*2.4. Lipidomics Analysis*

Oxidation of polyunsaturated fatty acid arachidonic, eicosapentaenoic, docosahexaenoic, linoleic, and dihomo-γ-linolenic generate bioactive lipids. The development of mass spectrometry platforms enabling quantification of diverse lipid species in human urine is of crucial importance to understand metabolic redox homeostasis in normal as well as pathophysiological conditions. Here we demonstrate clearly how administration of *Coriolus* to MD patients increases significantly the powerful anti-inflammatory eicosanoid LXA4 in plasma and lymphocytes as compared to untreated MD patients (Figure 8a,b). The same results were observed in urine, where a large increase in LXA4 was measured after *Coriolus* supplementation (Figure 8c). Consistently, analysis of urine levels of pro-inflammatory eicosanoids 11-dehydro TXB2, isoprostane PGF2α, and isoprostane iPF2α-VI showed the opposite results with significantly higher levels of these bioactive lipids in MD subjects than the levels found in *Coriolus* administered MD patients (Figure 9a–c).

**Figure 8.** Lipidomic analysis of bioactive lipids. Biolipids are synthesized by oxidation of polyunsaturated fatty acids, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, and dihomo-γ-linolenic acid. The development of enabling mass spectrometry platforms for the quantification of diverse lipid species in human urine is of paramount importance for understanding metabolic redox homeostasis in normal and pathophysiological conditions. Anti-inflammatory eicosanoid LXA4 were measured in plasma, lymphocytes (**a**,**b**) and in urine (**c**), as compared to untreated MD patients.

**Figure 9.** *Cont*.

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**Figure 9.** Lipidomic analysis of bioactive lipids. Analysis of urinay pro-inflammatory eicosanoids, 11-dehydro TXB2, isoprostane PGF2α, isoprostane iPF2α-VI, showing opposite results with significant higher levels of these bioactive lipids in MD subjects than the levels found in *Coriolus* administered MD patients are reported in (**a**–**c**).

Consistent with other findings showing that oxidative stress and altered thiol status in degenerating brain diseases correlates with systemic redox imbalance and oxidative stress, as in AD [31–34], the content of total GSH, reduced and oxidized glutathione and the GSH/GSSG ratio, was determined in the plasma of MD patients as a measure of the antioxidant status and compared with the levels of *Coriolus*-treated MD group (Table 4). We report the plasma from MD patients contained significantly lower levels of GSH as compared to *Coriolus*-supplemented patients, which paralleled to corresponding significantly higher GSSG levels (*p* < 0.05) (Table 4). These changes resulted in a plasma GSH/GSSG ratio which was significantly higher in the group of MD plus *Coriolus* subjects then the ratio found in the MD group alone (Table 4).


**Table 4.** Plasma and lymphocyte content of total, reduced (GSH) and oxidized (GSSG) glutathione in control and MD patients treated with Coriolus.

**\*** Significantly different from control (*p* < 0.05). **\*\*** Significantly different from MD alone (*p* < 0.05).

#### **3. Discussion**

MD is a chronic illness derived from combined neurodegenerative events occurring at level of spiral ganglion as well as hair cells of the inner ear, associated with a negative impact on the quality of life of individuals, presenting various symptoms, such as temporary hearing loss, dizziness, and tinnitus [7].

After its initial description by Prosper Meniere more than 150 years ago, the disease named after him is still at the center of scientific debate [1]. MD is a hydropic ear pathology, where episodic vertigo, tinnitus, and fluctuating hearing loss coexist with endolymphatic hydrops. [3]. Recent evidence indicates the involvement of oxidative stress in the development of endolymphatic hydrops associated with neuronal ganglion damage with apoptotic neuronal cell death as a prominent factor contributing to SNHL found in the later stages of MD [2]. Thus, it is conceivable that MD, as a systemic oxidant disorder [5], can be also considered, owing to its demonstrated neurodegenerative nature of the neuronal cochlear ganglion component, involved in its pathogenesis, a pursuable investigative model of neurodegeneration. Consistent with this possibility, the present study was undertaken to explore the hypothesis that changes in the redox status of glutathione, stress-responsive vitagenes, and lipidomics, the major determinants in the disruption of redox homeostasis affecting spiral ganglion neurons, may be positively impacted by nutritional intervention with *Coriolus*-MRL biomass supplementation.

