Idiopathic sudden sensorineural hearing loss (ISSNHL) is a common otological emergency, which refers to a hearing loss of more than 30 dB in three consecutive frequencies within 72 h (1). This disease is frequently diagnosed among people over 40 years old and is accompanied by tinnitus and vertigo (2). It is possible to develop into permanent deafness without timely treatment (3), which will seriously affect the life of patients.

The etiology of ISSNHL is still in the exploratory stage. At present, many scholars believed that it is mainly related to viral infection, endolymphatic hydrops, cochlear microcirculation, immune damage, and psychological factors (4). No matter what the pathogenic factors are, it is worth affirming that the cochlea is sensitive to ischemia and hypoxia (5). This change in the microenvironment aggravates the production of reactive oxygen species (ROS). Related studies suggest that oxidative stress is a risk factor for microcirculation damage (6). ROS are constantly produced during metabolism (7) and transformed into each other in four forms (8). The physiological concentration of ROS is beneficial to the body. They kill pathogenic microorganisms, maintain normal immune function (9), and act as a key second messenger in a variety of cellular signaling pathways (10). Once the balance of the oxidation-antioxidation system is broken, there are adverse effects. ROS directly attack proteins, lipids, and DNA. Furthermore, they change the phenotype of vascular smooth muscle cells, reduce the fluidity of cell membranes (11), and affect cochlear perfusion. There is evidence that endothelial dysfunction and impaired cochlea perfusion play a crucial role in the pathogenesis of ISSNHL (12).

Therefore, neutralizing excessive ROS may be an effective treatment for ISSNHL. At present, melatonin is one of the most investigated antioxidants. It is mainly produced by the pineal gland at night and participates in many physiological metabolic processes, such as regulating hormone secretion, controlling human growth, and aging (13). Compared with other antioxidants, melatonin can easily cross cell barriers due to its oleophilic and hydrophilic structure. Takumida et al. (14) found that melatonin and its receptors (melatonin receptor 1A; MT-1 and melatonin receptor 1B; MT-2) are present in the inner ear, which further supports the hypothesis that melatonin plays a physiological role in the inner ear. Recent studies have confirmed that melatonin has a protective effect on drug-induced hearing loss (DIHL) (15), noise-induced hearing loss (NIHL) (16), and age-related hearing loss (ARHL) (17). Thus, this research aimed to explore whether melatonin has a protective effect on hearing and verify the role of oxidative stress in ISSNHL. Multivariate logistic regression models were performed to assess the factors relevant to the occurrence and improvement of ISSNHL.

The time of drawing blood and the corresponding concentration of melatonin for each participant are shown in Figure 2. In addition, the intra-group comparison between the healthy controls group and the ISSNHL patients group showed that the p-values were 0.502 and 0.182, respectively, which was not statistically significant. The results show that melatonin secretion was stable during the period in which we drew blood. On admission, the comparison of related factors between the ISSNHL and the control groups is shown in Table 1. The mean ROS levels were significantly higher in the patients than in the controls (4.42 ± 4.40 vs. 2.30 ± 0.59 ng/mL; p = 0.031). There was no statistical difference in melatonin and TAC between the two groups, but the basic level of melatonin in the healthy control group was higher than that in the patient group (1841.87 ± 1336.57 vs. 1237.77 ± 742.35 pg/mL).

According to the significance level (Table 2), there was a linear relationship between the logi-conversion values of all the continuous independent variables and dependent variables. And there was no multicollinearity between each independent variable (Tolerance > 0.1, VIF < 10, Table 3). Subsequently, we used multivariable logistic regression to evaluate the effects of melatonin, ROS, and TAC levels on ISSNHL. Finally, the Logistic model was statistically significant (X² = 16.311 p < 0.05). The model could correctly classify 79.3% of the research objects. The sensitivity, specificity, positive predictive value, and negative predictive value of the model were 95.3%, 33.3%, 80.4%, and 71.4%, respectively. Among the three independent variables included in the model (Table 4), melatonin (OR = 0.999, 95% CI 0.998–1.000, p = 0.023) and ROS (OR = 3.248, 95% CI 1.109–9.516, p = 0.032) were statistically significant. The lower the level of melatonin, the higher the level of ROS, and the higher the risk of ISSNHL.

