卡馬西平對健康及癲雄性大鼠生殖系統(tǒng)相關(guān)指標(biāo)的影響

劉緒宏,張 輝,譚 梅,許笑天,伍春華

劉緒宏,張 輝,譚 梅,許笑天,伍春華

目的 探討卡馬西平單藥以及癲與卡馬西平共同作用下對雄性大鼠生殖系統(tǒng)相關(guān)指標(biāo)的影響，為癲男性患者臨床用藥提供基礎(chǔ)研究數(shù)據(jù)。方法 運(yùn)用氯化鋰-匹羅卡品建立Wistar雄性大鼠癲模型。選擇健康清潔的Wistar雄性大鼠設(shè)造模組、非造模組及對照組，再將造模組、非造模組隨機(jī)分為三個(gè)亞組，分別給予不同濃度卡馬西平灌胃60 d后，所有入組大鼠取血樣、睪丸、附睪、精子標(biāo)本，分別觀測血清性激素[(總睪酮(TT)、雌二醇(E2)、孕酮(P)、泌乳素(PRL)、黃體生成素(LH)、卵泡刺激素(FSH)]、臟器系數(shù)、精子計(jì)數(shù)、精子活力與形態(tài)等指標(biāo)，分析卡馬西平單藥及癲與卡馬西平協(xié)同作用對雄性大鼠生殖的影響。結(jié)果 造模組大鼠給予不同濃度卡馬西平灌胃后，LH、FSH、E2水平均較對照組明顯升高(P均<0.01)，各濃度組P水平均明顯低于對照組(P均<0.05)，各濃度組TT水平明顯低于對照組，PRL水平未見明顯改變。非造模組大鼠給予不同濃度卡馬西平藥物灌胃后, LH、FSH水平均較對照組明顯升高(P均<0.01)，TT水平均低于對照組(P均<0.05)，PRL水平中高濃度組明顯低于對照組(P均<0.05)， P、E2未見明顯改變。非造模組大鼠給予不同濃度卡馬西平藥物灌胃后, 各濃度組附睪重量及附睪臟器系數(shù)均較對照組明顯降低(P均<0.01)。結(jié)論 卡馬西平對于雄性大鼠生殖系統(tǒng)相關(guān)指標(biāo)存在明顯不良反應(yīng)，且有明顯藥物濃度關(guān)聯(lián)性，而卡馬西平與癲發(fā)作共同作用的不良反應(yīng)未見協(xié)同疊加效應(yīng)。

卡馬西平；癲；生殖；性激素

1 材料與方法

1.1.1 材料 6～8周齡(體重200～220 g)健康清潔級Wistar雄性大鼠100只(由揚(yáng)州大學(xué)實(shí)驗(yàn)動物中心提供)，并保持飼養(yǎng)環(huán)境室溫18～25 ℃、相對濕度50%～60%、晝夜循環(huán)照明環(huán)境中飼養(yǎng)，能自由攝食及飲水，每日定時(shí)清洗籠舍。

1.1.3 分組 設(shè)造模組、非造模組及對照組。將造模成功大鼠隨機(jī)取24只作為造模組，再將其隨機(jī)分為3組，分別為灌藥高、中、低濃度組，每組8只；非造模組為相同飼養(yǎng)環(huán)境下的健康雄性Wistar大鼠24只，亦隨機(jī)分成3組(將給予不同濃度灌藥)，每組8只；對照組10只(將給予灌注鹽水)。

1.1.4 給藥劑量及方法 按照低劑量組30 mg/(kg· d)，中劑量組60 mg/(kg·d)，高劑量組120 mg/(kg·d)，分別給予造模組及非造模組大鼠相應(yīng)濃度卡馬西平連續(xù)灌藥60 d，對照組給予生理鹽水連續(xù)灌胃60 d，1次/d，大鼠體重每3 d稱1次,以調(diào)整給藥劑量。

1.2 標(biāo)本取材與制備

1.2.1 血清性激素測定 所有入組大鼠給予連續(xù)灌胃60 d后，于同日早晨7：00-8:00采集外周血1次，經(jīng)放射免疫法 (RIA) 測定 TT、E2、P、PRL、LH、FSH。放射免疫分析試劑盒購自北京北方生物技術(shù)研究所，F(xiàn)T放射免疫分析試劑盒購自比利時(shí)BIOCODE-HYCEL公司，采用科大創(chuàng)新股份有限公司中佳分公司GC-911γ放射免疫計(jì)數(shù)器，嚴(yán)格按照說明書要求進(jìn)行測定。

1.2.2 性器官臟器系數(shù)測定 將所有大鼠脫臼處死，取雙側(cè)睪丸、附睪稱重后計(jì)算性器官臟器系數(shù)[臟器系數(shù)=臟器重量(g)/體重(g)×100%]，以百克體重計(jì)算。

