental mice were examined by an auditory brain stem response (ABR) test before noise exposure to confirm normal hearing. We used the normal hearing mice ABR threshold level of ≤30 dB at 4 and 8 kHz. SB extract was prepared from S. baicalensis supplied from the Kyongdong Oriental Market (Seoul, Korea). Baicalin and baicalein were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dried rhizome of SB (1 kg) was sonicated in 70% ethanol solution for 2 h (three times). Following filtration, the solution was evaporated to dryness in vacuo. The dried SB extract (88.0 g) was used as the SB extract sample in the experiment. SB was standardized by
T.H. Kang et al. / Neuroscience Letters 469 (2010) 298–302
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Fig. 1. HPLC chromatogram of baicalin and baicalein in Scutellaria baicalensis extract.
HPLC, as described by Gao et al. [6]. An analytical HPLC unit (Shimazu, Kyoto, Japan) equipped with a pump and set up for UV–vis detection at 277 nm, was used for analysis of the SB extract. Chromatographic conditions were as follows: column, Capcell Pak C18, 4.6 mm ID × 250 mm (Shiseido Fine Chemicals, Tokyo, Japan); eluent, 0.5% trifloroacetic acid (v/v), acetonitrile. The linear gradient was 0–40 min, 80–40% A. The flow rate was 0.8 ml/min at 40 ◦ C and the injection volume was 10 l. We designed two experiments to examine the otoprotective effect. First, we evaluated the protective effects of SB at different doses. Experimental mice were divided into five groups (n = 10/group) and were treated orally once daily with 0.5 ml distilled water (control), SB 100 mg/kg/day (SB 100), SB 300 mg/kg/day (SB 300), or SB 500 mg/kg/day (SB 500). One group was treated orally once daily with 0.5 ml distilled water without noise exposure (normal). The second experiment evaluated the protective effects of baicalein and baicalin at different doses. Experimental mice were divided into five groups (n = 10/group) and were treated orally once daily with 0.5 ml distilled water (control), baicalin 30 mg/kg/day (baicalin 30), baicalein 15 mg/kg/day (baicalein 15), baicalein 30 mg/kg/day (baicalein 30), or SB 300 mg/kg/day (SB 300). Preparations of SB, baicalein, and baicalin in distilled water were made daily just prior to treatment. Treatment groups were administered doses 30 min before noise exposure and once daily for 35 days. Doses in all groups were kept constant throughout the experimental period. Experimental mice were exposed to a single, continuous 4-kHz pure tone at a level of 120-dB SPL for 3 h to induce NIHL. During noise exposure, four animals were kept in a cage in a soundproof room. The pure tone of 4 kHz was produced by an audiometer (model GSI-61; Grason-Stadler Inc., Milford, NH, USA) and was presented via a set of loudspeakers on both sides of the cage. The overall noise level was measured at the center of the cage using a sound level meter (NA-24; Rion, Tokyo, Japan). Auditory function tests were performed prior to noise exposure (0 day), then 2, 7, 14, 21, 28, and 35 days after noise exposure. Prior to testing, all mice were anesthetized with xylazine (0.43 mg/kg) and ketamine (4.57 mg/kg), which were administered intramuscularly. Core temperature was maintained at 37.5 ± 1.0 ◦ C using a thermostatically controlled heating lamp in conjunction with a rectal probe. Hearing thresholds, latencies, and amplitudes were determined based on auditory brain stem responses (ABRs) and auditory middle latency responses (AMLRs) using two-channel recordings (GSI Audera; Viasys Healthcare, Conshohocken, PA, USA). A differential active needle electrode was placed s.c. below the test ear and a reference electrode at the vertex; a ground electrode was positioned just above the hind limb. For ABR recordings, alternating 4- and 8-kHz tone bursts (TBs; rise-plateau-fall; 2-1-2 cycles) were performed for the analysis of ABR. For AMLR recordings, rarefaction clicks (0.1-ms duration) were performed. The stimuli were delivered via earphones (ER-3A; Etymotic Research, Elk Grove, IL, USA). Physiological filters were set to pass electrical activity of 100–3000 Hz for ABRs and 10–250 Hz for AMLRs. The averages for 1000 sweeps with a rate of 20.1 stimuli/s in a 20-ms
