【作者】 唐乾利；

【导师】 何清湖；

【作者基本信息】 湖南中医药大学 ， 外科学（专业学位）， 2013， 博士

【摘要】 [目的]男性不育症是影响人类健康及子孙繁衍的重大疾病,常常表现为少弱精子症,其中过度氧化与能量代谢障碍可能是其关键病理生理学机制。目前,缺乏对少弱精子症的有效治疗药物。前期研究发现,具有健脾益肾功效的中药验方强精煎可抑制生精功能障碍大鼠生精细胞凋亡,并恢复睾丸的生精功能,但具体机制尚未清楚。本项目基于中医学对少弱精子症脾肾两虚的病机认识,在前期研究的基础上临床观察强精煎治疗脾肾两虚少弱精子症患者的疗效,并应用奥硝唑诱导建立少弱精子症大鼠模型,进一步探讨强精煎对该模型大鼠精子质量的影响,观察强精煎对少弱精子症模型大鼠睾丸及附睾组织病理组织、超微结构、抗氧化作用、能量代谢、氧化损伤调控效应因子(Nrf2)和能量代谢调控酶琥珀酸脱氢酶(SDH)基因表达的影响,探讨强精煎治疗少弱精子症的作用机制。[方法]1.临床观察：将符合脾肾两虚的男性不育少弱精子症患者120例随机分为对照组(黄精赞育胶囊组)与治疗组(强精煎组)。治疗组给予口服强精煎治疗,对照组给予口服黄精赞育胶囊治疗,疗程3个月。治疗结束后检测精子密度、活力、活率、精子形态率、精子膜表面抗精子抗体等指标,以及评价其安全性。2.动物实验：将SPF级实验动物100只雄性大鼠随机分为正常组、模型组、黄精赞育胶囊组、左卡尼汀口服溶液组、强精煎组,每组20只。正常组每天给予生理盐水灌胃；模型组予奥硝唑ORN(800mg/kg·d)灌胃；黄精赞育胶囊组予ORN(800mg/kg·d)+黄精赞育胶囊(200mg/kg·d)灌胃；左卡尼汀口服溶液组大鼠给予ORN(800mg/kg·d)+左卡尼汀口服溶液(100mg/kg·d)灌胃；强精煎组大鼠每天给予ORN (800mg/kg-d)+强精煎配方颗粒(10g/kg·d)灌胃。黄精赞育胶囊、左卡尼汀口服溶液、强精煎均溶于生理盐水,疗程4周。治疗结束后,观察大鼠睾丸、附睾组织病理及其超微结构,分析附睾精子密度和活率,以及检测附睾组织超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、乳酸脱氢酶(LDH)、a-葡糖苷酶、果糖浓度等指标,并采用Real-time PCR法检测大鼠附睾组织Nrf2和SDHmRNA水平。[结果]1.临床研究：①与对照组相比,强精煎显著提高a级和a+b级精子活力、精子密度及精子形态率(P<0.05),总有效率也明显高于对照组(P<0.05)。②强精煎未见明显毒副作用及不良反应。2.动物实验：①模型组大鼠附睾的精子密度及活动率明显低于正常组(P<0.05),而强精煎组、黄精赞育胶囊组、左卡尼汀口服溶液组的精子密度和活动率则明显高于模型组(P<0.05),并且强精煎组与正常组相类似(P<0.05)；②病理研究显示,模型组大鼠睾丸生精小管管腔内可见脱落的各级生精细胞,附睾管腔中精子数目明显减少,有较多的非精子细胞成分,而强精煎组、黄精赞育胶囊组及左卡尼汀口服溶液组大鼠睾丸、附睾均未见明显改变,睾丸生精小管内及附睾管腔均可见大量精子；超微结构显示模型组附睾管壁变薄,管腔内精子明显减少,不能充满管腔,排列紊乱,附睾管腔问间隙增大；与模型组相比,强精煎组、黄精赞育胶囊组及左卡尼汀口服溶液组附睾管壁较厚,精子充满管腔,排列较为规则,呈团簇样,管腔间隙较紧密,均与正常组类似。③模型组SOD和GSH-Px含量明显低于正常组(P<0.05),而MDA含量则明显高于正常组(P<0.05)；与模型组相比,黄精赞育胶囊组、左卡尼汀口服溶液组和强精煎组SOD、GSH-Px明显高于模型组(P<0.05),而MDA含量明显低于模型组(P<0.05)；强精煎组SOD、MDA和GSH-Px含量与正常组相类似(P>0.05)。Real-time PCR结果显示,模型组Nrf2mRNA比正常组明显下降(P<0.05)；与模型组比较,黄精赞育胶囊组、左卡尼汀口服溶液细和强精煎组Nrf2mRNA明显升高(P<0.05),也高于正常组(P<0.05),并且强精煎组表达量明显高于黄精赞育胶囊组或左卡尼汀口服溶液组(P<0.05)。④与正常组相比,模型组a-葡糖苷酶、果糖浓度明显下降(P<0.05),而LDH明显高于正常组(P<0.05)；与模型组相比,黄精赞育胶囊组、左卡尼汀口服溶液资组和强精煎组a-葡糖苷酶、果糖浓度明显高于模型组(P<0.05),而LDH则明显低于模型组(P<0.05)；强精煎组LDH、a-葡糖苷酶和果糖浓度都与正常组相类似(P>0.05)。Real-time PCR结果显示,模型组SDHmRNA明显低于正常组(P<0.05)；与模型组比较,黄精赞育胶囊组、左卡尼汀口服溶液细和强精煎组SDHmRNA明显升高(P<0.05),也高于正常组(P<0.05),并且强精煎组表达量明显高于黄精赞育胶囊组或左卡尼汀口服容液组(P<0.05)。[结论]1.强精煎可以明显提高人精子质量,改善精子活力及活率、精子形态率,并且无毒副作用。2.奥硝唑可诱导大鼠产生弱少精子症,并且与氧化过度和能量代谢障碍密切相关。3.强精煎可以改善甚至逆转奥硝唑诱导产生的弱少精子症。4.强精煎可以改善奥硝唑诱导附睾和睾丸的病理组织结构与超微结构。5.抗氧化作用和改善能量代谢很可能是强精煎治疗弱少精子症的机制。更多还原

【Abstract】 ObjectiveMale infertility, a critical disease involving human health and the generations of descendants, often shows oligospermia and asthenospermia, and the key pathophysiology mechanisms may refer to excessive oxidation and dysfunction of energy metabolism. At present, the effective drugs for oligospermia and asthenospermia are lack. Previous studies revealed that Qianjing decoction, a Chinese prescription performing to invigorate spleen and kidney, inhibits apoptosis of spermatogenic cells in rats with spermatogenic dysfunction and recovers the function, but the potential mechanisms remain unclear. Based on the interpretation of asthenia of both the spleen and kidney for oligospermia and asthenospermia according to traditional Chinese medicine (TCM) and the previous researches, the present study aimed to investigate the curative effects of Qianjing decoction on the patients with oligospermia and/or asthenospermia who were consistent with asthenia of both the spleen and kidney, and on the sperm