Protective Effects of Silymarin Against Age-Related Hearing Loss in an Aging Rat Model

Abstract

Age-related hearing loss (ARHL) is one of the most common chronic degenerative disorders. Several studies have indicated that supplementation with some antioxidants can slow down the progression of ARHL. Despite several lines of evidence about the potent antioxidant and anti-aging effects of silymarin, its protective effect against ARHL has not evaluated yet. The aim of the current study was to investigate the effects of silymarin in prevention of ARHL in a d-Galactose-induced aging rat model for the first time. 45 male wistar rats aged 3-month old were divided into 5 groups: group 1, 2 and 3 received 500 mg/kg/day d-Gal plus 100, 200 and 300 mg/kg/day silymarin respectively for 8 weeks, placebo group received 500 mg/kg/day d-Gal plus propylene glycol as placebo, and control group received normal saline during this period of time. Auditory brainstem responses were measured at several frequencies (4, 6, 8, 12 and 16 kHz) before and after the intervention. Placebo group and group 3 showed significant ABR threshold increase across frequencies of 4, 6, 16 kHz compared with the other groups (P < 0.05). However, rats treated with silymarin 100 and 200 mg/kg/day plus d-Gal did not show any significant ABR threshold shifts. Similarly, ABR amplitude of P2 at 4, 8 kHz and P1, P4 at 4 kHz in the placebo group and group 3 were decreased significantly compared with other groups (P < 0.05). However, no significant differences are found in ABR absolute and inter-peak latencies between groups (P > 0.05). The findings indicates that silymarin with doses of 100 and 200 mg/kg/day has protective effect against ARHL and it can be supplemented into the diet of older people to slow down the progression of age-related hearing loss.

Introduction

Aging is the common physiological condition with characteristic progressive degenerative changes in many living organs [1] including ionic changes, vascular insufficiency, increases in DNA damage as well as decrease in mitochondrial function, cellular water concentrations and elasticity of cellular membranes [2, 3]. This process is often accompanied by slow age-related degeneration of the auditory function that is known as presbycusis (age-related hearing loss; ARHL) [4–6]. Age-related hearing loss (ARHL) is one of the most prevalent sensory conditions in the aged population [5, 7–10]. This condition occurs in 25–40% of people aged 65 years and older, 40–66% of people older than 75 years and more than 80% of people older than 85 years [11, 12].

The etiology of ARHL is not yet well understood. Several lines of evidence indicate that many factors including extrinsic (e.g. exposures to environmental ototoxic agents and noise, vascular insufficiency, metabolic and hormonal changes, diet and immune system), and intrinsic factors (genetic-based physiological ageing process) contribute to the pathophysiology of ARHL [4, 8, 13–15]. Among the potential underlying mechanisms, mitochondrial dysfunction has been proposed to be a major contributor of aging. It has been demonstrated that aging is result from accumulated mitochondrial damage over time caused mainly by reactive oxygen/nitrogen species (ROS/RNS) [1, 3, 9, 16–23]. The malfunction of mitochondria is often associated with mitochondrial DNA damage (mtDNA deletion) [16–23].

Numerous studies have been demonstrated that like many stress-induced otological disorders such as noise-induced hearing loss (NIHL) [24–29], ototoxicity [29–33], tinnitus [34, 35], mtDNA deletion and oxidative stress play a crucial role in pathophysiology of presbycusis [3, 8–10, 31, 36–42]. Several studies have shown that ROS productions can directly damage major components of cells such as DNA and cell membranes that collectively lead to permanent hair cell dysfunction [25]. Accordingly, Benkafadar et al. [40] investigated the contribution of the oxidative stress to ARHL. By exposing the p3 mouse cochlear explants to H₂O₂ in vitro, they found a premature occurrence of cochlear sensory hair cell senescence and apoptosis in the cochlea [40]. Therefore, it seems that pharmacologic or genetic interventions that target oxidative stress might be an effective way to rescue cell damages and in turn slow down the progression of ARHL. Antioxidants are agents that are able to scavenge the ROS productions and consequently to prevent the possible damages leading to ARHL [31, 43]. Like other age-related conditions, several attempts have been conducted to prevent or slow the progression or onset of ARHL, which mainly include enhancing the antioxidant defenses of auditory system with exogenous antioxidants. However, conflicting findings have been reported about the effect of antioxidants in prevention of ARHL.