Mushrooms have been present in traditional medicine for thousands of years, and are reportedly endowed with immunomodulatory actions, associated with antioxidant, anticancer, antiviral, bacteriostatic, and hepatoprotective properties [43]. Mushroom-derived therapeutics, mainly polysaccharopeptides isolated from *Coriolus versicolor*, are well characterized and commercially available. Here we tested the hypothesis that neurotoxicity is an important causative factor involved in MD pathogenesis, which can be evaluated by measuring markers of oxidative stress and cellular stress response proteins in the peripheral blood of patients with MD. We evaluated in the present study systemic oxidative stress and cellular stress response in 40 patients suffering from MD in the absence and in the presence of treatment with mushroom biomass preparation from *Coriolus*. Systemic oxidative stress was estimated in plasma and urines of patients with MD or MD plus *Coriolus*, by measuring protein carbonyls, HNE, and ultraweak luminescence, as well as active biolipids such as lipoxin A4 and F2- isoprostanes, whereas in the lymphocyte heat shock proteins (HSP) heme oxygenase-1 (HO-1), Hsp70 and thioredoxin (Trx) levels were measured to evaluate the systemic cellular stress response. Increased levels of DNPH, HNE, ultraweak luminescence, and F2-isoprostanes were found in all the samples from MD patients with respect to the MD plus *Coriolus*-treated group. This was paralleled by a significant induction of lymphocyte HO-1, Hsp70, TrxR-1 as well as Sirtuin-1 and by a significant increase in the plasma ratio-reduced glutathione GSH) vs. oxidized glutathione (GSSG).

It is suggested that genetic factors may contribute partly to the etiologies of MD, as some associations have been reported for polymorphisms related to gene coding for protein involved in inflammation, circulation, and blood vessels, such as interleukin 1A (–889C/T), interleukin 6–572C/G), protein kinase C beta type (1425G/A), matrix metalloproteinase-1 (–1607G/2G), methylenetetrahydrofolate reductase (MTHFR) (C677T), prothrombin (G20210A), and complement factor H [44–51], and genes involved in free-radical processes. Although the initial causative factors triggering the disease have not been clarified, various genes and variants have been confirmed to be related to MD, which also suggests a specific family of genetic predisposition and implies genetic factors as key players in the initiation and progression of MD [52]. Consistent with this scenario, inflammation and oxidative stress-induced endolymphatic hydrops have been identified as a secondary pathogenesis of the disease [53]. Thus, MD etiology and pathogenesis appears to be an aberrant response of the adaptive or innate immune system, ultimately mediated by pro-inflammatory and oxidative processes underlying its physiopathological determinism [54]. Several mechanisms are involved in the development of immune-mediated inner-ear pathology, including (a) similarity with potentially harmful component of virus or bacteria, such as cross-reactive epitope inducing inner-ear damage; and (b) generation of pro-inflammatory Interleukin 1 β (IL-1B) or Tumor necrosis factor α (TNF) cytokines and transcriptional nuclear factor kB (NF-kB) [55]. Toll-like receptor coding genes, including TLR3, TLR7, TLR8, and TLR10, are widely reported to contribute to the disease, being directly related to the initiation and progression of MD, thus implying a specific role for the immune system during the pathological processes [56]. This is confirmed by recent findings highlighting the relationships between increased serum levels of IL6 and IL1 with vertigo, a specific complication of MD.

To survive different types of injuries and adapt to environmental changes, neuronal cells have evolved networks of responses capable of detecting and controlling different forms of stress [26–62]. As such, integrated survival mechanisms exist in the brain based on the activity of redox-dependent genes, called vitagenes, capable of sensing stress and including HSP (Hsps), thioredoxin, γ-GC ligase, and sirtuin family proteins, which together with bioactive lipids represent the last step in the "omic" cascade starting from genome, through transcriptome, proteome, and finally to metabolome. Lipid mediators as signaling factors play a fundamental role in the initiation, amplification, and resolution of inflammation [33,34]. Thus, use of urine sample for lipidomic analysis enables reproducible quantification of several lipid metabolites generated by lipoxygenase, cyclooxygenase, and cytochrome P450 activities, such as octadecanoids, eicosanoids, and docosanoids. Lipidomic analysis of urine reveals quantitative data that reflects the alterations in in eicosanoids levels seen in MD patients as compared to normal controls. Lipoxin A4, in particular, is a metabolic product of arachidonic acid, acting as an endogenous "breaking signal" towards inflammatory processes, actively operating in the detection and control of diverse forms of stress in the brain. Owing to its potent anti-inflammatory properties LXA4 positively influences the outcome in many inflammatory disorders, such as nephritis, periodontitis, arthritis, and inflammatory bowel disease [63,64]. Chronic inflammation sustains the progression of neurodegenerative pathologies, including AD and Parkinson's disease, but also in specific neuronal districts, as in the cochleovestibular apparatus and the spiral ganglion neural cells. Thus, identification of mechanisms capable of favorably impacting the pro-inflammatory environment generated in the MD pathology represents an area of active investigation. Consistent with this notion, the activation of the LXA4 pathway could therefore serve as a potential therapeutic target to treat MD-associated inflammation and cochleovestibular dysfunction. As LXA4 action is mediated by LXA4 receptor (ALX), a formyl-peptide receptor-like 1 (FPRL1) present on cellular membrane [33], the discovery of agents with the potential of increasing Lipoxin A4 (LXA4), and consequently of reducing inflammatory-mediated endolymphatic hydrops, can be relevant to therapeutics of this disease. Eicosanoid lipoxin A4 (LXA4) decreases toxic compounds such as ROS, inhibits recruitment of activated neutrophils and blocks accumulation of pro-inflammatory cytokines, thereby promoting resolution of inflammation [65].

