劉淑云 鄧力強(qiáng),2 楊燁 殷澤登

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·實(shí)驗(yàn)研究·

電針耳穴對(duì)年齡相關(guān)性聽力損失豚鼠聽覺中樞β-catenin表達(dá)的影響△

劉淑云1鄧力強(qiáng)1,2楊燁3殷澤登1

目的 探討電針耳穴對(duì)D-半乳糖所致的年齡相關(guān)性聽力損失豚鼠模型聽覺中樞β-鏈蛋白(β-catenin)表達(dá)的影響。方法 取3月齡豚鼠30只，隨機(jī)分成三組：D-半乳糖模型組、D-半乳糖+電針組和對(duì)照組，每組各10只；18月齡豚鼠10只作為自然老化組。D-半乳糖模型組給予D-半乳糖(300 mg·kg-1·d-1)頸背部皮下注射，每天1次，連續(xù)6周；對(duì)照組給予等量生理鹽水注射相同部位、相同頻次、持續(xù)相同時(shí)間；D-半乳糖+電針組給予相同劑量的D-半乳糖頸背部皮下注射，30 min后給予電針聽宮穴和翳風(fēng)穴治療15分鐘，每日1次，共6周。自然老化組豚鼠常規(guī)飼養(yǎng)。上述實(shí)驗(yàn)結(jié)束后采用蛋白質(zhì)印跡(Western blot)方法檢測(cè)四組豚鼠下丘和聽皮層β-catenin蛋白的表達(dá)變化。結(jié)果 ①與對(duì)照組相比，D-半乳糖模型組和自然老化組豚鼠下丘β-catenin蛋白表達(dá)量下降；與D-半乳糖模型組相比，D-半乳糖+電針組下丘β-catenin蛋白表達(dá)量增加；②D-半乳糖模型和自然老化組豚鼠聽皮層β-catenin蛋白表達(dá)量下降，D-半乳糖+電針組其表達(dá)量增加。結(jié)論 β-catenin蛋白可能通過Wnt/β-catenin信號(hào)通路，調(diào)控細(xì)胞生長(zhǎng)、分化及凋亡，參與下丘和聽皮層的老化過程；電針聽宮穴和翳風(fēng)穴可能通過增加下丘和聽皮層β-catenin蛋白表達(dá)，經(jīng)Wnt/β-catenin信號(hào)通路延緩豚鼠年齡相關(guān)性聽力損失的發(fā)生。

電針； 年齡相關(guān)性聽力損失； 聽覺中樞； β-鏈蛋白

據(jù)統(tǒng)計(jì)，老年性聾(又稱年齡相關(guān)性聽力損失)的發(fā)病率約在30%～60%之間[1]，聽力障礙嚴(yán)重影響老年人的生活質(zhì)量和社會(huì)交往，因此對(duì)老年性聾的機(jī)制及防治研究具有現(xiàn)實(shí)意義。研究發(fā)現(xiàn)Wnt信號(hào)通路參與內(nèi)耳多個(gè)環(huán)節(jié)，包括耳部結(jié)構(gòu)、前庭器官的形成和耳蝸的發(fā)育[2]。Wnt信號(hào)通路能夠誘導(dǎo)出生后小鼠耳蝸感覺前體細(xì)胞增殖[3]，可作為感音神經(jīng)性聽力損失后耳蝸植入引導(dǎo)軸突生長(zhǎng)的候選通路[4]。β-catenin作為經(jīng)典Wnt信號(hào)通路中最關(guān)鍵的信息分子，對(duì)細(xì)胞生長(zhǎng)、分化及凋亡起著調(diào)控作用[5]；Jacques等[6]研究發(fā)現(xiàn)Wnt/β-catenin信號(hào)在耳蝸發(fā)育中具有調(diào)節(jié)毛細(xì)胞增殖和分化的雙重作用；Ren等[7]研究顯示胚鼠神經(jīng)干細(xì)胞移植組的老年鼠聽力閾值降低，達(dá)到改善聽功能的作用。但β-catenin在年齡相關(guān)性聽力損失豚鼠聽覺中樞表達(dá)及電針耳穴對(duì)其表達(dá)是否有影響及作用機(jī)制尚不清楚，因此，本研究擬通過建立年齡相關(guān)性聽力損失豚鼠模型，檢測(cè)其下丘和聽皮層β-catenin蛋白的表達(dá)并觀察電針耳穴對(duì)其表達(dá)的影響，探討β-catenin在年齡相關(guān)性聽力損失發(fā)病中的作用，及電針耳穴對(duì)年齡相關(guān)性聽力損失防治的作用及機(jī)制。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物及分組 選取耳廓反射靈敏、無噪聲暴露及耳毒性藥物使用史的3月齡雜色豚鼠30只，隨機(jī)分為D-半乳糖模型組、D-半乳糖+電針組、對(duì)照組，每組10只；另選取18月齡豚鼠10只作為自然老化組。豚鼠購(gòu)買于西南醫(yī)科大學(xué)(原瀘州醫(yī)學(xué)院)實(shí)驗(yàn)動(dòng)物中心。