The general status of the patients in the effective and ineffective groups is shown in Table 5. The effective group had lower basic ROS levels (3.05 ± 1.81 vs. 5.62 ± 5.56 ng/mL; p = 0.042) and higher melatonin levels (1400.83 ± 784.89 vs.1095.97 ± 689.08 pg/mL; p = 0.046). Furthermore, there were statistical differences in vertigo (X² = 6.702, p = 0.010) between these two groups.

Again, we performed the same verification as the steps above. Multivariable logistic regression was used to analyze the relationship between melatonin, ROS, vertigo, and the prognosis of ISSNHL. Finally, the Logistic model was statistically significant (X² = 16.499, p < 0.05). The model could correctly classify 76.7% of the research objects. The sensitivity, specificity, positive predictive value, and negative predictive value of the model were 78.3%, 75.0%, 75.0%, and 78.3%, respectively. Among the three independent variables included in the model (Table 6), melatonin (OR = 0.999, 95% CI 0.997–1.000, p = 0.049), ROS (OR = 1.154, 95% CI 1.025–2.236, p = 0.037), and vertigo (OR = 6.011, 95% CI 1.339–26.983, p = 0.019) were statistically significant. The lower the level of melatonin, the higher the level of ROS, accompanied by vertigo, and the higher the risk of poor prognosis for the patients with ISSHNL.

After systemic treatment, blood samples were collected again in 31 patients. Comparing the three indicators before and after treatment (Table 7), the melatonin levels were significantly reduced (1279.83 ± 691.29 vs. 425.24 ± 69.14 pg/mL; p < 0.01), the TAC was enhanced (0.69 ± 0.08 vs. 0.83 ± 0.09 Mm; p < 0.01), and the body’s state of high oxidative stress had been alleviated (4.50 ± 5.02 vs. 3.32 ± 2.54 ng/mL; p = 0.034). Compared with the control group (Table 8), the level of ROS in the patients with ISSNHL decreased to normal after treatment. For more information on the intuitive comparison of various indicators, please refer to Figure 3.

The influence of ISSNHL on the personal life of patients promotes the continuous development of research on the pathogenesis of ISSNHL. This study suggests that the level of melatonin and ROS may be related to the occurrence of ISSNHL. The logistic regression analysis indicated that melatonin, ROS, and vertigo were significantly correlated to hearing improvement after ISSNHL treatment. Therefore, improving the state of oxidative stress may become an effective method for the treatment of ISSNHL in the future.

The results show that the ROS levels of patients with ISSNHL were higher than that of the healthy controls (Figure 3B), which is consistent with the results in the literature (22, 23). This is not a coincidence as Guo et al. (24) speculated that the generation of superoxide anion radicals and the decreased activity of nitric oxide synthase led to endothelial dysfunction. Finally, hair cell loss, vascular intimal thickening, and luminal narrowing of spiral arteries were detected in the mouse cochlea. Merchant et al. (25) pointed out that the pathological activation of nuclear factor NF-kappa-B (NF-kB) induces oxidative stress, which causes ISSNHL. In a prospective study, total oxidative state (TOS) levels in 50 patients with ISSNHL were compared with those in 50 normal controls, and the corresponding global oxidative stress index was calculated. The patients with ISSNHL showed significantly higher TOS levels and oxidation index (26). Excessive ROS depletes intracellular nitric oxide levels and increases the release of adhesion molecules, lipid-inflammatory mediators, and cytokines, leading to endothelial damage (6) and blood flow disorders. The cochlear labyrinthine artery is sensitive to ischemia as it has no branches. Subsequently, impairment of perfusion leads to an immediate dysfunction of the organ of Corti, resulting in irreversible hearing loss. ROS also have toxic effects on cell functions, especially hydroxyl radicals, which directly damage various organelles and pathways. As the main source of ROS, mitochondria may be damaged while producing ROS, which affects mitochondrial membrane potential and energy metabolism (27). Moreover, Becatti et al. (28) used fluorescent markers to evaluate the fluidity of red blood cell membranes. The results showed that lipid peroxidation of red blood cell membranes in patients with ISSNHL resulted in decreased deformability and increased blood viscosity, affecting the cochlear microcirculation.