1.2.3 精子懸液制備及計(jì)數(shù) 將所有大鼠處死后分離附睪，立即取雙側(cè)附睪尾放入盛有12 ml、37 ℃的M199培養(yǎng)液中，沿附睪尾縱切4～5刀，放入37 ℃培養(yǎng)箱中擴(kuò)散10 min，讓精子從附睪尾中游離出，得到精子懸液(2.0×107/ml)。將制備的精子懸液用吸管取少量滴入白細(xì)胞計(jì)數(shù)板計(jì)數(shù)精子數(shù)[7]。

1.2.4 精子活動率的測定 取精子懸液1～2 滴于清潔玻片上，在(20±2)℃的室溫下，20 min 內(nèi)在光鏡下進(jìn)行觀察，每只大鼠分析200個(gè)精子，并對精子的活動率進(jìn)行分級。分級標(biāo)準(zhǔn)如下：(Ⅰ)不活動；(Ⅱ)原地顫動或原地轉(zhuǎn)動；(Ⅲ)慢速直線或轉(zhuǎn)動向前運(yùn)動；(Ⅳ)快速直線向前運(yùn)動。統(tǒng)計(jì)活動的精子總數(shù)，計(jì)算精子的活動率[活動精子率=(Ⅱ+Ⅲ+Ⅳ)/(Ⅰ+Ⅱ+Ⅲ+Ⅳ)×100%]。睪精子形態(tài)觀察：用吸管吸取少量上述精子懸液，滴于載玻片上，立即用另一玻片均勻推開，干燥后用甲醇固定5 min，干后再用2%伊紅水溶液染色1 h，在400倍光學(xué)顯微鏡下觀察精子形態(tài)。

2 結(jié) 果

2.1 血清性激素 造模組高、中、低濃度卡馬西平灌胃后，LH、FSH、E2均較對照組明顯升高(P均<0.01)，但各濃度組間未見統(tǒng)計(jì)學(xué)差異。各濃度組P值均明顯低于對照組(P<0.05)，造模組間比較，高濃度組降低更為顯著，差異有統(tǒng)計(jì)學(xué)意義。各濃度組TT值明顯低于對照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)，而各濃度組間未見明顯差異，PRL與對照組比較無統(tǒng)計(jì)學(xué)差異。非造模組高、中、低濃度卡馬西平灌胃后，LH、FSH均較對照組顯著升高(P均<0.01)，但各濃度組間未見統(tǒng)計(jì)學(xué)差異。TT各濃度組均低于對照組(P均<0.05)，其中高濃度組明顯低于對照組(P均<0.01)，與中低濃度組存在統(tǒng)計(jì)學(xué)差異。PRL水平中、高濃度組明顯低于對照組(P均<0.05)，其余組別無統(tǒng)計(jì)學(xué)差異(表1)。

2.2 性器官臟器系數(shù) 造模組大鼠給予高、中、低濃度卡馬西平藥物灌胃后，體重及臟器系數(shù)檢測值與對照組比較無統(tǒng)計(jì)學(xué)意義。非造模組中各濃度組附睪重量及附睪臟器系數(shù)均較對照組降低，差異具有統(tǒng)計(jì)學(xué)意義(P<0.01)。余各濃度組體重、睪丸重量及睪丸臟器系數(shù)與對照組比較無統(tǒng)計(jì)學(xué)意義(表2)。

2.3 精子計(jì)數(shù)及精子活動率 造模組和非造模組大鼠各濃度組間精子數(shù)均未見明顯異常，但精子活動率均明顯低于對照組，具有顯著統(tǒng)計(jì)學(xué)差異(P均<0.01)，其中均以高濃度組精子活動率減低最為明顯(表3)。

表1 造模組和非造模組大鼠給予高、中、低濃度卡馬西平藥物灌胃血清性激素檢測結(jié)果 ±s)

注：與對照組比較，①P<0.01；②P<0.05

表2 造模組和非造模組大鼠給予高、中、低濃度卡馬西平灌胃體重及臟器系數(shù)檢測結(jié)果 ±s)

注：與對照組比較，①P<0.01；②P<0.05

表3 造模組和非造模組大鼠給予高、中、低濃度卡馬西平灌胃精子計(jì)數(shù)及精子活動率檢測結(jié)果 ±s)

注：與對照組比較，①P<0.01；②P<0.05

3 討 論

[1] 李 云,徐艷惠,劉麗芬,等. 18F-FDGPET/CT腦顯像在癲發(fā)作期及發(fā)作間期致灶定位中的應(yīng)用價(jià)值[J].武警醫(yī)學(xué)，2013, 26(9): 800-803.

[2] Mattson R H, Cramer J A. Epilepsy, sex hormones and antiepileptic drugs[J]. Epilepsia, 1985,26(suppl1):40.

[3] Xu Xiaotian, Zhang Hengzhong, Xu Yao. Effects of antiepileptic drugs on reproductive endocrine function, sexual function and sperm parameters in Chinese Han men with epilepsy[J] .J Clin Neu , 2013,20:1492-1497.