Previous studies showed that supplementation with certain antioxidants can slow the progression of ARHL [37, 44–59], but not always [60–65]. Among them, most studies have conducted on animals and a few studies have investigated the effect of antioxidants on prevention of ARHL in humans [47, 48, 52, 53, 58, 63, 64]. So far, antioxidants such as vitamin A, E, C, α-lipoic acid [61], lecithin [54], a combination of L-NAME, iron, B12, L-CYSSG, ribose-cysteine and ascorbic acid [48], l-carnitine [56] and Q10 [57] have been studied for prevention of age-related hearing loss. However, the use of antioxidants for prevention of ARHL is presently inconclusive and controversial and discovery of new antioxidants has potential for future researches. One of potent antioxidants that has multiple function is silymarin. Silymarin is the extract of flavonoid isolated from plant fruits milk thistle which has anti-inflammatory, anticancer and antioxidant effects [66–68]. According to studies, silymarin has no significant side effects and has been used for different diseases including liver disease, cancer, diabetes, hepatitis and skin disorders [67, 69–72]. Furthermore, new applications of silymarin are discovered day by day and the use is growing [67, 70]. One of such applications might be the use of silymarin for the disorders of auditory system, which has been evaluated in a few studies until now. Mohammadkhani et al. [25] demonstrated that silymarin can protect against temporary and permanent noise-induced hearing loss. In addition, protective effects of silymarin against ototoxic drugs such as cisplatin [73] and gentamicin [74, 75] have been also reported. In gentamicin ototxixcity, silymarin can reduce the amount of MDA (malondialdehyde) and increase catalase activity in the cochlear tissues, through the recovery of the nerve growth factor or NGF (nerve growth factor), thus preserving the survival of hair cells and significantly improve responses the auditory brainstem [74]. Recently, it has been demonstrated that silymarin has anti-aging effect [76, 77]. However, there have been no studies about the potential effect of silymarin in prevention of age-related hearing loss.

Because of gradual nature of age-related hearing loss in humans, investigation of the effectiveness of antioxidants require a relatively long period of time. However, development of animal models has provided useful research tool to a better understanding of the underlying mechanisms of aging recently. Among the animal models, chronic administration of d-galactose (d-gal) has been widely used as an effective tool to mimic the natural aging process. The underlying mechanism of d-gal is mainly based on that the oversupply of d-galactose accelerates the generation of free radicals by its conversion to galactose oxidase, hydrogen peroxide and aldose. Therefore, it has been well established that d-gal can cause oxidative stress by accumulation of free radicals in cells which in turn may account for the acceleration of aging [77, 78]. Hence rodents treated with d-gal (100–500 mg/kg) for the specified period (consecutive 6–8 weeks) has been widely used to study oxidative-induced aging or anti-aging therapeutic interventions [77, 78]. This model exhibits accelerated aging in several tissues such as brain and liver [77]. In the auditory system, a mimetic rat model of aging using d-gal has been also established previously to study the mechanism of ARHL [6, 17–19, 22, 23, 79–84].

Objectives

Given the important role of oxidative stress in aging and especially in ARHL and also potent multi-function effects of silymarin, it may have potential for prevention of ARHL. For this purpose, the present study investigated the protective effects of silymarin against ARHL in rats chronically injected with d-gal using auditory brain stem responses.

Method

Animals

45 male 3-month-old wistar rats (280 ± 10 g) were prepared from Faculty of Pharmacology of Tehran University of Medical Sciences (Tehran, Iran). Because of sex differences in ability to detoxify ROS, only male rats were used [25]. The rats were caged in an animal facility within the standard polypropylene cages at 23 ± 1 °C temperature, 55 ± 10% humidity and an altering 12-h light–dark cycle. They had free access to standard food and water during the experiments. All ethical issues regarding the use and care of rats were considered carefully. Ethics committee of Tehran University of Medical Sciences approved the study protocol. For acclimation to the new living condition, animals kept in a quiet room for 7 days before starting the experiment.