quality in ornidazole-induced rats with oligospermia and asthenospermia. Moreover, the study was conducted to observe the effects of the decoction on the testicle and epididymal tissue pathology, ultrastructure, antioxidation, energy metabolism, and the gene expression of SDH and Nrf2which involve in antioxidation and energy metabolism respectively in rats with oligospermia and asthenospermia, which would reveal the mechanisms of Qiangjing decoction in treating the disease.Methods1. Clinical trial:One hundred and twenty patients with oligospermia and/or asthenospermia who were consistent with asthenia of both the spleen and kidney according to TCM were randomly assigned to the control (Huangzanyu capsule, n=60) and the treated(Qiangjing decoction, n=60) groups. The former was received Huangzanyu capsule, while the latter was given Qiangjing decoction. The course lasted3months. After treatment, the indexes including sperm concentration, motility and viability, the rate of teratosperm, and antisperm antibody were checked, and the safety was also evaluated.2. Animal experimental design:One hundred SPF male rats were randomly divided into5groups:the normal group (n=20) receiving saline solution by intragastric administration, the model group (n=20) receiving ornidazole (800mg/kg.d), the huangjingzanyu capsule group (n=20) receiving ornidazole (800mg/kg.d) and huangjingzanyu capsule (200mg/kg.d), the levocarnitine group (n=20) receiving ornidazole (800mg/kg.d) and levocarnitine (100mg/kg.d), and the Qiangjing decoction group (n=20) receiving ornidazole (800mg/kg.d) and Qiangjing decoction (10g/kg.d). The treatment continued4weeks. After treatment, the pathology and ultrastructure of testis and epididymal tissue were investigated, sperm concentration and motility were detected, and the indexes including superoxide dismutase (SOD), malonaldehyde (MDA), glutathione peroxidase (GSH-Px), lactate dehydrogenase (LDH), a-glucosidase, and fructose in the epididymal homogenate were also determined.Moreover, the mRNA levels of Nrf2and SDH in the epididymal tissue were measured using Real-time PCR.Results1. Clinical trial:1) Compared to the control group, Qiangjing decoction efficiently enhanced class a and a+b sperm motility and sperm concentration, improved sperm morphology (all P<0.05), and total effective rate in Qiangjing decoction was higher than that in the control group (P<0.05).2) Qiangjing decoction had few side effects and adverse reactions.2. Animal trial:1) Both of sperm concentration and motility in the model group were obvious lower than that in the normal group (P<0.05), while the indexes in the huangjingzanyu capsule group, the levocarnitine group, and the Qiangjing decoction group were significant higher than that in the model group (P＜0.05), and the indexes in the Qiangjing decoction group were similar to the normal group (P>0.05).2) According to the pathology studies, there were different spermatogenic cells which were dropped in the seminiferous tubules of the testis, and a lot of non-sperm cell components in the epididymal lumens with decreased sperm cells in the model group. Compared to the normal group, there were few changes in the testis and epididymis with a great many of sperm cells in the Qiangjing decoction