老年性聽損之臨床與基礎研究：肥胖與聽力退化之關聯性

Clinical and basic studies of age-related hearing impairment: relationship of obesity and hearing degeneration

黃俊豪(Juen-Haur Hwang)

指導教授 ： 楊偉勛

共同指導教授 ： 劉殿楨 ； 許權振

國立臺灣大學/醫學院/臨床醫學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.02063

摘要

背景：老年性聽損是複雜性疾病，受到基因與環境因子的影響。以往的研究顯示肥胖相關的併發症，例如：血脂異常、糖尿病、高血壓等，和聽力退化有顯著相關。但是，肥胖本身是否可以直接造成聽力退化，目前並不清楚！另一方面，缺氧、氧化壓力傷害、粒線體功能異常、耳蝸內細胞凋亡等等，被認為是產生老年性聽損的機制，也和肥胖有關。但是，肥胖相關聽力退化的詳細機制，目前也不清楚。
目的：探討肥胖和老年性聽損之關聯性，探討血漿中脂締素(adiponectin)濃度與氧化壓力，與老年性聽損的關聯性，以及探討肥胖相關聽力退化的詳細機制。
假設：腰圍或中央型肥胖是聽力退化的獨立預測因子。血漿中脂締素濃度和聽力閾值呈現顯著負相關。血漿中活氧分子濃度和聽力閾值呈現顯著負相關。高脂飼料引起之肥胖，可以經由增強缺氧、發炎反應，並活化半胱胺酸蛋白酶相關與不相關的細胞凋亡訊息傳遞路徑，造成耳蝸內細胞凋亡。
臨床重要性：若本假說成立，則我們將找到聽力退化全新的獨立預測因子，開闢老年性聽損研究的新領域。預計將對聽力老化的預防與治療，做出些許貢獻。
實驗方法：
第一部份：人類腰圍和聽力閾值之相關性。
以橫斷式研究方法，邀集40歲以上台大健檢中心民眾與大林慈濟醫院志工，進行病史問卷調查、身體檢查、聽力檢查、抽血檢查。納入聽力正常或患有對稱性神經感覺性聽力損傷之成年人，並排除早發型聽力障礙病史或家族史、明顯認知功能障礙、高度噪音曝露病史等等條件，最後有954名納入本研究，分析腰圍與聽力閾值之相關性。其中，聽力閾值方面，本研究以年齡、性別不相關之平均聽力Z值來代表，並將聽力分為低頻區(250與500 Hz)、中頻區(1000與2000 Hz)、高頻區(4000與8000 Hz)。
第二部份：人類血漿中脂締素(adiponectin)濃度和聽力閾值之相關性。
此部份延續第一部份之研究，分析同一批人血漿中脂締素濃度和平均聽力Z值之相關性。
第三部份：人類血漿中活氧分子濃度和聽力閾值之相關性。
此部份延續第一部份之研究，以化學冷光法分析其中302名受試者血漿中活氧分子濃度和平均聽力Z值之相關性。
第四部份：飲食造成肥胖與小鼠聽力退化的因果關係與機制。
採用60隻、4週大、雄性、聽性腦幹反應8000 Hz斷音(tone burst)閾值小於50 dB之ICR/CD1小鼠，隨機分成兩組，對照組餵食一般飼料，飲食造成肥胖組餵食60%脂肪熱量飼料，共餵食16週。在4週大與20週時，分別測量其體重、聽性腦幹反應閾值、血漿生化分析，並在20週時犧牲後，進行耳蝸組織染色分析。
結果：
第一部份：人類腰圍和聽力閾值之相關性。。
本研究納入954名受試者，平均年齡是56.5歲(標準差為8.7)，女性有535名、男性有419名。在校正冠心症、糖尿病、高血壓、高血脂症、慢性腎病、抽煙、喝酒、噪音曝露、甚至體質指數等因子後，腰圍大小仍然與中頻區與高頻區平均聽力Z值呈現顯著正相關。換言之，腰圍越大，中頻區與高頻區聽力越差。