Our results obtained with a nutritional approach based on a *Coriolus versicolor* biomass supplementation are relevant to innovative therapeutic anti-inflammatory strategies aimed to minimize consequences associated with neurodegeneration and oxidative stress of cochleovestibular system pathologies including not only MD but also sudden sensorineural hear loss (SSNHL) where it has recently demonstrated a critical role played by NLRP3 inflammasome [41]. NLRP3 is a sensor of the intracellular innate immune response expressed in immune cells, including monocytes and macrophages. Activation of the NLRP3 inflammasome results in augmentation of IL-1β secretion and cochlear autoinflammation. [66,67].

Due to different biological routes of actions, ranging from anticancer, antiviral, bacteriostatic, and regulation of of immune function, as well as antioxidant and protectant of hepatocytes [29,68], relevant to the inflammatory disease pathogenesis, mushrooms in the past have been diffusely applied for therapeutic use in traditional medicine [27,28]. It has been shown, in fact, that cytokine response triggered by activated immune cells occurs after stimulation with immunostimulatory molecules derived from mushroom preparations, which are mainly β-glucans [30,57,69–71]. Despite this, however, the active ingredients are not fully characterized, which makes mushroom extracts very difficult to reconcile with current pharmaceutical practices involving highly purified compounds and, therefore, difficult to patent, as they are complex mixtures of molecules of unknown concentrations to be administered for therapeutic purposes. In addition, mushroom-derived polysaccharides are complex molecules that cannot be synthesized, as the mass production of these compounds would require timely and costly extraction processes. Consequently, most research efforts have focused on low molecular weight compounds, such as cordycepin [72], which is a cytotoxic nucleoside analog inhibitor of cell proliferation. However, polysaccharopeptides isolated from *Coriolus versicolor* are well characterized and their commercial diffusion well established. In addition to its medical applications, *Coriolus versicolor* is widely used to degrade organic pollutants such as pentachlorophenol (PCP) [73]. Notably, as previously mentioned, several studies have demonstrated significant ultrastructural reductions in dendritic innervation densities, at level of cochlear ganglion neurons, pointing the possibility that neurotoxicity plays an important role in the pathology of MD [6]. Interestingly, a recent study in mice has found that *Coriolus versicolor* biomass promotes significant increases in dendritic length and branching and total dendritic volume of immature neurons, suggesting a positive effect of oral *Coriolus versicolor* administration on hippocampal neurogenic reserve [74]. Taking all this into account and given the inflammatory pathogenesis of MD degenerative damage, our findings of reduction in oxidative stress and inflammatory mediators associated with increased anti-inflammatory metabolites in mushroom-treated patients has innovative therapeutic potential.

Moreover, increasing evidence suggests that alteration of redox status, overloading of peroxidative product hydroxynonenals (HNE) or protein carbonyls can severely alter redox homeostasis [1]. Thus, the ensuing oxidative stress is a primary causative factor underlying endolymphatic hydrops pathogenesis, associated with cellular degenerative damage and apoptotic cell death affecting vulnerable cells of cochleovestibular apparatus, and thus contributing to the SNHL and vestibular dysfunction found in later stages of MD.

Moreover, it is known that normal auditory function depends on maintenance of the unique ion composition in the endolymph. Hence, reduction of microvascular alterations due to decreased oxidative stress in the inner ear after mushroom treatment has relevant implications [34]. Our data on the modulation of the stress-responsive protein involved in stress tolerance and cell survival are relevant as a potential target of mushrooms therapeutics and nutritional redox approaches, as the ability of neurons to cope with stressful conditions relies upon the capability to activate stress-responsive pro-survival pathways that normally function at a very low level and that result generally in increased synthesis of antioxidant and anti-apoptotic molecules. Among the cellular pathways conferring protection against oxidative stress, a key role is played by vitagenes, which include HSP (Hsps) Hsp70, heme oxygenase-1, and small Hsps, together with thioredoxin, enzymes of Meister cycle for the synthesis of glutathione and sirtuins [10,24]. Given the broad cytoprotective properties of the heat shock response there is now emerging interest in developing pharmacological agents able to potentiate

neuroprotective stress responses [26]. When appropriately activated, cellular stress response can restore redox equilibrium and neuronal homeostasis.