Drugs Administration

Rats were anesthetized with ketamine (40 mg/kg) and xylazine (10 mg/kg) given intraperitoneally. Baseline ABRs were recorded at all rats and then they were randomly divided into 5 groups (n = 9, each). Rats were received the intervention as follows:

Group 1 500 mg/kg/day d-galactose (intraperitoneally [i.p.], Merck) plus 100 mg/kg/day silyamrin (dissolved in propylene glycol, Goldaru co.) for 8 consecutive weeks [d-Gal + 100 SIL group],

Group 2 500 mg/kg/day d-galactose plus 200 mg/kg/day silyamrin for 8 consecutive weeks [d-Gal + 200 SIL group],

Group 3 500 mg/kg/day d-galactose plus 300 mg/kg/day silymarin for 8 consecutive weeks [d-Gal + 300 SIL group],

Group placebo 500 mg/kg/day d-galactose plus propylene glycol as placebo for 8 consecutive weeks; and

Group control 0.9% saline for 8 consecutive weeks

Pure silymarin powder at doses of 100, 200 and 300 mg/kg/day were carefully dissolved in propylene glycol and gavaged to rats in group 1, 2 and 3. After 30 min, they were intraperitoneally injected with d-galactose for 8 consecutive weeks. In the placebo group, the animals were gavaged with propylene glycol and then injected intraperitoneally with d-galactose. Control group received same volume of normal saline during this period of time.

ABR Testing

Baseline ABRs measurement with tone burst stimuli were performed using Biologic Navigator pro system (Natus, USA) for all animals before and after the intervention at the frequencies of 4, 6, 8, 12, 16 kHz. Because middle ear effusions can interfere with the ABR results, animals with delayed latency and increased threshold were excluded. In addition, to identify animals with otitis media at the end of intervention, they were killed after the last ABR testing and middle ear was carefully observed to exclude the infected ears.

For ABRs measurement, inverting needle electrode was inserted subdermally below the test ear and a reference electrode at the vertex. The ground electrode was placed subdermally below the contralateral ear. The responses of the electrodes were filtered (100–3000 Hz) and amplified (100,000×). The impedance of electrode was determined to be less than 5 Ω. Tone-burst stimuli were delivered in a free-field condition through a speaker which was placed directly above the rat’s right ear at a height of 10 cm. One thousand and twenty-four tone stimuli were presented at the rate of 23.1 and averaged to record a waveform at each level. The tone intensity was varied in a decreasing 10 dB SPL steps from the 80 dB SPL and then 5 dB SPL steps near threshold. Threshold measurements were performed with P2 wave. ABR threshold was determined as the lowest level at which 2 recognizable P2 response can be detected. In addition to threshold, amplitude and absolute and inter-peak latency were also analyzed. The wave amplitude was defined as the peak-to-peak amplitude from the positive peak to the subsequent negative trough of each wave. Amplitude and latency for each frequency were measured at 80 dB SPL. Two latencies were measured for each ABR wave: (1) absolute latency: the latency comprising the time between the stimulus onset and the positive peak, and (2) the inter-peak latencies between I–II, II–IV, and I–IV waves.

The data obtained before and after intervention was analyzed using SPSS Software version 20.0. Comparisons between groups were performed using two-way analysis variance (ANOVA). P < 0.05 was statistically considered significant.

Results

The data from one rat in each silymarin groups and two rats in each placebo and control groups were excluded because of otitis media. A sensitivity analysis showed their measurements could not change the results. In baseline ABR measurement, no significant difference was found between the groups for any of the test frequencies (P > 0.05).

To examine the effects of d-Galactose and silymarin on the ABR, changes in the experimental groups (d-Gal + 100, 200 and 300 mg/kg/day silymarin) were compared to that of the placebo and control groups. Among ABR parameters, both absolute and inter-peak latencies was not significantly different between groups across the all frequencies. In contrast, a significant increase in ABR threshold were observed when placebo group and group 3 was compared with other groups across frequencies of 4, 6 and 16 kHz (P < 0.05).

Among groups, placebo group showed maximum increase in ABR threshold which was equal or greater than 5 dB SPL across all frequencies. Interestingly, rats treated with d-Gal plus 300 mg/kg/day silymarin also showed significant threshold increase that was lower than 5 dB SPL. As shown in Fig. 1, for both of these groups, maximum threshold shift was observed at 4 and 16 kHz. In contrast, rats treated with d-Gal plus 100 and 200 mg/kg/day silymarin as well as control group did not show any significant ABR threshold shifts. Threshold shift in control group was minimal compared with other groups. The mean threshold shift was small and comparable in group 1 (1.92, 0.6, 1.67, 1.73, 0.47 dB SPL) and group 2 (0.07, 0.12, 3.75, 3.41, 0.42 dB SPL) at 4, 6, 8, 12 and 16 kHz respectively and there was no significant difference between them (P > 0.05).

Fig. 1.