time window for ABRs and for 250 sweeps with a rate of 9.1 stimuli/s in a 70-ms time window for AMLRs were determined. Cochlear function was determined based on transient evoked otoacoustic emissions (TEOAEs) using ILO v6 (Otodynamics, Hatfield, Hertfordshire, UK). TEOAEs were evoked by 80-s clicks of 90-dB SPL intensity, with a masking noise in the opposite ear, according to the standard nonlinear ILO protocol. TEOAE responses were evaluated in the frequency domain (FFT) by estimating the S/N ratio at 1, 1.5, 2, 3, and 4 kHz. Data were analyzed using the SigmaPlot software (Systat Software, Chicago, IL, USA). All data were expressed as means ± standard error of the mean (SEM). Statistical comparisons between groups were performed using two-way ANOVA with Tukey’s post hoc multiple comparison. P values <0.05, 0.01, and 0.001 were deemed to be statistically significant. We examined the HPLC profile of the SB extract to standardize the crude extracts of the natural product. An external calibration method was used for quantitative analysis with HPLC. Whole chromatograms compared to the standard compounds baicalin and baicalein provided a useful means of identifying and assessing SB. Calibration curves were obtained by plots of the peak area vs. the concentrations of calibration standards. The chromatogram of SB was identified to contain baicalin and baicalein at retention times of 15.3 and 23.8 min, respectively (Fig. 1). The contents of baicalin and baicalein in the SB extract were 15.2% and 2.01%, respectively. To examine the protective effect of the SB extract against the severity of hearing loss from noise exposure, ABR tests were performed prior to noise exposure (0 day), then 2, 7, 14, 21, 28, and 35 days after noise exposure. The increased hearing threshold in the control group showed hearing threshold shift of 49 dB at 4 kHz and 32 dB at 8 kHz of ABR, at 2 days after noise exposure. The SB 100, 300, and 500 groups showed suppressed hearing threshold shifts for 4- and 8-kHz TBs compared to the control group, at each test time for 35 days (p < 0.05, p < 0.01, and p < 0.001). The SB 100, 300, and 500 groups showed decreased hearing threshold shifts in a dose-dependent manner for 35 days (Fig. 2A and B). These data indicated that SB may protect against hearing threshold shifts from noise exposure in the NIHL mouse model. To identify the protective constituent(s) from the SB extract, ABR tests were performed for 35 days after noise exposure. The baicalein 15 and 30 groups showed suppressed hearing threshold shifts compared to the control group at each test time for 35 days (p < 0.05, p < 0.01, and p < 0.001). The baicalein 15 and 30 groups showed decreased hearing threshold shifts in a dose-dependent manner at later test times. The baicalein 30 group was similar to the normal group in hearing threshold shifts at 8-kHz TB after 21 days. The baicalin 30 group was similar to the control group in hearing threshold shifts for 21 days. Hearing threshold shifts in the baicalin 30 group decreased at later test times, but not significantly. Hearing threshold shifts in the SB 300 group were to a degree similar to those in the baicalein 15 group (Fig. 2C and D). These data indicated that the preventative efficacy of SB in the NIHL mouse model may be related to baicalein, and not baicalin.
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Fig. 2. Results obtained for the no noise exposure (normal), SB 100 mg/kg-treated (SB 100), SB 300 mg/kg-treated (SB 300), SB 500 mg/kg-treated (SB 500), or no treatment (control) groups of (A) and (B) graph and baicalin 30 mg/kg-treated (baicalin 30), baicalein 15 mg/kg-treated (baicalein 15), baicalein 30 mg/kg-treated (baicalein 30), SB 300 mg/kg-treated (SB 300), or no treatment (control) groups of (C) and (D) graph with regard to increased hearing thresholds of ABR with 4, and 8 kHz TBs stimulation for 35 days after noise induction. Data are presented as the mean ± S.E.M., N = 10 each groups. * p < .05, ** p < .01, *** p < .001 vs. control group. Waveforms of the auditory brainstem response with ABR in control (control), SB 300 mg/kg treatment (SB 300), and baicalein 30 mg/kg treatment (baicalein 30) mice at 0, 2, and 35 days after the noise induction of (E) graph. ABR waveforms are plotted in the latency domain of the x-axis (ms) and in the amplitude domain of the y-axis (V). ABR waveforms were recorded from variable intensities. The baseline in ABR is regarded under 0.12 V of each peak amplitude, which is the optimal signal-to-noise ratio to discriminate neural activity in the auditory pathway following the sound from the background electrical activity and other bioelectric signals.