第二部份：人類血漿中脂締素(adiponectin)濃度和聽力閾值之相關性。
此部份延續第一部份之研究，血漿中脂締素(adiponectin)的濃度，平均為10.2 μg/ml (標準差為5.0)。在校正冠心症、糖尿病、高血壓、高血脂症、慢性腎病、抽煙、喝酒、噪音曝露、甚至腰圍等因子後，人類血漿中脂締素濃度仍然與高頻區平均聽力Z值呈現顯著負相關。換言之，人類血漿中脂締素濃度越高，聽力越好。另外，在校正年齡、性別、腰圍大小、慢性疾病、抽菸、喝酒、噪音曝露、其他因子後，人類血漿中脂締素濃度每升高一個單位(μg/ml)，發生高頻區老年性聽損(平均純音聽力閾值大於25 dB)的機會就會減為0.965倍。
第三部份：人類血漿中活氧分子濃度和聽力閾值之相關性。
在校正中央型肥胖、冠心症、高血壓、糖尿病、高血脂病、慢性腎病、抽菸、喝酒、噪音曝露等因子後，Luminol相關化學冷光訊號(主要反應過氧化氫(H2O2)、次氯酸(HOCl/OCl–)、氫氧基(•OH)等強度)，和低頻區、中頻區、高頻區平均聽力Z值，分別呈現顯著正相關。Lucigenin相關化學冷光訊號(主要反應過氧陰離子(O2•–)強度)，在校正各項因子後，和低頻區平均聽力Z值呈現顯著正相關。
第四部份：飲食造成肥胖與小鼠聽力退化的因果關係與機制。
ICR/CD1小鼠在餵食16週(4週齡至20週齡)高脂飼料後，飲食引起肥胖組之平均體重、空腹血漿中三酸甘油脂濃度、腸系膜脂肪重量較對照組明顯增加。聽性腦幹反應之閾值，在16000 Hz與32000 Hz處也較對照組顯著提高。不過，空腹之血糖與血漿高密度膽固醇之濃度，在兩組間無顯著差異。
在小鼠內耳切片H&E染色方面，飲食引起肥胖組耳蝸基底迴血管紋處血管內徑較對照組明顯變小，且血管壁厚度與血管半徑比值顯著提高。飲食引起肥胖組耳蝸基底迴處之螺旋神經節與螺旋韌帶纖維細胞密度較對照組明顯減少。但是，耳蝸柯氏體之內外毛細胞退化程度，在兩組間無顯著差異。
在小鼠內耳切片免疫組織染色方面，在耳蝸基底迴處之螺旋神經節與螺旋韌帶兩處，缺氧引發因子(hypoxia-induced factor-1α,HIF-1α)、腫瘤壞死因子alpha(tumor necrosis factor alpha, TNF-α)、核因子kappa B (nuclear factor-kappa B, NF-κB)、半胱胺酸蛋白酶第三型(caspase 3)、聚腺嘌呤二磷酸核醣具核酶第一型(poly(adenosine diphosphate-ribose) polymerase-1, PARP-1)、引發凋亡因子(apoptosis inducing factor, AIF)之染色強度，飲食引起肥胖組都比對照組顯著增加。
結論：人類腰圍或中央型肥胖是老年性聽損全新的獨立危險因子，高頻區的聽力損傷和腰圍大小有顯著正相關。人類血漿中脂締素濃度與高頻區損傷呈現顯著負相關，脂締素對於高頻區的聽力可能有保護作用。血漿活氧化物濃度和人類老年性聽損嚴重程度呈現顯著正相關。不同種類活氧化物可能對於不同頻率的聽覺功能有不同影響。飲食引起之肥胖會加速CD/1小鼠聽力退化，最明顯的組織學發現為耳蝸血管紋血管變窄、增加螺旋神經節與螺旋韌帶之發炎反應與細胞凋亡。此外，高脂飼料會增強活化半胱胺酸蛋白酶相關與不相關的細胞凋亡訊息傳遞路徑，增加耳蝸內螺旋神經節與螺旋韌帶纖維細胞凋亡。