group, the huangjingzanyu capsule group, and the levocarnitine group. In ultrastructure, the epididymal lumens wall was thin with decreased sperm cells in epididymal lumens in the model group, and the epididymal lumens were in disorder, the gap between epididymal lumens were increased. Compared to the model group, the epididymal lumens wall was thick with full sperm cells in epididymal lumens in the Qiangjing decoction group, the huangjingzanyu capsule group, and the levocarnitine group, and the epididymal lumens were in order, the gap between epididymal lumens were smaller, which were all similar to the normal group.3) SOD and GSH-Px in the model group were much lower than that in the normal group (P＜0.05), while MDA higher than normal group (P＜0.05). Compared to the model group, the indexes of SOD and GSH-Px in the huangjingzanyu capsule group, the levocarnitine group, and the Qiangjing decoction group were obviously increased (P<0.05), but MDA was lower than that in the model group (P＜0.05). The three indexes in the Qiangjing decoction group were all similar to the normal group (P>0.05). Real-time PCR revealed that the mRNA level of Nrf2was significantly decreased compared to the normal group (P＜0.05). And the mRNA level in the huangjingzanyu capsule group, the levocarnitine group, and the Qiangjing decoction group was markedly increased when compared to the model group or the normal group (P＜0.05). Moreover, the mRNA level in the Qiangjing decoction group was much higher than that in the huangjingzanyu capsule group or the levocarnitine group (P＜0.05).4) Compared to the normal group, a-glucosidase and fructose in the model group were decreased (P＜0.05), while LDH higher than normal group (P＜0.05). Compared to the model group, the indexes of a-glucosidase and fructose in the huangjingzanyu capsule group, the levocarnitine group, and the Qiangjing decoction group were obviously increased (P＜0.05), but LDH was lower than that in the model group (P＜0.05). The three indexes in the Qiangjing decoction group were similar to the normal group (P>0.05). Real-time PCR revealed that the mRNA level of SDH was markedly decreased compared to the normal group (P＜0.05). And the mRNA level in the huangjingzanyu capsule group, the levocarnitine group, and the Qiangjing decoction group was significantly increased when compared to the model group or the normal group (P＜0.05). Moreover, the mRNA level in the Oiangjing decoction group was much higher than that in the huangjingzanyu capsule group or the levocarnitine group.ConclusionsX.Qiangjing decoction significantly enhances sperm quality, improves the sperm motility and viability, and the rate of teratosperm in the patients with oligospermia and/or asthenospermia.2.Ornidazole induces oligospermia and asthenospermia in rats, which has close relationship with excessive oxidation and dysfunction of energy metabolism.3.Qiangjing decoction improves and even reverses ornidazole-induced oligospermia and asthenospermia in rats.4.Qiangjing decoction improves the pathology structure and ultrastructure of testis and epididymal tissue in ornidazole-induced rats.5.Antioxidation and improvement of energy metabolism are likely the mechanisms of Qiangjing decoction in treating oligospermia and asthenospermia.更多还原