The protective effects of the SB extract and baicalein were quantitatively different in proportion to the hearing threshold shift level. The control group at 4- and 8-kHz TB showed a hearing threshold shift of over and below 40 dB, respectively. The suppression ratios of hearing threshold shifts at 4- and 8-kHz TBs were 62% and 87% in the SB 500 group and 64% and 95% in the baicalein 30 group, respectively, compared to the control group. The hearing threshold shift in the SB 500 and baicalein 30 groups recovered significantly at 8-kHz TBs. This indicates that SB or baicalein administration could be more protective in mild rather than in severe NIHL. Waveforms of ABRs in the control, SB 300, and baicalein 30 groups at 0, 2, and 35 days are shown in Fig. 2E. Hearing threshold was defined as positive peaks at the lowest intensity of stimulation. Positive peaks in ABR waveforms appeared at the lower stimulus intensity in SB 300 and baicalein 30 groups from 2 to 35 days into the study. However, positive peaks in ABR waveforms of the control group appeared at the higher stimulus intensity over the course of the study. To investigate whether the protective effects of SB or baicalein in central auditory function could be demonstrated in NIHL, AMLR tests were performed for 35 days. SB 100, 300, and 500 protected against Pa latency delays and amplitude decreases compared to the control group at each test time for 35 days. The SB 100, 300, and 500 groups showed decreased Pa latencies and increased Pa amplitudes in a dose-dependent manner in AMLRs for 35 days (Fig. 3A and B). The baicalin 30 group was similar to the control group in Pa latencies and amplitudes of AMLRs for 35 days. Pa latencies in the SB 300, baicalein 15, and 30 groups were similar from 21 to 35 days postexposure. However, the Pa amplitudes of the baicalein 15 and 30 groups increased compared to the SB 300 group (Fig. 3C and D). These data indicate that SB may protect against central auditory function impairment by noise exposure in the NIHL mouse model. Also, the preventive efficacy against central auditory function of SB in the NIHL mouse model may be related to baicalein.
To assess abnormality in the cochlear response in the NIHL mouse model, TEOAE tests were performed at 35 days. The S/N ratio in the control group was the lowest at 4 kHz, the noise exposure frequency. S/N ratios in the SB 100, 300, and 500 groups increased significantly compared to the control group in TEOAE responses at 1, 1.5, 2, 3, and 4 kHz at 35 days (p < 0.05, p < 0.01, and p < 0.001). The S/N ratios in the SB 100, 300, and 500 groups increased dosedependently at 2-, 3-, and 4-kHz TEOAEs. The S/N ratios in the baicalein 15, 30, or baicalin 30 groups increased compared to the control group in TEOAE responses at 35 days. The S/N ratios in the baicalein 30 group increased significantly compared to the control group at all frequencies (p < 0.05 and p < 0.001). Results of TEOAEs in the control, SB 100, 300, 500, baicalein 15, 30 and baicalin 15 groups corresponded to hearing threshold shift tendencies in ABRs (Fig. 4). These results indicated that SB or baicalein may protect the organ of Corti against such injury in this NIHL mouse model. After loud sound stimulation, an increase in iNOS expression in the cochlea and isolated outer hair cells of noise exposed mice and Guinea pigs using immunohistochemical staining and quantitative analysis of captured images [18]. Increased iNOS expression and production of both NO and ROS following noise stress may lead to marginal cell pathology, and the dysfunction of cochlear microcirculation by inducing blood vessel wall damage [19]. Thus, the inhibition of NO production may be a useful strategy for the treatment of NIHL. SB has major bioactive flavonoid constituents and possesses a wide range of biological properties, including antioxidant [2], and inhibits NO production [9]. Baicalein is known to be a selective inhibitor of 12-lipooxygenase, which is responsible for the production of ROS in arachidonic acid metabolism [17]. Increasing evidence indicates that baicalein possesses the ability to reduce free radical production and to protect cells against damage induced by ROS [4]. Additionally, baicalein has been found to suppress NO production in peripheral tissues [3]. Recently, Suk et al. reported that
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Fig. 3. Auditory middle latency responses (AMLRs) from the no noise exposure (normal), the SB 100 mg/kg-treated (SB 100), SB 300 mg/kg-treated (SB 300), SB 500 mg/kgtreated (SB 500), or no treatment (control) groups of (A) and (B) graphs and the baicalin 30 mg/kg-treated (baicalin 30), baicalein 15 mg/kg-treated (baicalein 15), baicalein 30 mg/kg-treated (baicalein 30), SB 300 mg/kg-treated (SB 300), or no treatment (control) groups of (C) and (D) graphs with regard to Pa latencies and Na–Pa amplitudes for 35 days after noise induction. Data are presented as the mean ± S.E.M., N = 10 each groups. * p < .05, ** p < .01 vs. control group.