關鍵字

老年性聽損 ； 腰圍 ； 中央型肥胖 ； 血漿 ； 脂締素 ； 活氧分子 ； 缺氧 ； 發炎 ； 細胞凋亡路徑

並列摘要

Background：Age-related hearing impairment (ARHI) is a complex disease, and could be affected by genetic and environmental factors. Previous studies showed that
obesity-related co-morbidities, such as: dyslipidemia, diabetes mellitus, and hypertension, were associated with ARHI. But, it is still unclear whether obesity per sec could lead to ARHI. On the other hand, hypoxia, oxidative stress, mitochondrial dysfunction, cochlear cell apoptosis, etc, were supposed to be the mechanisms of ARHI and were also associated with obesity. But, the detailed mechanisms of obesity-related hearing degeneration are still unclear.
Purpose：To investigate the relationship of obesity, plasma adiponectin levels, oxidative stress and ARHI, and to investigate the detailed mechanisms of obesity-related hearing degeneration.
Hypothesis：Waist circumference (WC) or central obesity was a novel independent risk factor for ARHI. Plasma adiponectin concentration might be negatively associated with hearing thresholds in humans. Plasma reactive oxygen species concentrations were positively associated with hearing thresholds in humans. Obesity might exacerbate hearing degeneration via enhancing hypoxia, inflammation, caspase-dependent and –independent apoptosis signaling pathways.
Clinical significance: If the hypotheses were true, we will find novel risk factors for ARHI, and we also offer a brand new avenue for the research in ARHI. We expect that our studies could make a contribution on the prevention and treatment for hearing degeneration.
Methods:
Part one: Association of WC and hearing thresholds in humans.
By cross-sectional design, we invited subjects, who were older than 40 years old, from the Health department of National Taiwan University Hospital and volunteer helpers from Buddhist Dalin Tzu Chi General Hospital. All eligible subjects received medical questionnaire, physical examination, audiometry, and blood biochemistric study. Nine hundred and fifty-four subjects with normal or symmetric sensorineural hearing impairment, but without personal or family histories of early-onset hearing loss, obvious cognitive dysfunction, high occupational noise exposure, were included in this study. Association of WC and hearing thresholds were analyzed. The hearing thresholds were presented with age- and sex-independent Z-scores of low (250 and 500 Hz), middle (1000 Hz and 2000 Hz) and high (4000 Hz and 8000 Hz) frequencies, which were transformed from individual pure tone thresholds.
Part two: Association of plasma adiponectin concentration and hearing thresholds in humans.
This part was a subsequent study of Part one. The association between plasma adiponectin concentration and hearing thresholds was analyzed in the same study population of Part one.
Part three: Association of plasma reactive oxygen species (ROS) concentration and hearing thresholds in humans.
This part was also a subsequent study of Part one. The association between plasma ROS concentrations, which were detected by chemiluminescence method, and hearing thresholds was analyzed in 302 subjects within the study population of Part one.
Part four: Effects and mechanisms of diet-induced obesity on hearing degeneration.
Sixty 4-weeks-old male CD/1 mice with auditory brainstem response (ABR) thresholds lower than 50 decibel sound pressure level (dB SPL) by 8000 Hz tone burst were randomly and equally divided into two groups. Diet-induced obesity (DIO) group fed with high fat diet and control group fed with normal diet for 16 weeks. Morphometry, biochemistry, ABR thresholds, omental fat weight and histopathology of the cochlea were compared at the end of study.
Results：
Part one: Association of WC and hearing thresholds in humans.
There were 954 subjects (535 were female; 419 were male) in this study. The mean age of all subjects were 56.5 years old (standard deviation was 8.7). After adjusting for coronary artery disease (CAD), diabetes mellitus (DM), hypertension (HTN), dyslipidemia, chronic kidney disease (CKD), smoking, drinking, noise exposure and even body mass index (BMI), WC still showed significant positive association with Z scores of middle or high frequencies. In other words, the larger the WC is, the worser the hearing of middle and high frequencies is.
Part two: Association of plasma adiponectin concentration and hearing thresholds in humans.
The general characteristics of subjects were the same as those in Part one. The mean concentration of plasma adiponectin was 10.2 μg/ml (standard deviation was 5.0). After adjusting for CAD, DM, HTN, dyslipidemia, CKD, smoking, drinking, noise exposure, and even WC, the plasma adiponectin concentration still showed significant negative association with Z scores of high frequencies. In other words, the larger the plasma adiponectin concentration is, the better the hearing of high frequencies is. In addition, the risk of hearing impairment for high frequencies (pure tone average≧25 dB hearing level) decreased by 0.965-fold for every 1 unit increase in adiponectin concentration, after adjusting for age, gender, WC, systemic diseases, and smoking, alcohol consumption, and noise exposure.
Part three: Association of plasma ROS concentration and hearing thresholds in humans.
Luminol-dependent chemiluminescence signals, which reflect hydrogen peroxide (H2O2), hypochlorite (HOCl/OCl–), and hydroxyl radicals (•OH) levels, showed significant positive association with Z-low, Z-middle, or Z-high after adjusting central obesity, systemic diseases, habits, and noise exposure. Lucigenin-dependent chemiluminescence signals, which mainly reflect superoxide anion (O2•–) level, showed significant positive association with Z-low, but not with Z-middle or Z-high after adjusting other variables.
Part four: Effects and mechanisms of diet-induced obesity on hearing degeneration.
Compared to control group, the diet-induced obesity (DIO) group had significant higher body weight, fasting plasma triglyceride concentration, and omental fat weight after ICR/CD1 mice were fed with high fat diet for 16 weeks. The auditory brainstem response thresholds at 16000 Hz and 32000 Hz were significantly higher in the DIO group than the control group. But, the fasting plasma sugar and high-density lipoprotein cholesterol concentration were not significantly different in both groups.
Cohlear H&E stain showed that DIO group had thinner diameter with higher vessel wall to radius ratio in the stria vascularis of cochlear basal turn. The cell density of spiral ganglion or spiral ligament was significantly lower in the DIO group than in the control group. But, the degeneration of Organ of Corti was similar in both groups.
Cohlear immunohistochemistric stain showed that hypoxia-induced factor-1α, tumor necrosis factor α, nuclear factor-kappa B, caspase 3, poly(adenosine diphosphate-ribose) polymerase-1, apoptosis inducing factor stains were significantly denser in the DIO group than in the control group.
Conlcusions: Waist circumference or central obesity was a novel independent risk factor for age-related hearing impairment. Plasma adiponectin concentration showed negative association with hearing of of high frequencies. Adiponectin might play a protective role on hearing, especially for high frequencies. Plasma ROS levels were associated with severity of age-related hearing impairment in humans. Various ROS might have differential impacts on auditory dysfunctions. Diet-induced obesity could exacerbate the hearing degeneration in CD/1 mice. The most notable histological findings were narrower blood vessels in the stria vascularis, increased inflammatory responses, increased cell loss of spiral ganglion and spiral ligament. And, the high fat diet could increase cell apoptosis of spiral ganglion and spiral ligament via activation of both caspase-dependent and caspase-independent apoptosis signaling pathways.

