Mechanism Of HDAC2 On Glucocorticoid Resistant Formation In Sudden Sensorineural Hearing Loss Patients

Objective: Glucocorticoid (GC) is an efficient treatment for patients with sudden sensorineural hearing loss (SSNHL). However, minority of the patients show insensitive to GC. Authors investigated GC sensitivity by GC proliferation inhibition (GCPI) of PBMCs in vitro and examined the correlation between PBMCs proliferation inhibition rate and PTA improvement after the treatment, evaluated expression of histone deacetylase 2 (HDAC2) in peripheral blood mononuclear cells (PBMCs) from patients with refractory SSNHL and identified the relationship between the level of HDAC2 and glucocorticoid insensitivity.Methods: Part 1:GC proliferation inhibition of PBMCs:50 patients received intratympanic methylprednisolone perfusion (IMP) from july 2013 to January 2015 in Nanjing Drum Tower Hospital, Medical School of Nanjing University, China, were selected in this study. Before IMP peripheral venous blood were collected and used to isolate PBMC for GCPI. PBMCs were divided into four groups: A: 1640 Cell culture medium, B: 1640 + LPS (1μg/ml), C:1640+LPS+dexamethasone (DEX) (10"6 M), D: blank control. PBMCs were cultured in 5% CO2 incubator for 24h at 37℃, the proliferation of cells was observed by MTT assay, the absorbance in the 490nm wavelength can reflect the cell proliferation inhibition. Hearing improvement was defined as a decrease of PTA (0.25 - 8 kHz). PBMC proliferation inhibition rate, the time period from onset to IMP and the hearing improvement after perfusion were compared with linear correlation degree, The patients were divided into GC sensitive group (PTA decrease≥ 15dB) (n= 28) and GC insensitive group (PTA decrease< 15dB) (n= 22), PBMC proliferation inhibition rate and the time period from onset to IMP were compared between the two groups. Multivariable analysis was used to compare the patients’gender, affected ear side, audiometric curve and so on between the two groups. PBMC proliferation inhibitions were observed after culturing 24 hours.Part 2:the expression and activity of HDAC2 in the PBMCs:PBMCs were collected 42 SSNHL patients who failed to a conventional treatment before and after 10 day intratympanic methylprednisolone perfusion (IMP) and systemic Ginkgo biloba extract treatment. The SSNHL patients were assigned into two groups (GC sensitive and insensitive groups) according to their hearing recovery after IMP. Real time PCR and HDAC2 Assay Kit were used to detect the expression level of HDAC2 mRNA and HDAC2 activity in PBMCs. PBMCs collected from17 volunteers (patients with deviated nasal septum) were used as normal controls.Results:Part 1:GC proliferation inhibition of PBMCs:The results showed that there was a great difference in the inhibition rates among all patients. The rate and hearing improvement of each patient was compared with linear correlation degree, there was a significantly positive correlation between PBMC proliferation inhibition rate and the hearing improvement (R = 0.688). However time period from onset to IMP was minus correlation to hearing improvement(R = -0.372). The average PBMC proliferation inhibition rate in GC sensitive group was significantly higher than GC insensitive group. Multivariable analysis indicated that factors like gender, affected ear side, audiometric curve and so on may not affect the hearing improvement.Part 2:the expression and activity of HDAC2 in the PBMCs:Compared to the control group, the level of HDAC2 activity was significantly depressed while the HDAC2 mRNA expressing was much higher in all refractory SSNHL patients before IMP. GC treatment and antioxidant significantly increased the expression of HDAC2 mRNA and HDAC2 activity in the GC sensitive group while no change was observed in the GC insensitive group.Conclusions:Time period from onset to IMP was minus correlation to hearing improvement. However there was a more significantly positive correlation between PBMC proliferation inhibition rate and the hearing improvement. PBMC proliferation inhibition rate in GC sensitive group was much higher than it in GC insensitive group. PBMC IVPGCIT can predict the sensitivity to GC of SSNHL patients, and it can be used to guide clinical individualized treatment of SSNHL. Reduced HDAC2 activity may be one of the mechanisms of corticosteroid in-sensitivity in SSNHL. Glucocorticoids plus antioxidant treatment can increase the expression of HDAC2 mRNA and activity in GC sensitive patients. HDAC2 activity can be regulated through post-translational modifications.