baicalein could attenuate LPS-activated microglial death through suppression of cytotoxic NO production via iNOS inhibition [22]. These findings attracted our interest and led us to examine whether baicalein may have beneficial effects against auditory toxicity in NIHL. We thus believe that the preventative efficacy of SB in NIHL is related to baicalein. We suggest that SB or baicalein may have the capacity to protect against impairment due to noise exposure by inhibiting the production of NO. In the subcortical auditory pathway, the highest levels of NOS are found in the inferior colliculus
(IC), and may be related to the AMLR generator [5]. After loud sound stimulation, the amplitude decreases in AMLRs in this study indicate central auditory dysfunction, perhaps caused by increased NO concentrations. We investigated the protective effects of SB or baicalein in NIHL in a mouse model. The similarity in findings with SB 300 mg/kg/d and biacalein 15 mg/kg/d relative to the HPLC data indicating that biacalein comprised 2.01% of SB by area in the HPLC assay. We thought that the prevention efficacy of the SB in the NIHL might
Fig. 4. Results obtained for the no noise exposure (normal), the SB 100 mg/kg-treated (SB 100), SB 300 mg/kg-treated (SB 300), SB 500 mg/kg-treated (SB 500), or no treatment (control) groups of (A) graph. Results obtained for the baicalin 30 mg/kg-treated (baicalin 30), baicalein 15 mg/kg-treated (baicalein 15), baicalein 30 mg/kg-treated (baicalein 30), SB 300 mg/kg-treated (SB 300), or no treatment (control) groups of (B) graphs. All groups estimated the signal-to-noise intensity of TEOAE with 2, 3, and 4 kHz at 35 days after noise induction. Data are presented as the mean ± S.E.M., N = 10 each groups. * p < .05, ** p < .01, *** p < .001 vs. control group.
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be both mainly related to baicalein and partly related to other constituents. Also, the protective effects of SB were quantitatively different in proportion to the hearing threshold shift degree. The hearing threshold shifts at 8-kHz TBs were lower than at 4-kHz TBs of noise exposure frequency in control group. The protective efficacy of SB or baicalein at 8-kHz TBs was better than 4-kHz TBs. This indicates that SB or baicalein administration could be more protective in mild rather than in severe NIHL. We suggest that SB or baicalein may be useful as a therapeutic for the prevention of NIHL. In this study, however, the precise mechanism of the preventative efficacy of SB or baicalein was not determined. Moreover, inhibition of NO by SB or baicalein was not demonstrated in the preventative efficacy. Thus, future studies should seek to ascertain the mechanism(s) of otoprotection by SB or baicalein.
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[9]
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Acknowledgement This work was supported by the Ministry of Education, Science & Technology (MEST)/Korea Science & Engineering Foundation (KOSEF) through the Vestibulocochlear Research Center (VCRC) at Wonkwang University (R13-2002-055-00000-0).
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Effect of baicalein from Scutellaria baicalensis on prevention of noise-induced hearing loss Tong Ho Kang a , Bin Na Hong b , Channy Park b , Se Young Kim a , Raekil Park c,∗ a b c
College of Life Sciences, Kyung Hee University, Gyeonggi 446-701, Republic of Korea Department of Audiology, Nambu University, Gwangju 506-824, Republic of Korea Vestibulocochlear Research Center & Department of Microbiology, College of Medicine Wonkwang University, 344-2 shinyong-dong, Iksan, Jeonbuk 570-749, Republic of Korea
a r t i c l e
i n f o
Article history: Received 14 October 2009 Received in revised form 2 December 2009 Accepted 3 December 2009 Keywords: Noise-induced hearing loss Scutellaria baicalensis Baicalein Mouse Auditory
a b s t r a c t Noise-induced hearing loss (NIHL) has been thought to primarily involve damage to the sensory hair cells of the cochlea via mechanical and metabolic mechanisms. This study examined the effects of baicalin, baicalein, and Scutellaria baicalensis (SB) extract against NIHL in a mouse model. Mice received oral treatment with SB, baicalin, baicalein beginning 30 min prior to noise exposure and continuing once daily throughout the study. Hearing threshold shift was assessed by auditory brain stem responses for 35 days following noise exposure. Central auditory function was evaluated by auditory middle latency responses. Cochlear function was determined based on transient evoked otoacoustic emissions. SB significantly reduced threshold shift, central auditory function damage, and cochlear function deficits, suggesting that SB may protect auditory function in NIHL and that the active constituent may be a flavonoid, baicalein. © 2009 Elsevier Ireland Ltd. All rights reserved.