Mean ABR threshold shifts in the study groups at different frequencies

Generally, average threshold shift ranged from 0.07 dB SPL (in group 2) to 6.25 dB SPL (in placebo group). In placebo group, mean ABR threshold shift was highest at 16 kHz and lowest at 8 kHz. Similarly, in the group 3, response of 8 kHz showed minimum threshold shift compared to other frequencies and was nearly identical to threshold shift at 6 kHz. However, in contrast to placebo group, group 3 showed maximum threshold shift at 4 kHz. Group 1 and 2 were only groups that showed threshold decrease (improvement) which was at 16 and 6 kHz respectively. At these groups, mean threshold shift varied across frequencies and ranged from − 0.47 to 1.92 dB SPL and 0.07 to 3.75 dB SPL in the group 1 and group 2 respectively. Maximum threshold shift was observed at 4 kHz in group 1 and 8 kHz in group 2. Control group also showed slight but non-significant threshold increase that ranged from at least 0 to maximum 1.87 dB SPL at 6–16 kHz respectively. Interestingly, in control group threshold shift was identical (1.25 dB SPL) for all frequencies of 4, 8 and 12 kHz.

Mean (SD) ABR thresholds at 4, 6, 8, 12, 16 kHz are displayed on Table 1 before and after intervention.

Table 1.

Average ABR thresholds before and after the intervention

Group	Frequency
	4 kHz	6 kHz	8 kHz	12 kHz	16 kHz
d-Gal + SIL 100
Before	27.78 (2.63)	28.57 (3.87)	28.33 (2.50)	28.89 (2.20)	28.33 (2.50)
After	27.50 (3.78)	29.17 (2.04)	30.00 (3.78)	30.62 (3.20)	27.86 (4.88)
P (within groups)	1.00	.15	.26	.18	.70
d-Gal + SIL 200
Before	25.56 (3.90)	27.00 (4.47)	25.00 (4.33)	27.22 (3.63)	28.33 (2.50)
After	25.63 (3.20)	26.88 (5.93)	28.75 (5.17)	30.63 (4.95)	28.75 (6.40)
P (within groups)	1.00	.56	.23	.13	.78
d-Gal + SIL 300
Before	25.00 (2.50)	26.11 (3.33)	26.67 (3.33)	26.67 (3.33)	26.67 (3.33)
After	30.00 (3.53)	28.89 (2.20)	29.44 (3.00)	30.56 (3.00)	31.11 (3.33)
P (within groups)	.02*	.05	.02*	.02*	.01*
d-Gal + placebo
Before	24.38 (4.17)	26.43 (3.78)	26.25 (2.31)	26.25 (2.31)	26.25 (2.31)
After	30.00 (4.47)	31.67 (2.58)	30.83 (4.91)	31.67 (2.58)	32.50 (2.73)
P (within groups)	.04*	.05	.03*	.02*	.02*
Control
Before	28.13 (4.58)	30.00 (3.78)	29.38 (3.20)	30.00 (3.78)	29.38 (3.20)
After	29.38 (4.17)	30.00 (3.78)	30.63 (3.20)	31.25 (3.53)	31.25 (4.43)
P (within groups)	.15	1.00	.41	.15	.08
P (between groups)	.01*	.04*	.40	.07	.02*

The data are expressed as average (standard deviation)

* P value < 0.05

In addition to threshold, a significant decrease in ABR amplitude was observed in placebo group and group 3 at the frequencies of 4 and 8 kHz compared with other groups. In both these groups amplitude of P1, P2, P4 ABR waves of 4 kHz and P2 ABR wave of 8 kHz decreased significantly(P < 0.05). Similarly, P2 amplitude at 12 kHz showed marginal decrease in the both of thses groups (P = 0.058). Maximum amplitude decrease (1.91 μv) was attributed to group 3. Control group showed non-significant decrease in P2 amplitude. In contrast, a significant increase was observed in ABR amplitude in the group 1 and 2 at the frequencies of 4 and 8 kHz). A sample of ABR waves at placebo group is illustrated in Fig. 2 before and after the d-GAL injection from 80 dB SPL to around the threshold level. As shown, there is a clear difference between the amplitude of ABR wave at 4 kHz before and after the d-gal injection.

Fig. 2.

Sample of ABR wave at 4 kHz in placebo group before (a) and after (b) the d-GAL injection

These results indicate that pretreatment with silymarin at the doses of 100 and 200 mg/kg/day protect against d-galactose-induced threshold shift that was observed in placebo group. However, increasing silymarin dose to 300 mg/kg/day was not effective and did not result in any significant result. This suggests the dose-dependent effects of silymarin in the auditory system.