According to other authors, oxidative stress is thought to play a role in sensorineural hearing loss (SHL) (29). ROS have been identified as the main cause of aminoglycoside antibiotics-induced hearing loss (30). Besides, ROS induce the reduction of nicotinamide adenine dinucleotide phosphate (NADPH) by binding to the sulfhydryl group of the enzyme and affecting glutathione peroxidase (GPX) and glutathione reductase (GRD) activity (31), resulting in the conversion of hydrogen peroxide to hydroxyl radicals instead of water. Furthermore, the antioxidant defense mechanism of hair cells is weakened, which leads to the release of cytochrome c from mitochondria and activates the caspase pathway to trigger cell apoptosis (32) and causes bilateral high-frequency SHL. A guinea pig model (33) showed that ROS transduced the mitogen-activated protein kinase (MAPK) pathway to induce hair cell apoptosis after sound injury. It is uncertain whether these verified pathways apply to ISSNHL and the molecular mechanism of the role of ROS in ISSNHL is still being explored.

We found that the ROS levels in the ineffective group were higher than those in the effective group (Figure 3D), and regression analysis showed that the higher the ROS, the worse the prognosis of ISSNHL. Higher concentrations of ROS may cause more hair cell damage and at a certain peak level, endothelial cells undergo “sudden death” (34). For this reason, the hearing loss that has already occurred is hard to recover, even when prompt and comprehensive treatment is administered. Furthermore, the basal region of the cochlea responsible for high-frequency hearing is more metabolically active than the apical region. Therefore, the basal region generates more ROS while consuming energy, and is more susceptible to oxidative stress damage (35). This may also be the reason for the prognosis of the all-frequency and high-frequency hearing loss being worse than other types of ISSNHL.

There was no significant difference in TAC between the patients with ISSNHL and the healthy controls (Table 8). We believe that the normal antioxidant system could not remove the excess ROS, and the balance had been broken. After systemic treatment, the level of ROS dropped to the same level as the healthy controls (Table 8) and the antioxidant capacity was enhanced. Consistent with the results of previous findings (36), the state of oxidative stress in vivo was significantly improved after steroid treatment. Glucocorticoids (GCs) effectively improve the microcirculation of the inner ear, decrease the edema of vascular endothelial cells, and improve the state of ischemia and hypoxia (37). After treatment, the level of ROS decreased (Figure 3F), which further shows that cochlear microcirculation is related to ROS. Unfortunately, only a part of the patients had improved hearing. We postulate the following reasons: first, ROS are not the only causative agent, and many other factors are involved in the pathogenesis of ISSNHL; second, hair cells could not regenerate after being destroyed by ROS; third, it takes a longer period for the hearing improvement of ISSNHL patients.

It is certain that oxidative stress causes damage by inducing endothelial dysfunction within inner ear micro-circulation. To eliminate excessive ROS to improve the hearing of patients, we chose to observe serum melatonin concentrations as it, as an antioxidant, has been proven to protect hair cells in SHL. Demir et al. (15) injected melatonin into the tympanic chamber of rats and found that melatonin has an antagonistic effect on cisplatin ototoxicity due to its antioxidant and immunomodulatory functions. Serra et al. (17) obtained the same results in a study on melatonin and ARHL. Mice with orally administered melatonin maintained a higher distortion product otoacoustic emissions (DPOAE) amplitude and signal-to-noise ratio at the end of the experiment. Melatonin is an indoleamine hormone secreted by the pineal gland controlled by the hypothalamic suprachiasmatic nucleus (SCN). Compared with other traditional antioxidants, melatonin has certain advantages. Its structural properties enable it to shuttle cells freely, effectively reducing high levels of ROS.