Noise-induced hearing loss (NIHL) is a major cause of deafness worldwide and has been thought to primarily involve damage to the sensory hair cells of the cochlea through mechanical injury and/or metabolic deficits [21]. Over the past decade, it has become clear that oxidative stress, induced by noise overexposure, can lead to cell injury, sensory cell death, and permanent NIHL [7]. Hair cell death, induced by noise exposure, arises through the overproduction of free radicals such as reactive oxygen species (ROS), reactive nitrogen species (RNS), and other free radicals through the action of oxidative stress generators [20,23]. Pharmacological protection against NIHL by chemicals has been well reviewed by Lynch and Kil [14]. Successful approaches have included a wide variety of antioxidants [13], glutamate antagonists [1], nitric oxide synthase (NOS) inhibitors [15]. In response to acoustic overexposure, cochlear GSH levels initially increase and then decline steeply. GSH-related enzymes such as gamma glutamyl cysteine synthase, glutathione reductase, and glutathione peroxidase are modulated by loud noise exposure [10,16]. The replenishment of GSH with a glutathione prodrug such as NAC, Dmethionine, or an ester of GSH could reduce hearing loss from loud noise [8,12]. Treatment with ebselen after noise exposure reduced both OHC loss and the swelling of the stria vascularis. This might be due to the preservation of endogenous glutathione peroxidase from ROS/RNS [11].
∗ Corresponding author. Tel.: +82 63 850 6777; fax: +82 63 852 0220. E-mail address: [email protected] (R. Park). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.12.009
Scutellaria biacalensis Georgi (SB), commonly known as Skullcap, is rich in polyphenols and flavonoids; baicalin, baicalein, oroxylin A, and wogonin. Baicalin and baicalein, major flavonoids from SB, have attracted considerable attention because it has a variety of interesting activities such as antitumor, anti-inflammatory, and antioxidant effects [2]. However, no protective effect of a natural product in NIHL in a mouse model has been previously reported. In this study, we evaluated the otoprotective effects of SB, at different orally administered doses, in a NIHL mouse model. We also determined the most effective component from SB (i.e., baicalein or baicalin) in the NIHL mouse model. Seven-week-old male ICR mice (Jung-Ang Lab Animal, Seoul, Korea) were used. They were housed under a 12/12-h light/dark cycle, with food and water provided ad libitum. All experimental procedures were performed in accordance with the Principles of Laboratory Animal Care (NIH publication, #80-23, revised in 1996) and the Animal Care and Use Guidelines of Nambu University, Korea. Experimental mice were examined by an auditory brain stem response (ABR) test before noise exposure to confirm normal hearing. We used the normal hearing mice ABR threshold level of ≤30 dB at 4 and 8 kHz. SB extract was prepared from S. baicalensis supplied from the Kyongdong Oriental Market (Seoul, Korea). Baicalin and baicalein were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dried rhizome of SB (1 kg) was sonicated in 70% ethanol solution for 2 h (three times). Following filtration, the solution was evaporated to dryness in vacuo. The dried SB extract (88.0 g) was used as the SB extract sample in the experiment. SB was standardized by
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Fig. 1. HPLC chromatogram of baicalin and baicalein in Scutellaria baicalensis extract.
HPLC, as described by Gao et al. [6]. An analytical HPLC unit (Shimazu, Kyoto, Japan) equipped with a pump and set up for UV–vis detection at 277 nm, was used for analysis of the SB extract. Chromatographic conditions were as follows: column, Capcell Pak C18, 4.6 mm ID × 250 mm (Shiseido Fine Chemicals, Tokyo, Japan); eluent, 0.5% trifloroacetic acid (v/v), acetonitrile. The linear gradient was 0–40 min, 80–40% A. The flow rate was 0.8 ml/min at 40 ◦ C and the injection volume was 10 l. We designed two experiments to examine the otoprotective effect. First, we evaluated the protective effects of SB at different doses. Experimental mice were divided into five groups (n = 10/group) and were treated orally once daily with 0.5 ml distilled water (control), SB 100 mg/kg/day (SB 100), SB 300 mg/kg/day (SB 300), or SB 500 mg/kg/day (SB 500). One group was treated orally once daily with 0.5 ml distilled water without noise exposure (normal). The second experiment evaluated the protective effects of baicalein and baicalin at different doses. Experimental mice were divided into five groups (n = 10/group) and were treated orally once daily with 0.5 ml distilled water (control), baicalin 30 mg/kg/day (baicalin 30), baicalein 15 mg/kg/day (baicalein 15), baicalein 30 mg/kg/day (baicalein 30), or SB 300 mg/kg/day (SB 300). Preparations of SB, baicalein, and baicalin in distilled water were made daily just prior to treatment. Treatment groups were administered doses 30 min before noise exposure and once daily for 35 days. Doses in all groups were kept constant throughout the experimental