*4.3. Ethical Permission*

The study was approved by the local Ethics Committee (prot. N. 76/2018/PO, 16 April 2018) and informed consent was obtained from all patients.

## *4.4. Patients*

We enrolled 40 patients (22 males and 18 females, with an average age of 49.5 +/− 14.6 years; range 29–60 years) with MD according to the diagnostic scale of the Committee on Hearing and Equilibrium of the American Academy of Otolaryngology—Head and Neck Surgery published in 1995 for MD [13,58] (two or more definitive spontaneous episodes of vertigo 20 min or longer, audiometrically documented hearing loss on at least one occasion, tinnitus or aural fullness in the treated ear). Patients were divided into two groups, A and B. Group A consisted of 22 patients suffering from cochlear sensorineural hearing loss (SNHL) that was been subjected to treatment with biomass preparation from *Coriolus versicolor* mushroom (MRLs), administered orally in tablets of 500 mg (3 tablets every 12 h, morning and evening, for 2 consecutive months), while Group B, formed of 18 patients, also suffering from cochlear SNHL, was not subjected to any treatment. Constituted exclusion criteria: (i) older than 60 years; (ii) presence of cardiovascular diseases; (iii) presence of metabolic disorders and/or parts; (iv) the presence of external ear pathologies and/or medium; (v) presence of alterations of state-acoustic nerve; (vi) prior learning and/or recent treatment with antioxidant drugs or otherwise active in the compartment cochlear.

All patients, after targeted anamnestic investigation, underwent the T0 initial phase, where the Profile of Mood States (POMS) questionnaire was administered, to assess the emotional and degree of psychological stress status, indexed on the basis of specific elements, such as: Tension–Anxiety (TA), Depression–Discouragement (D), Anger–Hostility (AH), Vigor–Activity (V), Fatigue (F), Confusion–Loss (C), in relation to the impairment caused in each subject from hearing impairment. The POMS original scale contains 65 self-report items using the 5-point Likert Scale. Participants can choose from 0 (not at all) to 4 (extremely). In addition, all subjects were given a tinnitus questionnaire consisting of 40 multiple choice questions to define the impact of symptoms on the patient life. In the groups the grade of severity for each patient was established on the basis of the vertigo attack frequency over a year (from 2 to 8 crisis), the intensity and the duration of symptoms (from a few days, to some weeks, to a month in the most severe case). In addition, the hearing loss degree was assessed instrumentally, allowing staging of the disease in MD patients.

Enrolled patients were also examined to define the qualitative and quantitative characteristics of auditory function: (a) examination ENT; (b) test tone audiometry; (c) speech audiometry; (d) impedenzometry examination. Such instrumental examinations were aimed at defining not only the extent of hearing, but also the location of the SNHL, to define it whether cochlear or retrocochlear. Each patient, either in Group A or Group B, was subjected to blood and urine sampling for biochemical analysis in plasma and lymphocytes, and in urines of specific markers of cellular oxidative stress, lipid and protein oxidative metabolism, cellular stress response (vitagenes), glutathione status (reduced glutathione (GSH), oxidized glutathione (GSSG), and GSH/GSSG ratio and lipoxin A4. Phase T1 in Group A, the mushroom-treated group of patients, was accomplished by oral administration of MRL *Coriolus versicolor* biomass compound for 2 consecutive months, to assess its neuroprotective, anti-neuroinflammatory potential, and thus test the possible protection against the cellular degeneration in general and, particularly, in the inner ear. At 2 months from the beginning of treatment (T1 phase) we evaluated in all patients the degree of evolutive trend of auditory function as well as cellular oxidative stresses, redox status, cellular stress response, and Lipoxin A4, in the blood. The correlative analysis, aimed at highlighting the antioxidant properties of the compound administered and its effects at the cellular level as well as at the clinical level, audiological function, will define a neurobiological clinical model to assess the effectiveness of pharmacological compounds in counteracting oxidative stress and neuroinflammatory damage associated with MD.

#### *4.5. Sampling*

Blood (6 mL) was collected after an overnight fast by venopuncture from an antecubital vein into tubes containing ethylenediamine tetraacetic acid (EDTA) as anticoagulant. Immediately after sampling, two blood aliquots were separated: first 2 mL were centrifuged at 10.000× *g* for 1 min at 4 ◦C to separate plasma from red blood cells; the remaining aliquot (4 mL) was used for lymphocytes purification. All samples were stored at −80 ◦C until analysis.
