奥硝唑诱导弱精子症与少弱精子症大鼠模型的建立.pdf

.1208.生殖医学杂志2 0 2 3年8 月第32 卷第8 期DO1:10.3969/j.issn.1004-3845.2023.08.011奥硝唑诱导弱精子症与少弱精子症大鼠模型的建立刘胜京，晏斌，安晓静，耿强，韩强，赵丰，张继伟1，王福，郭军1*（1.中国中医科学院西苑医院，北京10 0 0 9 1;2.天津中医药大学第一附属医院，天津30 0 19 3;3.首都医科大学附属北京中医医院，北京10 0 0 10）【摘要】目的通过不同剂量的奥硝唑（ORN)制备弱精子症及少弱精子症大鼠模型，并探讨其对附睾、睾丸及超微结构的损伤，以及对精子线粒体的影响。方法48 只雄性SD大鼠随机分为4组，每组12 只，实验组分别给予ORN200mg/kg、ORN400mg/kg、O RN8 0 0 m g/k g 灌胃，空白对照组给予1%羧甲基纤维素钠灌胃，连续灌胃2 8 d。观察记录各组大鼠体重、附睾及睾丸脏器指数，检测附睾精子浓度及活力，采用苏木素-伊红（HE)染色方法观察附睾头、附睾尾及睾丸病理损伤情况，透射电镜观察附睾、睾丸及精子超微结构，统计分析4组间各项指标差异。结果与空白对照组相比，ORN200mg/kg剂量组附睾及睾丸脏器指数、精子浓度及活力均无显著变化（P0.05）,H E染色及透射电镜结果均显示附睾及睾丸组织无明显损伤；ORN400mg/kg剂量组附睾脏器指数显著降低（P0.05），精子活力显著降低（16.3士7.2)%vs.（36.1士6.6%，P0.05），H E染色观察附睾及睾丸组织损伤不明显，透射电镜超微结构可见附睾及睾丸线粒体出现部分空泡化，精子线粒体出现损伤，但仍可见较为完整的精子线粒体结构；ORN800mg/kg剂量组附睾及睾丸脏器指数均显著降低（P0.05），精子浓度（15.3士9.2）10%/mlvs.（6 0.3土10.2）10%/ml,P0.001及活力（7.3士5.2）%vs.（36.1士6.6%，P0.001均显著下降，HE染色可见附睾及睾丸组织损伤明显，透射电镜下可观察到附睾及睾丸线粒体空泡化明显，精子线粒体明显肿胀断裂。结论ORN400mg/kg灌胃2 8 d可用于弱精子症大鼠模型的制备，其对附睾、睾丸组织损伤及精子线粒体结构损伤不明显；ORN800mg/kg灌胃2 8 d可用于少弱精子症大鼠模型的制备，可明显造成附睾及睾丸组织损伤，破坏精子线粒体结构。【关键词】奥硝唑；男性不育症；弱精子症；少弱精子症；动物模型【中图分类号】R698十.2Establishment of ornidazole-induced rat models with asthenozoospermia and oligoasthenozoospermiaLIU Sheng-jing,YAN Bin,AN Xiao-jing,GENG Qiang”,HAN Qiang,ZHAO Feng?,ZHANG Ji-wei,WANG Fu,GUOJun!*1.Xiyuan Hospital of China Academy of Chinese Medical Sciences,Beijing 1000912.First Teaching Hospital of Tianjin University of Traditional Chinese Medicine,Tianjin 3001933.Beijing Chinese Medicine Hospital Affiliated to Capital Medical University,Beijing 100010Objective:To establish rat model with asthenozoospermia and oligoasthenozoospermia byadministrating the different doses of ornidazole(ORN),and to explore its effects on the ultrastructuraltissue damage of the epididymis and testis,and sperm mitochondria.Methods:Forty-eight male SD rats were randomly divided into 4 groups,12 rats for each group.Therats in experimental groups were given ORN 200 mg/kg,400 mg/kg and 800 mg/kg by gavage,respectively,and the negative control group was given 1%sodium carboxymethyl cellulose by gavage,continuous gavage for 28 days.The body weight,organ index of epididymis and testis,sperm concentration【文献标识码】A【A b s t r a c t【收稿日期】2 0 2 3-0 2-0 7；【修回日期】2 0 2 3-0 3-2 3【基金项目】国家自然科学基金面上项目（8 2 17 439 2，8 2 17 42 17）；国家中医药管理局中医药传承与创新“百千万”人才工程岐黄学者资助项目（国中医药人教函【2 0 2 2 6 号）；中国中医科学院科技创新工程重大攻关项目重点项目（CI2021A02201）【作者简介】刘胜京，男，山东聊城人，博士研究生，生殖男科学专业.