period. Experimental mice were exposed to a single, continuous 4-kHz pure tone at a level of 120-dB SPL for 3 h to induce NIHL. During noise exposure, four animals were kept in a cage in a soundproof room. The pure tone of 4 kHz was produced by an audiometer (model GSI-61; Grason-Stadler Inc., Milford, NH, USA) and was presented via a set of loudspeakers on both sides of the cage. The overall noise level was measured at the center of the cage using a sound level meter (NA-24; Rion, Tokyo, Japan). Auditory function tests were performed prior to noise exposure (0 day), then 2, 7, 14, 21, 28, and 35 days after noise exposure. Prior to testing, all mice were anesthetized with xylazine (0.43 mg/kg) and ketamine (4.57 mg/kg), which were administered intramuscularly. Core temperature was maintained at 37.5 ± 1.0 ◦ C using a thermostatically controlled heating lamp in conjunction with a rectal probe. Hearing thresholds, latencies, and amplitudes were determined based on auditory brain stem responses (ABRs) and auditory middle latency responses (AMLRs) using two-channel recordings (GSI Audera; Viasys Healthcare, Conshohocken, PA, USA). A differential active needle electrode was placed s.c. below the test ear and a reference electrode at the vertex; a ground electrode was positioned just above the hind limb. For ABR recordings, alternating 4- and 8-kHz tone bursts (TBs; rise-plateau-fall; 2-1-2 cycles) were performed for the analysis of ABR. For AMLR recordings, rarefaction clicks (0.1-ms duration) were performed. The stimuli were delivered via earphones (ER-3A; Etymotic Research, Elk Grove, IL, USA). Physiological filters were set to pass electrical activity of 100–3000 Hz for ABRs and 10–250 Hz for AMLRs. The averages for 1000 sweeps with a rate of 20.1 stimuli/s in a 20-ms
time window for ABRs and for 250 sweeps with a rate of 9.1 stimuli/s in a 70-ms time window for AMLRs were determined. Cochlear function was determined based on transient evoked otoacoustic emissions (TEOAEs) using ILO v6 (Otodynamics, Hatfield, Hertfordshire, UK). TEOAEs were evoked by 80-s clicks of 90-dB SPL intensity, with a masking noise in the opposite ear, according to the standard nonlinear ILO protocol. TEOAE responses were evaluated in the frequency domain (FFT) by estimating the S/N ratio at 1, 1.5, 2, 3, and 4 kHz. Data were analyzed using the SigmaPlot software (Systat Software, Chicago, IL, USA). All data were expressed as means ± standard error of the mean (SEM). Statistical comparisons between groups were performed using two-way ANOVA with Tukey’s post hoc multiple comparison. P values <0.05, 0.01, and 0.001 were deemed to be statistically significant. We examined the HPLC profile of the SB extract to standardize the crude extracts of the natural product. An external calibration method was used for quantitative analysis with HPLC. Whole chromatograms compared to the standard compounds baicalin and baicalein provided a useful means of identifying and assessing SB. Calibration curves were obtained by plots of the peak area vs. the concentrations of calibration standards. The chromatogram of SB was identified to contain baicalin and baicalein at retention times of 15.3 and 23.8 min, respectively (Fig. 1). The contents of baicalin and baicalein in the SB extract were 15.2% and 2.01%, respectively. To examine the protective effect of the SB extract against the severity of hearing loss from noise exposure, ABR tests were performed prior to noise exposure (0 day), then 2, 7, 14, 21, 28, and 35 days after noise exposure. The increased hearing threshold in the control group showed hearing threshold shift of 49 dB at 4 kHz and 32 dB at 8 kHz of ABR, at 2 days after noise exposure. The SB 100, 300, and 500 groups showed suppressed hearing threshold shifts for 4- and 8-kHz TBs compared to the control group, at each test time for 35 days (p < 0.05, p < 0.01, and p < 0.001). The SB 100, 300, and 500 groups showed decreased hearing threshold shifts in a dose-dependent manner for 35 days (Fig. 2A and B). These data indicated that SB may protect against hearing threshold shifts from noise exposure in the NIHL mouse model. To identify the protective constituent(s) from the SB extract, ABR tests were performed for 35 days after noise exposure. The baicalein 15 and 30 groups showed suppressed hearing threshold shifts compared to the control group at each test time for 35 days (p < 0.05, p < 0.01, and p < 0.001). The baicalein 15 and 30 groups showed decreased hearing threshold shifts in a dose-dependent manner at later test times. The baicalein 30 group was similar to the normal group in hearing threshold shifts at 8-kHz TB after 21 days. The baicalin 30 group was similar to the control group in hearing threshold shifts for 21 days. Hearing threshold shifts in the baicalin 30 group decreased at later test times, but not significantly. Hearing threshold shifts in the SB 300 group were to a degree similar to those in the baicalein 15 group (Fig. 2C and D). These data indicated that the preventative efficacy of SB in the NIHL mouse model may be related to baicalein, and not baicalin.