並列關鍵字

age-related hearing impairment ； waist circumference ； central obesity ； plasma ； adiponectin ； reactive oxygen species ； hypoxia ； inflammation ； apoptosis signaling pathways

參考文獻

Cueva RA . Auditory brainstem response versus magnetic resonance imaging for the evaluation of asymmetric sensorineural hearing loss . Laryngoscope 2004 Oct ; 114 : 1686 – 92 .

Pan WH, Yeh WT, Weng LC. Epidemiology of metabolic syndrome in Asia. Asia Pac J Clin Nutr. 2008; 17 Suppl 1: 37-42.

Karnchanakas T, Tantanavat A, Sinsakontavat J. Inner ear problems of Thai priest at Priest Hospital. J Med Assoc Thai. 2008; 91: S63-7.

Erdem T, Ozturan O, Miman MC, Ozturk C, Karatas E. Exploration of the early auditory effects of hyperlipoproteinemia and diabetes mellitus using otoacoustic emissions. Eur Arch Otorhinolaryngol. 2003 Feb; 260: 62-6.

Tabata M, Kadomatsu T, Fukuhara S, Miyata K, Ito Y, Endo M, Urano T, Zhu HJ, Tsukano H, Tazume H, Kaikita K, Miyashita K, Iwawaki T, Shimabukuro M, Sakaguchi K, Ito T, Nakagata N, Yamada T, Katagiri H, Kasuga M, Ando Y, Ogawa H, Mochizuki N, Itoh H, Suda T, Oike Y. Angiopoietin-like protein 2 promotes chronic adipose tissue inflammation and obesity-related systemic insulin resistance. Cell Metab. 2009 Sep; 10(3): 178-88.