Our research found that the melatonin levels in the effective group were higher than those in the ineffective group (Figure 3C) before treatment. We speculate that a higher concentration of basal melatonin may have a positive effect on patients with ISSNHL, as shown by the logistic regression analysis. As previously described, the erythrocyte membranes of patients with ISSNHL are accompanied by lipid peroxidation and functional alterations. Melatonin can directly eliminate ROS when in contact with the cell membrane. At the same time, melatonin reduces intracellular malondialdehyde (MDA, excessive MDA alters membrane fluidity) in a concentration-dependent manner (38), which protects the morphology and function of cell membranes from ROS attack and improves the blood and oxygen supply to the inner ear. Melatonin is then metabolized in vivo through enzymatic and chemical reactions such as oxidation to several derivatives, including N1-acetyl-N2-formyl-methoxykynuramine (AFMK) and N2-acetyl-5-methoxykynuramine (AMK) (39). These possess even greater antioxidative capacities than melatonin. In other words, melatonin can scavenge multiple free radicals. In addition, AFMK and AMK exhibit anti-inflammatory and immunomodulatory activities (40), inhibit the formation of prostaglandins, tumor necrosis factor-α (TNF-α), and interleukin-8 (IL-8) (8), and protect hair cells from inflammatory mediators.

The balance of the oxidation-antioxidant system is inseparable from the action of antioxidant enzymes, which is the first line of defense against free radicals. Superoxide dismutase (SOD) is a key antioxidant enzyme that converts superoxide anion radicals into hydrogen peroxide (41). Previous studies have shown that the level of SOD in rats increased significantly after injection with melatonin (42), indicating that melatonin enhanced its activity to achieve indirect antioxidation. In the presence of transition metals, hydrogen peroxide generates highly toxic hydroxyl radicals (43), while catalase (CAT) and glutathione peroxidase (GPX) catalyze peroxidation hydrogen to produce water and oxygen (44). The concentration of GPX is higher at night, which is consistent with the circadian rhythm of melatonin. Melatonin maintains the activity of these enzymes in a receptor-dependent manner. Similarly, Baydas et al. (45) observed a significant decrease in GPX activity in multiple tissues after the removal of the pineal gland in rats. Another study found the activity of CAT is weakened under strong oxidative stress conditions, and when melatonin is present, the high activity of CAT can be maintained (46). The antioxidants can accelerate ROS degradation, while antioxidant enzymes reduce ROS production. Therefore, melatonin not only removes the generated ROS but also avoids the generation of ROS at the source, a characteristic that other antioxidants do not possess.

Mitochondria are the major source of cellular ROS (47). ROS are formed continuously by electron leakage from the respiratory chains (48). Over time, mitochondria may be affected by severe oxidative stress. Therefore, mitochondria may be a potential target for melatonin to eliminate ROS. First, melatonin stabilizes the inner membrane and maintains the integrity of the mitochondria. It then upregulates the expression and activity of antioxidant enzymes in the mitochondria (49) and inhibits pro-oxidant enzymes. Most importantly, melatonin reduces the production of free radicals during respiration by stimulating complexes I and IV of the mitochondrial respiratory chain. Finally, melatonin regulates mitochondrial gene expression and maintains a high level of glutathione, which greatly enhances the antioxidant capacity of mitochondria (50). In short, melatonin could reduce ROS generation and protect the mitochondria from ROS-induced damage.

Most treatments for ISSNHL are based on two etiological theories: circulatory disorders and inflammatory response. Interestingly, the systematic treatment we use seems to have the same effect as melatonin (Table 9). Hargunani et al. (51) confirmed that GCs plays a therapeutic role by binding to glucocorticoid receptors (GRs). GRs are widely found in the human inner ear, especially the spiral ligament in the cochlea (52). Studies have shown that melatonin plays a role in delaying cell loss in spiral ligaments (53). In other words, the concentration of melatonin affects the efficacy of GCs in the treatment of ISSNHL. In addition to GCs as a first-line treatment, most doctors in our country use vasodilators as a combination therapy (54). The best arterial blood supply and adequate oxygenation are prerequisites for the full function of the inner ear, which is the reason for choosing vasodilators. However, melatonin seems to have a superior performance in dilating blood vessels. Melatonin regulates the central nervous system and coordinates the activity of multiple organs to maintain more regular and stable hemodynamics. It can also promote the production of nitric oxide (NO) and reduce peripheral resistance and ischemia–reperfusion injury (55, 56). When to use HPOT is still controversial. Our view is consistent with some recent studies (57, 58) that additional therapeutic benefit can be provided when HPOT is used in combination with GCs to treat ISSNHL. It is undeniable that HBOT has the function of increasing the oxygen tension in the blood and nourishing nerve elements, whether as a routine treatment or as a remedial measure (59). Besides, some domestic scholars have pointed out that HPOT can activate the superoxide free radical scavenging system and reduce the damage to the inner ear caused by ROS. However, the use of hyperbaric oxygen (HPO) may also accelerate the production of ROS and lead to lipid peroxidation, which is harmful to the body (60). Surprisingly, it has been reported that melatonin could effectively prevent HPO-induced oxidative stress, even physiologically secreted melatonin (61). Animal experiments have shown that the combination of melatonin and HPO was superior to either one in vascular events (62). Based on these findings and our regression analysis, we boldly speculate that melatonin can cooperate with these treatment measures to play a more active role and avoid some side effects.