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Fig. 2. Results obtained for the no noise exposure (normal), SB 100 mg/kg-treated (SB 100), SB 300 mg/kg-treated (SB 300), SB 500 mg/kg-treated (SB 500), or no treatment (control) groups of (A) and (B) graph and baicalin 30 mg/kg-treated (baicalin 30), baicalein 15 mg/kg-treated (baicalein 15), baicalein 30 mg/kg-treated (baicalein 30), SB 300 mg/kg-treated (SB 300), or no treatment (control) groups of (C) and (D) graph with regard to increased hearing thresholds of ABR with 4, and 8 kHz TBs stimulation for 35 days after noise induction. Data are presented as the mean ± S.E.M., N = 10 each groups. * p < .05, ** p < .01, *** p < .001 vs. control group. Waveforms of the auditory brainstem response with ABR in control (control), SB 300 mg/kg treatment (SB 300), and baicalein 30 mg/kg treatment (baicalein 30) mice at 0, 2, and 35 days after the noise induction of (E) graph. ABR waveforms are plotted in the latency domain of the x-axis (ms) and in the amplitude domain of the y-axis (V). ABR waveforms were recorded from variable intensities. The baseline in ABR is regarded under 0.12 V of each peak amplitude, which is the optimal signal-to-noise ratio to discriminate neural activity in the auditory pathway following the sound from the background electrical activity and other bioelectric signals.
The protective effects of the SB extract and baicalein were quantitatively different in proportion to the hearing threshold shift level. The control group at 4- and 8-kHz TB showed a hearing threshold shift of over and below 40 dB, respectively. The suppression ratios of hearing threshold shifts at 4- and 8-kHz TBs were 62% and 87% in the SB 500 group and 64% and 95% in the baicalein 30 group, respectively, compared to the control group. The hearing threshold shift in the SB 500 and baicalein 30 groups recovered significantly at 8-kHz TBs. This indicates that SB or baicalein administration could be more protective in mild rather than in severe NIHL. Waveforms of ABRs in the control, SB 300, and baicalein 30 groups at 0, 2, and 35 days are shown in Fig. 2E. Hearing threshold was defined as positive peaks at the lowest intensity of stimulation. Positive peaks in ABR waveforms appeared at the lower stimulus intensity in SB 300 and baicalein 30 groups from 2 to 35 days into the study. However, positive peaks in ABR waveforms of the control group appeared at the higher stimulus intensity over the course of the study. To investigate whether the protective effects of SB or baicalein in central auditory function could be demonstrated in NIHL, AMLR tests were performed for 35 days. SB 100, 300, and 500 protected against Pa latency delays and amplitude decreases compared to the control group at each test time for 35 days. The SB 100, 300, and 500 groups showed decreased Pa latencies and increased Pa amplitudes in a dose-dependent manner in AMLRs for 35 days (Fig. 3A and B). The baicalin 30 group was similar to the control group in Pa latencies and amplitudes of AMLRs for 35 days. Pa latencies in the SB 300, baicalein 15, and 30 groups were similar from 21 to 35 days postexposure. However, the Pa amplitudes of the baicalein 15 and 30 groups increased compared to the SB 300 group (Fig. 3C and D). These data indicate that SB may protect against central auditory function impairment by noise exposure in the NIHL mouse model. Also, the preventive efficacy against central auditory function of SB in the NIHL mouse model may be related to baicalein.