（*通讯作者，Email：g u o j u n 112 6 12 6.c o m）生殖医学杂志2 0 2 3年8 月第32 卷第8 期and motility were observed.HE staining was used to observe the pathological damage of epididymis head,epididymis tail and testis.The ultrastructure of epididymis,testis and sperm was observed by transmissionelectron microscope.The differences in various indicators among the four groups were analyzed.Results:Compared with the negative control group,there was no significant change in the epididymisand testis organ index,sperm concentration and motility in the ORN 2o0 mg/kg group,and there was noobvious tissue damage of epididymis and testis tested by HE staining and transmission electronmicroscopy.The epididymal organ index of ORN 400 mg/kg group significantly decreased(P0.05),andthe testicular organ index did not change significantly.Sperm motility decreased significantly(16.37.2)%vs.(36.16.6)%,P0.05).HE staining showed that there was no obvious tissue damage to the epididymis and testis.Transmissionelectron microscopy revealed partial vacuolization of the epididymis and testicular mitochondria,and spermmitochondria was damaged,but relatively complete sperm mitochondrial structure was still visible.In theORN 800 mg/kg group,the epididymal and testicular organ indexes were significantly reduced,the spermconcentration(15.39.2)X10%/ml vs.(60.310.2)X10%/ml,P0.001 and motility(7.35.2)%vs.(36.1 6.6)%,P0.001J were significantly decreased.HE staining showed significant damage to theepididymis and testis.Under transmission electron microscopy,obvious vacuolization of mitochondria in theepididymis and testis can be observed,and the sperm mitochondria were significantly swollen and fractured.Conclusions:Adiministration of ORN 400 mg/kg by gavage for 28 days can be used for theestablishment of rat model with asthenozoospermia,but it does not significantly damage the epididymis,testis and sperm mitochondrial structure.Adiministration of ORN 800 mg/kg by gavage for 28 days can beused for the preparation of rat model with oligoasthenozoospermia,which significantly damages theepididymis,testis and destroys the sperm mitochondrial structure.Key words:Ornidazole;Male infertility;Asthenozoospermia;Oligoasthenozoospermia;Animal model(JReprod Med 2023,32(08):1208-1215)男性不育症是指夫妇备孕1年以上，性生活规作用部位及原因进行探讨，以期为弱精子症及少弱律且未采取避孕措施，由于男方因素导致女方不能精子症的研究提供理论基础。怀孕的一类疾病1。其中,弱精子症与少弱精子症材料与方法是男性不育症最常见的原因2 ，然而目前临床尚缺乏治疗弱精子症及少弱精子症的有效药物3-4，通过构建稳定的弱精子症及少弱精子症动物模型，对探索弱精子症及少弱精子症的发病机制及新药研发均具有重要的意义。奥硝唑（Ornidazole,ORN)是继甲硝唑、替硝唑之后的第3代硝基咪唑类抗生素，具有良好的抗厌氧菌和抗原生质（如滴虫等）感染作用，常用于泌尿生殖道感染等疾病。目前生殖毒性结果显示ORN对雄性大鼠生殖系统具有可逆性影响，其影响程度与给药剂量有关5。有研究证实其可用于诱导弱精子症及少弱精子症模型，但是其影响环节及影响