1.2 實(shí)驗(yàn)方法

1.2.1 年齡相關(guān)性聽力損失豚鼠模型的建立 D-半乳糖模型組給予D-半乳糖(300 mg·kg-1·d-1)頸背部皮下注射，每天1次，連續(xù)6周；對(duì)照組給予等量生理鹽水注射相同部位、相同頻次、持續(xù)相同時(shí)間；D-半乳糖+電針組給予相同劑量的D-半乳糖頸背部皮下注射，30 min后給予電針聽宮穴和翳風(fēng)穴治療15分鐘，每日1次，共6周。自然老化組豚鼠常規(guī)飼養(yǎng)。

1.2.2 電針聽宮穴和翳風(fēng)穴 D-半乳糖+電針組豚鼠清醒狀態(tài)下，在注射D-半乳糖30 min后，用自制裝置固定，取雙側(cè)聽宮穴和翳風(fēng)穴[8，9]，用HM6805-I型經(jīng)穴治療儀(HM6805-I型，1100801072，四川恒明科技開發(fā)有限公司)刺激，疏密波，頻率14 Hz，強(qiáng)度0.4～0.6 mA，連續(xù)輸出，9次/秒，連續(xù)刺激15 min，每天1次，共6周。刺激強(qiáng)度以豚鼠針刺部位顫動(dòng)、四肢輕輕抖動(dòng)但不掙扎嘶叫為度[10]。

1.2.3 雙側(cè)下丘及聽皮層取材及樣本制備 完成上述實(shí)驗(yàn)(6周)后各組豚鼠麻醉下，斷頭、冰上取雙側(cè)下丘和聽皮層，立即放入-80 ℃冰箱中保存?zhèn)溆?。?80 ℃冰箱中每組取出豚鼠下丘和聽皮層，稱重、混合、剪碎、移入勻漿器，加入裂解液(裂解母液300 μl+TBP0.3 μl+Biolyte1.5 μl)，勻漿15次，置冰上震蕩裂解15 min，4 ℃，12 000 rpm離心40 min，收集上清液即為蛋白質(zhì)提取物；用Bradford法測(cè)定樣品的總蛋白含量。

1.2.4 Western blot法檢測(cè)β-catenin蛋白表達(dá) 采用4%濃縮膠和10%分離膠分離蛋白質(zhì)樣品，濃縮膠電壓80 V，分離膠電壓120 V；采用半干式轉(zhuǎn)膜方法轉(zhuǎn)膜、脫脂牛奶進(jìn)行封閉、 β-catenin抗體和內(nèi)參一抗(β-actin)室溫孵育2小時(shí)或4 ℃過夜，二抗(anti-mouse IgG-HRP)和內(nèi)參二抗(anti-rabbit IgG-HRP)室溫孵育1小時(shí)。底物發(fā)光，柯達(dá)X-OMAT膠片壓片，并用Quantity One軟件分析條帶灰度值；為避免人為因素造成的實(shí)驗(yàn)誤差，結(jié)果采用內(nèi)參(β-actin)對(duì)上樣量加以校正，每組實(shí)驗(yàn)結(jié)果采用目的蛋白灰度值/內(nèi)參灰度值的比值的相對(duì)灰度值進(jìn)行比較。

1.3 統(tǒng)計(jì)學(xué)方法 采用SPSS13.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析，運(yùn)用單因素方差中LSD檢驗(yàn)方差是否齊性，SNK分析組間差異，P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 各組豚鼠下丘β-catenin蛋白表達(dá) 與對(duì)照組相比，D-半乳糖模型組和自然老化組豚鼠下丘β-catenin蛋白表達(dá)降低(P<0.05)；與D-半乳糖模型組相比，D-半乳糖+電針組豚鼠下丘β-catenin蛋白表達(dá)增加(P<0.05)；D-半乳糖模型組和自然老化組之間差異無統(tǒng)計(jì)學(xué)意義(P>0.05)(表1，圖1)。