Discussion

The present study evaluated the effectiveness of silymarin as an otoprotective agent against age-related hearing loss in wistar rats. Significant preservation of tone-bursts ABR thresholds and amplitude increase at most of the frequencies was observed after a 8 week pretreatment with silyamrin at the doses of 100 and 200 mg/kg/day that was administered 60 min before the i.p. d-galactose injection.

The mechanism of age-related hearing loss has been investigated in numerous animal models and several recent studies have shown that mtDNA deletion, a biomarker of aging, can be induced by some agents. d-galactose injection is one of them. It has shown that d-Gal can induce oxidative stress in several portions of auditory system [6, 17–19, 22, 23, 79–81]. According to studies, it seems daily injection of 500 mg/kg/day d-Gal for consecutive 8 weeks, could be an appropriate model to imitate age-related hearing loss [1, 18, 81]. This is because d-Gal can leads to several histopathological changes in the peripheral and central portions of auditory system, which is very similar to natural age-related hearing loss process. These age-related oxidative changes include increased mitochondrial DNA deletion (mtDNA 4834 bp deletion), increased levels of lipids peroxidation and in turn malondialdehyde (MDA) marker, increased apoptosis rate of neurological and neurodegenerative changes in auditory cortex, inferior colliculus, the cochlear nucleus and the inner ear of d-galactose animals that was similar to changes in normal aged animals [6, 18, 22]. Other changes which have been found in d-GAL rats include increased expression levels of uncoupling proteins, NOX, cleaved caspase-3 and number of TUNEL-positive cells in the inner ear of d-Gal rats [17, 82], the reduced expression level of cytochrome c oxidase subunit III in the auditory cortex [81], the increased expression of NOX₂, 8-hydroxy-2-deoxyguanosine, a biomarker of DNA oxidative damage, and uncoupling protein 2, together with a decrease in the mitochondrial total antioxidant capabilities in the auditory cortex [83] and the increased p66Shc protein (an age-related adaptor protein) in the cochlear lateral wall [79]. Despite these line of evidence, that suggest d-galactose can eliminate mitochondrial DNA in the auditory system, similar to changes in age-related hearing loss, there are conflicting results about the relationship between auditory brain stem response and d-galactose injection [6, 17–19, 46, 84, 85].

Similar to the present results, Du et al. [17] reported that d-gal can shift ABR threshold at several frequencies. However, in study of Zhong et al. [18], Kong [19, 84, 85], Wu et al. [79] and Peng et al. [46], ABR threshold remained unchanged. One of the likely reasons is that they used click stimuli for acquiring auditory brain stem responses while deterioration of hearing in aging process is more accurately measured with acute frequencies such as tone-burst stimuli [50]. Because age-related hearing loss primarily affects the high frequencies then spread to lower frequencies and processing of sounds in auditory system is frequency-specific from cochlea to auditory cortex. So, in the present study, tone burst stimuli were used to have an accurate study of the auditory system.

In contrast to results of the current study, a small but significant latency increase was observed in the study of Chen and colleagues [6]. They found significant greater absolute latencies of I, IV, V and I–IV waves of ABR and Pa wave of MLR in d-gal-induced aging rats compared with the control group that was similar to naturally aging rats [6]. The data about the latency and amplitude was not analyzed in previous studies.

As mentioned, d-Gal injection can increase the formation of free radicals, depletion of intracellular antioxidants, several histopathological changes that finally damage to different portions of auditory system. On the other hand, evidence has showed that antioxidants can protect against these oxidative stress conditions. However, all antioxidants do not have the same effect and strength of each antioxidant is definitely different. For this reason, several antioxidants have been evaluated to discover if they have any beneficial effects on the prevention of ARHL [86]. One of the antioxidants that has not been studied much in ear-related disorders is silymarin. Numerous evidence indicates that silymarin can decrease oxidative stress [66–68] and has anti-aging [76, 77, 87], anti-inflammatory and anti-cancer properties [72, 88, 89]. As a result, it seems that silymarin has many functions and potent antioxidant effects of silymarin are one of the proposed mechanisms for its beneficial pharmacological effect [90]. It has been suggested that the antioxidant properties of silymarin is mainly responsible for its protective actions [91]. It seems that is due to its ability to decreasing the oxidative stress by several mechanisms like scavenge free radicals or chelate the metal ions [25].