[4] 褚春民，張桁忠，朱亞琳，等.苯巴比妥治療對男性癲患者血清性激素水平的影響[J].中華男科學(xué)雜志，2007,13(8)：749-750.

[5] 許笑天，張桁忠，趙子攀，等．癲對雄性wistar 大鼠生殖系統(tǒng)的影響[J]．癲與神經(jīng)電生理學(xué)雜志，2012,21(1):1-4.

[6] Racine R J.Modification of Seizure Activity byElectrical Stimulation lI Motor Seizure[J].Electroencephogr Clin Neurophysiol，1972,32(3):281-294.

[7] 于 峰,姚寶玉,王 薏,等. 小鼠畸形精子分型[J]. 上海實(shí)驗(yàn)動物科學(xué),1997,17(4):234-236.

[8] JONES K L. Pattern of malformations in the children of women treated with carbamzepine during pregency[J]. N Engl J Med,1989,320,1661-1666.

[9] Crebelli.Threshold mediated mechanisms in mutagnesis:implications in the classification and regulation of chemical mutagens[J].Muta Res,2000,464(1):129-135.

[10] Shetty akhila,Jagadish,Narayana K.The effects of carbamazepine on sperm morphology in wistar rats[J].India J Phy Phar,2007,51(3):255-260.

[11] Penovjch P E．The effccts of cpilepsy and jts treaInlent on scxual andrcproductive function[J]．EpiIcpsia, 2000，4l(suppl 2)：53-61．

[12] 王 雁，譚 蘭. 卡馬西平對男性癲患者雄性激素水平的影響[J].神經(jīng)疾病與精神衛(wèi)生,2001,1(1)：38-39.

[13] Duncan S, Blacklaw J, Beastall G H,etal. Antiepileptic drug therapy and sexual function in men with epilepsy[J]. Epilepsia,1999,40:197-204.

[14] Rattya J, Turkka J, Pakarinen A J,etal. Reproductive endocrine effects of valproate, carbamazepine and oxcarbazepine in men with epilepsy[J]. Neurology, 2001,56:31-36.

[15] Isojarvi J I, Lofgren E, Juntunen K S,etal. Effect of epilepsy and antiepileptic drugs on male reproductive health[J].Neurology,2004,62:247-253.

[16] Roste L S, Tauboll E, Haugen T B,etal. Alterations in semen parameters in men with epilepsy treated with valproate or carbamazepine monotherapy[J]. Eur J Neurol,2003,10:501-506.

(2014-06-10收稿 2014-09-15修回)

(責(zé)任編輯 尤偉杰)

Effects of reproductive system in experimental male rats with carbamazepine

LIU Xuhong,ZHANG Hui,TAN Mei,XU Xiaotian, and WU Chunhua.

Department of Neurology，Jiangsu Provincial Corps Hospital, Chinese People’s Armed Police Forces, Yangzhou 225003, China

Objective To study the effects of CBZ and CBZ with epilepsy on reproductive system of male Wistar rats. Methods Male rat epilepsy models were established with lithium-pilocarpine and health male Wistar rats controls were used. They were given three different concentration carbamazepine infused into stomach. Blood, testis and epididymis, and sperms were sampled; serum sex hormones, viscera coefficient, sperm counts, sperm motility and morphology were observed for analyzing on effects male epileptrc rats’ reproductive system.Results In the epilepsy model rats receiving high, medium and low concentration CBZ showed that LH, FSH, E2 were significantly higher than in controls, there were but no significant differences between the three concentration groups. Progesterone (P) levels of three concentrations were obviously lower than in controls, but in low concentration group was significantly lower than in the other two concentration groups. Testosterone (T) levels in three concentration groups were significantly lower than in healthy group. No changes were found in PRL.In the health control Wistar rats receiving. high, medium and low concentration CBZ, LH and FSH were significantly higher than in controls, testosterone (T) levels in three concentration groups were significantly lower than in health group, especially the high concentration group,PRL levels in medium and low concentration group obviously lower than in controls. There was no difference in P and E2. There were no changes in weight and testicular weight and testicular viscera coefficient between male epilepsy rats and health group, but epididymis weight and epididymis viscera coefficient were obviously lower in the health rats with three concentration CBZ than in controls. No sperm count change in the three concentrations for male epilepsy rats and health group, but both of them had the lower sperm activity rate than in controls, especially in high concentration group. Conclusions Carbamazepine itself for male rats reproductive system has obvious side effects and obvious drug concentration relevance, both carbamazepine and epilepsy also present the part of the side effects of correlation concentration,but the mechanism is relatively complex, caused by many factors in joint action.

carbamazepine；epilepsy；reproduction；sex hormones

劉緒宏，本科學(xué)歷，主任醫(yī)師，E-mail:xuhlzy@163.com

225003揚(yáng)州，武警江蘇總隊(duì)醫(yī)院神經(jīng)內(nèi)科

R971.6；R742.1