2.2 各組豚鼠聽皮層β-catenin蛋白表達(dá) 與對(duì)照組相比，D-半乳糖模型組和自然老化組豚鼠聽皮層β-catenin蛋白表達(dá)降低(P<0.05)；與D-半乳糖模型組相比，D-半乳糖+電針組豚鼠聽皮層β-catenin蛋白表達(dá)增加(P<0.05)；D-半乳糖模型組和自然老化組之間差異無統(tǒng)計(jì)學(xué)意義(P>0.05)(表1，圖2)。

表1 各組豚鼠下丘及聽皮層β-catenin蛋白表達(dá)相對(duì)灰度值

注：★與對(duì)照組比較，P<0.05；▲與D-半乳糖模型組比較，P<0.05

圖1 各組豚鼠下丘β-catenin蛋白表達(dá)CG為對(duì)照組，EG為自然老化組，DG為D-半乳糖模型組，DEG為D-半乳糖+電針組

圖2 各組豚鼠聽皮層β-catenin蛋白表達(dá)CG為對(duì)照組，EG為自然老化組，DG為D-半乳糖模型組，DEG為D-半乳糖+電針組

3 討論

3.1 β-catenin在豚鼠年齡相關(guān)性聽力損失發(fā)生和發(fā)展中的作用 β-catenin作為機(jī)體組織重要的細(xì)胞黏附分子和信號(hào)轉(zhuǎn)導(dǎo)蛋白，是經(jīng)典Wnt信號(hào)通路中最關(guān)鍵的信息分子，對(duì)細(xì)胞生長(zhǎng)、分化及凋亡起著調(diào)控作用。研究發(fā)現(xiàn)β-catenin在耳蝸毛細(xì)胞分化和再生中起作用[11，12]，斑馬魚毛細(xì)胞的再生由Wnt/β-catenin信號(hào)通路控制[13]，且H3K9me2抑制可通過下調(diào)Wnt信號(hào)通路來減少毛細(xì)胞損害的斑馬魚毛細(xì)胞再生，結(jié)果表明，H3K9me2下調(diào)通過Wnt /β-catenin 信號(hào)傳導(dǎo)途徑的失活顯著降低新霉素誘導(dǎo)的毛細(xì)胞損傷后的毛細(xì)胞再生[14]。研究發(fā)現(xiàn)在老年大鼠腦中，β-catenin主要分布于室下層、皮層錐體細(xì)胞層、丘腦和海馬等區(qū)域，而且陽(yáng)性細(xì)胞數(shù)量和強(qiáng)度顯著低于成年大鼠[15]，老化小鼠腦室下區(qū)β-catenin蛋白表達(dá)比青年小鼠明顯降低[16]。本課題組前期研究發(fā)現(xiàn)D-半乳糖注射能致豚鼠ABR波III潛伏期延長(zhǎng)，文中結(jié)果顯示D-半乳糖模型組和自然老化組豚鼠下丘和聽皮層β-catenin蛋白表達(dá)明顯低于對(duì)照組，說明D-半乳糖可能通過Wnt/β-catenin信號(hào)通路調(diào)控豚鼠下丘和聽皮層細(xì)胞的再生、分化和凋亡，參與豚鼠聽覺中樞老化過程，從而參與豚鼠年齡相關(guān)性聽力損失的發(fā)生和發(fā)展過程。而老齡化可能會(huì)導(dǎo)致Wnt/β-catenin信號(hào)通路相關(guān)信號(hào)分子表達(dá)下降，增加多巴胺神經(jīng)元易損性[17]。