After treatment, the concentration of melatonin decreased significantly (Table 7), which may be related to our treatment. The link between steroids and melatonin was mentioned a long time ago. Demisch et al. (63) observed that melatonin plasma levels decreased significantly in adults treated with dexamethasone. They believed that dexamethasone could interact with the pineal gland to affect melatonin secretion. Another study showed that oral GCs can increase melatonin excretion in children (64). With the deepening of the study, the pineal gland was considered a neuroendocrine organ regulated by the central clock (SCN) and peripheral circadian clocks (65). Dexamethasone treatment changed the mRNA expression of several clock genes in the pineal gland. Furthermore, the synthesis of melatonin requires the participation of a variety of enzymes. Arylalkylamine N-acetyltransferase (AANAT) is the rate-limiting enzyme in the synthesis of melatonin (66). Dexamethasone reduces the synthesis of melatonin by reducing the activity of AANAT (67). Other studies have found that propranolol caused a dose-dependent decrease in melatonin levels (68). There are many kinds of vasodilators and their mechanisms are different, so we are not sure whether the vasodilators used to treat ISSNHL will affect the concentration of melatonin. In addition, as mentioned above, melatonin can antagonize the negative effects of HPOT, and endogenous melatonin is even more effective than exogenous administration (69), which may cause melatonin loss. Overall, under the current treatment, first, the synthesis of melatonin was reduced. Second, the excretion of melatonin increased. Third, we postulate that melatonin was involved in the clean-up of ROS and works with drugs. Finally, melatonin decreased significantly after systematic treatment. Of course, there is a lack of research on drug interaction, and the specific mechanism needs to be further studied.

A recent study shows that melatonin alleviates pyroptosis of hair cells via the MT-1 and MT-2/Nrf2 (NFE2L2)/ROS/NLRP3 pathway, which further supports the conjecture that melatonin can be used for the treatment of ISSNHL (70). In addition, melatonin also has an antidepressant function (71) and improves tinnitus (72). It is well known that depression and tinnitus afflict some patients with ISSNHL patients and eliminating these symptoms has a certain positive effect on the prognosis of patients.

Few studies have linked melatonin to ISSNHL. According to our results, appropriate melatonin supplementation may have a positive effect on hearing improvement in patients with ISSNHL.

The limitations of this study include that it was based on the physiological concentration of melatonin and the sample size was too small. Furthermore, the use of peripheral blood measurement indicators to evaluate the redox level of the inner ear was not accurate enough. In future studies, we will establish animal models to measure melatonin and ROS levels in the inner ear with more accurate data.

We conclude that low plasma melatonin and high ROS are significant in the occurrence and development of ISSNHL. Furthermore, the levels of melatonin and ROS, and vertigo appear to be predictive of the effectiveness of hearing improvement following ISSNHL treatment.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The project was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University [(2019) medical research review no. (100)]. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

JS and LF designed the experiment, analyzed the data, and wrote the article. FO recruited patients and collected blood samples. YX and QL guided the study. ZZ and HJ gave final approval for the version to be published. All authors contributed to the article and approved the submitted version.

This work was supported by the Fund Project of the Education Department of Jiangxi Province (GJJ190028) and the Health Commission Foundation Project of Jiangxi Province (202130198). Central Funds Guiding the Local Science and Technology Development (20221ZDG020066).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.