To assess abnormality in the cochlear response in the NIHL mouse model, TEOAE tests were performed at 35 days. The S/N ratio in the control group was the lowest at 4 kHz, the noise exposure frequency. S/N ratios in the SB 100, 300, and 500 groups increased significantly compared to the control group in TEOAE responses at 1, 1.5, 2, 3, and 4 kHz at 35 days (p < 0.05, p < 0.01, and p < 0.001). The S/N ratios in the SB 100, 300, and 500 groups increased dosedependently at 2-, 3-, and 4-kHz TEOAEs. The S/N ratios in the baicalein 15, 30, or baicalin 30 groups increased compared to the control group in TEOAE responses at 35 days. The S/N ratios in the baicalein 30 group increased significantly compared to the control group at all frequencies (p < 0.05 and p < 0.001). Results of TEOAEs in the control, SB 100, 300, 500, baicalein 15, 30 and baicalin 15 groups corresponded to hearing threshold shift tendencies in ABRs (Fig. 4). These results indicated that SB or baicalein may protect the organ of Corti against such injury in this NIHL mouse model. After loud sound stimulation, an increase in iNOS expression in the cochlea and isolated outer hair cells of noise exposed mice and Guinea pigs using immunohistochemical staining and quantitative analysis of captured images [18]. Increased iNOS expression and production of both NO and ROS following noise stress may lead to marginal cell pathology, and the dysfunction of cochlear microcirculation by inducing blood vessel wall damage [19]. Thus, the inhibition of NO production may be a useful strategy for the treatment of NIHL. SB has major bioactive flavonoid constituents and possesses a wide range of biological properties, including antioxidant [2], and inhibits NO production [9]. Baicalein is known to be a selective inhibitor of 12-lipooxygenase, which is responsible for the production of ROS in arachidonic acid metabolism [17]. Increasing evidence indicates that baicalein possesses the ability to reduce free radical production and to protect cells against damage induced by ROS [4]. Additionally, baicalein has been found to suppress NO production in peripheral tissues [3]. Recently, Suk et al. reported that
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Fig. 3. Auditory middle latency responses (AMLRs) from the no noise exposure (normal), the SB 100 mg/kg-treated (SB 100), SB 300 mg/kg-treated (SB 300), SB 500 mg/kgtreated (SB 500), or no treatment (control) groups of (A) and (B) graphs and the baicalin 30 mg/kg-treated (baicalin 30), baicalein 15 mg/kg-treated (baicalein 15), baicalein 30 mg/kg-treated (baicalein 30), SB 300 mg/kg-treated (SB 300), or no treatment (control) groups of (C) and (D) graphs with regard to Pa latencies and Na–Pa amplitudes for 35 days after noise induction. Data are presented as the mean ± S.E.M., N = 10 each groups. * p < .05, ** p < .01 vs. control group.
baicalein could attenuate LPS-activated microglial death through suppression of cytotoxic NO production via iNOS inhibition [22]. These findings attracted our interest and led us to examine whether baicalein may have beneficial effects against auditory toxicity in NIHL. We thus believe that the preventative efficacy of SB in NIHL is related to baicalein. We suggest that SB or baicalein may have the capacity to protect against impairment due to noise exposure by inhibiting the production of NO. In the subcortical auditory pathway, the highest levels of NOS are found in the inferior colliculus
(IC), and may be related to the AMLR generator [5]. After loud sound stimulation, the amplitude decreases in AMLRs in this study indicate central auditory dysfunction, perhaps caused by increased NO concentrations. We investigated the protective effects of SB or baicalein in NIHL in a mouse model. The similarity in findings with SB 300 mg/kg/d and biacalein 15 mg/kg/d relative to the HPLC data indicating that biacalein comprised 2.01% of SB by area in the HPLC assay. We thought that the prevention efficacy of the SB in the NIHL might
Fig. 4. Results obtained for the no noise exposure (normal), the SB 100 mg/kg-treated (SB 100), SB 300 mg/kg-treated (SB 300), SB 500 mg/kg-treated (SB 500), or no treatment (control) groups of (A) graph. Results obtained for the baicalin 30 mg/kg-treated (baicalin 30), baicalein 15 mg/kg-treated (baicalein 15), baicalein 30 mg/kg-treated (baicalein 30), SB 300 mg/kg-treated (SB 300), or no treatment (control) groups of (B) graphs. All groups estimated the signal-to-noise intensity of TEOAE with 2, 3, and 4 kHz at 35 days after noise induction. Data are presented as the mean ± S.E.M., N = 10 each groups. * p < .05, ** p < .01, *** p < .001 vs. control group.
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be both mainly related to baicalein and partly related to other constituents. Also, the protective effects of SB were quantitatively different in proportion to the hearing threshold shift degree. The hearing threshold shifts at 8-kHz TBs were lower than at 4-kHz TBs of noise exposure frequency in control group. The protective efficacy of SB or baicalein at 8-kHz TBs was better than 4-kHz TBs. This indicates that SB or baicalein administration could be more protective in mild rather than in severe NIHL. We suggest that SB or baicalein may be useful as a therapeutic for the prevention of NIHL. In this study, however, the precise mechanism of the preventative efficacy of SB or baicalein was not determined. Moreover, inhibition of NO by SB or baicalein was not demonstrated in the preventative efficacy. Thus, future studies should seek to ascertain the mechanism(s) of otoprotection by SB or baicalein.
[7] [8]
[9]
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Acknowledgement This work was supported by the Ministry of Education, Science & Technology (MEST)/Korea Science & Engineering Foundation (KOSEF) through the Vestibulocochlear Research Center (VCRC) at Wonkwang University (R13-2002-055-00000-0).
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