3.2 β-catenin在電針耳穴防治豚鼠年齡相關(guān)性聽力損失中的作用 針刺耳穴不僅可以通過對(duì)毛細(xì)胞和耳蝸血管紋細(xì)胞線粒體酶的活性保護(hù)，維持細(xì)胞能量代謝，保證細(xì)胞各種功能的活動(dòng)，減少細(xì)胞損傷[18]，而且對(duì)慶大霉素致聾豚鼠耳蝸螺旋神經(jīng)節(jié)細(xì)胞產(chǎn)生保護(hù)作用[19]。Wnt/β-catenin信號(hào)通路的失活能顯著降低斑馬魚毛細(xì)胞的再生[20]，且β-catenin基因突變小鼠表現(xiàn)為下丘畸形和中腦導(dǎo)水管狹窄，引起腦積水[21]；研究發(fā)現(xiàn)激活Wnt信號(hào)通路不僅能增加早期前體感覺細(xì)胞增殖，還能使支持細(xì)胞重新進(jìn)入細(xì)胞周期并分化為毛細(xì)胞，并能調(diào)節(jié)毛細(xì)胞束的方向[22]；Jan等[23]發(fā)現(xiàn) Wnt激動(dòng)劑能刺激鼓膜邊緣細(xì)胞增殖，而Wnt抑制劑抑制鼓膜邊緣細(xì)胞增殖；Lgr5+支持細(xì)胞作為耳蝸毛細(xì)胞的祖細(xì)胞[24]，由β-catenin過度表達(dá)參與新生小鼠毛細(xì)胞的生成[25]；Li等[26]研究證實(shí)Notch抑制通過激活Wnt信號(hào)通路并促進(jìn)Lgr5+祖細(xì)胞有絲分裂增加導(dǎo)致哺乳動(dòng)物耳蝸產(chǎn)生毛細(xì)胞；β-catenin表達(dá)增強(qiáng)不僅可降低活性氧的水平，還能抑制半胱天冬酶介導(dǎo)的細(xì)胞凋亡，從而保護(hù)新霉素導(dǎo)致小鼠耳蝸毛細(xì)胞的損失，成為預(yù)防毛細(xì)胞死亡的新的治療靶點(diǎn)[27]。本課題組前期研究發(fā)現(xiàn)電針耳穴能縮短D-半乳糖致年齡相關(guān)性聽力損失豚鼠ABR波III潛伏期，但具體機(jī)制尚不清楚；本研究發(fā)現(xiàn)D-半乳糖+電針組豚鼠下丘和聽皮層β-catenin蛋白表達(dá)明顯高于D-半乳糖模型組，說明電針耳穴能夠增加下丘和聽皮層β-catenin蛋白表達(dá)，從而可能通過Wnt/β-catenin信號(hào)通路調(diào)節(jié)聽覺中樞細(xì)胞的再生、分化來改善聽力，延緩年齡相關(guān)性聽力損失的發(fā)生。

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(2016-05-11收稿)

(本文編輯 李翠娥)

The Effects of Electroacupuncture at Tinggong and Yifeng on β-catenin Expression in Auditory Center of Guinea Pig with Age-related Hearing Loss

Liu Shuyun*, Deng Liqiang, Yang Ye, Yin Zedeng

(*Department of Otorhinolaryngology of the Affiliated Hospital of Southwest Medical University, Luzhou， 646000, China)

Objective To explore the effects of the application of electroacupuncture at Tinggong and Yifeng to the β-catenin expression in auditory center of guinea pigs with age-related hearing loss (AHL) induced by D-galactose.Methods There were four groups. Thirty 3-month-old guinea pigs were randomly divided into three groups including the D-galactose group (DG,n=10), D-galactose and electroacupuncture group (DEG,n=10), and the control group (CG,n=10). The elderly group (EG) included ten 18-month-old guinea pigs. The guinea pigs in the DG had been subcutaneously injected D-galactose (300 mg·kg-1·d-1) for 6 weeks. The CG received the same dose of physiological saline as D-galactose. Moreover, the guinea pigs in the DEG were subcutaneously injected the same dose of D-galactose, and electroacupunctured at Tinggong and Yifeng for 15 minutes half an hour later. Finally, the guinea pigs in the EG were conventionally raised. The expressions of the β-catenin in the auditory center of the guinea pigs were detected by Western blots.Results There was a significant decrease of β-catenin expression in the inferior colliculus of the DG and the EG (P<0.05, compared with CG) and a significant increase in the DEG (P<0.05, compared with DG). There was a significant decrease of β-catenin expression in the auditory cortex of the DG and the EG (P<0.05, compared with CG) and a significant increase in the DEG (P<0.05, compared with DG).Conclusion Beta-catenin may participate in the aging process induced by D-galactose of the inferior colliculus and auditory cortex by regulating cell growth, differentiation and apoptosis by Wnt/beta-catenin signal pathway. Beta-catenin may play a role in inhibiting the occurrence of AHL by electroacupuncturing at Tinggong and Yifeng by Wnt/beta-catenin signal pathway.

Electroacupuncture; Age-related hearing loss; Auditory center; Beta-catenin

劉淑云，女，四川人，碩士，主要研究方向?yàn)槁犃εc言語(yǔ)疾病的基礎(chǔ)與臨床。

殷澤登(Email: landao0070450@sina.com)

10.3969/j.issn.1006-7299.2016.06.015

時(shí)間：2016-11-2 16:18

R764.43+6

A

1006-7299(2016)06-0593-04

△ 四川省衛(wèi)生廳課題(070248)和(100237)資助

1 西南醫(yī)科大學(xué)附屬醫(yī)院耳鼻咽喉頭頸外科(瀘州 646000)； 2 湖南省郴州市第一人民醫(yī)院兒童耳鼻喉科； 3 西南醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院生物化學(xué)教研室

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