Because application of silymarin for auditory disorders is relatively new area of research, main function of silyamrin in the auditory system is not clear yet. In our previous study, silymarin specifically protected against temporary and permanent noise-induced hearing loss [25]. It was attributed to that silymarin could inhibit DNA oxidation and lipid peroxidation in the cochlea [25]. There are also studies that suggest otoprotective effects of silymarin against cisplatin [73] and gentamicin ototoxicity [74]. It has been shown that in gentamicin ototoxicity, silymarin can regenerate NGF, reduce MDA level and increase catalase activity within the cochlear tissues and by which can improve the ABR responses [74]. In cisplatin ototoxicity, pre-treatment with silymarin decreased the expression level of cleaved caspase-3 and PARP in the auditory cells and therefore prevented from cisplatin ototoxicity [73]. Ototoxicity may be a disorder that share similar mechanism to age-related hearing loss in which endogenous antioxidants is decreased and MDA level and caspase-3 activation; a stress-induced apoptotic pathway in the ear; is increased [32].

Protective mechanisms of silymarin against ARHL is probably due to the effects of silymarin in stabilizing of the cell membranes [90, 92, 93], free radicals scavenging [90, 94–96], reduction of cell membranes lipoproxidation [67, 90, 92, 97], protection from depletion of glutathione [90, 97] as well as increasing intracellular glutathione reserves [95, 97], inhibition of iNOS [92, 97] that suppress the adverse effects of free radicals. Silymarin can also restore SOD, CAT and GSH in d-gal rats and through which decrease the oxidative stress [66, 67, 92].

Furthermore, we believe that other mechanism actions of silymarin have been also involved in the auditory system in addition to a simple action. This is because that silymarin is a multi–target and multi-function agent. It can not only inhibit mtDNA damage induced by d-GAL [91, 98] but also has protective activity in ameliorating DNA damages [91]. In addition, silymarin can inhibit the activation of c-Jun N-terminal protein kinase (JNK), MAPK kinase (MEK) and caspase apoptotic pathways [99, 100] which are apoptosis-related genes involved in age-related hearing loss [101]. It has been also shown that silymarin can inhibit NADPH oxidase activity [91, 102], which was found to increase after d-GAL injection [1].

On the other hands, silyamrin has anti-inflammatory properties [72, 88, 89, 92] that is particularly important because it has been demonstrated that expression of pro-inflammatory cytokines, is increased in d-Gal aging rats [93] as well as other aging animal models [103]. It has been also demonstrated that there is relation between markers of inflammatory status and hearing thresholds in older people [104]. In addition, silymarin can activate and synthesize and increase expression of protective molecules like sirtuins, thioredoxins, heat shock proteins (HSPs), etc. and can regulate SIRT1 and Bcl-2 family [91]. It should be noted that some of these agents change during the aging process. For example it has been found that HSPs expression, which plays a protective role against cochlear damage, is altered during the age-related hearing loss [8] and low levels of Bcl-2 have been reported in d-Gal-treated mice [77] as well as naturally aged gebrils [8]. There is also finding that suggest that the simultaneous decreases of NAD+/NADH ratio and SIRT1 expression with age may play a major role in etiology of age-related hearing loss [105]. Therefore, silymarin can be useful in ARHL by regulating Bcl-2 and SIRT1.

Therefore, it appears that mechanism actions of silymarin in the auditory system are more than the simple radical scavenging function. This has been summerized in Fig. 3. In should be noted that in studies that used antioxidants to prevent disease, lack of effect with a dose of an antioxidant, and a significant effect with another dose of same antioxidant indicate that the use of antioxidants is highly dose-dependent and the dose is an important factor when antioxidants have been used. Administered dose of silymarin varies across studies. The present study showed a dose–response manner of silymarin in the auditory system such that 100 and 200 mg/kg/day silymarin had beneficial effect but increasing silymarin dose to 300 mg/kg/day was not effective. The beneficial effect of silymarin is thus related with dose, as demonstrated in the present study, particularly at doses which are usually used in other animal studies (i.e. 100 and 200 mg/kg/day) [25, 76, 106–108].

Fig. 3.

Silymarin multi-function mechanism and its role in prevention of age-related hearing loss

Conclusion

This study indicates the otoprotective effect of silymarin with doses of 100 and 200 mg/kg/day against age-related hearing loss. As far as we know, the current study is the first report about the effects of silymarin on age-related hearing loss that is expanding our knowledge about the mechanism actions of silymarin in the aging auditory system. Since silymarin is already found safe in human, therefore, it can be supplemented into the diet of older people to slow age-related hearing loss. Investigating the effective duration of treatment with silymarin for ARHL is among the plans of future.

Funding

The article was supported by Tehran University of Medical Sciences.

Compliance with Ethical Standards

Conflict of interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.