【作者】 杨凯；

【导师】 曾庆琪；

【作者基本信息】 南京中医药大学 ， 中医外科学（男科方向）（专业学位）， 2020， 博士

【摘要】 目的本文首先对曾庆琪教授治疗男性不育症的学术思想和临证经验进行分析总结;在前期理论的基础上采用单盲、随机、阳性对照药物的临床研究方法初步探讨润精汤对特发性少弱精子症患者的临床有效性和安全性;然后在运用奥硝唑诱导建立的少弱精子症SD雄性大鼠模型的基础上,通过观察润精汤对少弱精子症模型大鼠精液质量、性激素水平、大鼠睾丸组织病理形态变化、睾丸组织细胞凋亡、睾丸波形蛋白表达和ERK信号通路表达的变化,初步探讨润精汤治疗少弱精子症的作用靶点和作用机制,为润精汤治疗特发性少弱精子症的临床应用提供科学依据。方法1.经验总结:对曾庆琪教授治疗男性不育症的学术思想和临证经验进行分析总结,从病因病机、辨病论治、诊断治法、方药配伍、优生助孕和预防调护等方面进行了详细论述。2.临床研究:采用单盲、随机、阳性对照药物的临床研究方法,将符合诊断标准和纳入标准的72例特发性少弱精子症患者随机分为试验组(润精汤)和对照组(还少胶囊),每组36例,分别治疗3个月,记录两组患者治疗前后的精液质量(精液量、精子浓度、精子总数、精子前向运动百分率、精子总活动力)、性激素水平、中医症状评分、安全性指标和不良反应情况,评价润精汤的临床有效性和安全性。3.实验研究:将32只SD雄性大鼠采用单纯随机抽样方法分成四组,每组8只,分别为空白组、模型组、低剂量组和高剂量组。空白组予以生理盐水灌胃;模型组予以奥硝唑ORN(400mg/kg·d)灌胃;低剂量组予以润精汤(14g/kg)+奥硝唑ORN(400mg/kg·d)灌胃;高剂量组予以润精汤(56g/kg)+奥硝唑ORN(400mg/kg·d)灌胃,疗程四周。实验结束后,检测大鼠血清性激素指标(FSH、LH和T)、大鼠精液质量(精子浓度、精子前向运动百分率、精子总活动力和精子畸形率),采用Western Blot和免疫组织化学法测定大鼠睾丸波形蛋白表达和睾丸ERK信号通路表达,采用TUNEL法检测大鼠睾丸组织细胞凋亡情况。结果1.临床研究:①试验组(润精汤)和对照组(还少胶囊)均能提高精液质量(精液量、精子浓度、精子总数、精子前向运动百分率、精子总活动力)、调节性激素水平和改善中医临床症状,从而提高患者配偶妊娠率和临床有效率,且试验组(润精汤)改善更为明显。②润精汤未见明显毒副作用及不良反应。2.实验研究:①精液质量:模型组大鼠精子浓度、精子总活动力、精子前向运动百分率低于空白组(P<0.05),精子畸形率高于空白组(P<0.05);低剂量组、高剂量组的精子总活动力高于模型组(P<0.05),精子畸形率低于模型组(P<0.05);低剂量组的浓度和精子前向运动百分率轻度高于模型组(P>0.05);高剂量组的浓度和精子前向运动百分率高于模型组(P<0.05)。②性激素:各组黄体生成素(LH)无明显变化(P>0.05);模型组卵泡刺激素(FSH)高于空白组(P<0.05),低剂量组和高剂量组低于模型组(P<0.05);模型组睾酮(T)低于空白组(P<0.05),低剂量组和高剂量组高于模型组(P<0.05)。③睾丸组织病理形态变化:空白组大鼠睾丸组织中各曲细精管生精上皮形态规则、排列整齐和结构完整,生精小管管腔内可见各级分裂活跃、形态规则、排列整齐和结构完整的生精细胞和大量形态正常且成熟的精子。模型组大鼠睾丸组织中各曲细精管生精上皮形态紊乱、排列不整齐和结构松散,生精小管管腔内各级生精细胞排列紊乱,且大量减少,甚至脱落,精子和生精细胞数量明显减少,且形态明显异常,生精上皮部分变性可见大量空泡。低剂量组大鼠睾丸组织中各曲细精管生精上皮形态的规则性、排列的整齐性和结构的完整性较模型组有所改善,生精小管管腔内正常生精细胞数和形态正常且成熟的精子数较模型组也有所增加,脱落生精细胞数量有所减少。高剂量组大鼠睾丸组织病理形态结构较低剂量组进一步有所改善,接近于空白组。④睾丸组织细胞凋亡的变化:模型组生精小管管腔内可见大量生殖细胞凋亡,大鼠睾丸组织凋亡阳性细胞率明显高于空白组(P<0.01)。低剂量组和高剂量组生精小管管腔内可少量生殖细胞凋亡,低剂量组大鼠睾丸组织凋亡阳性细胞率明显低于模型组(P<0.05),高剂量组大鼠睾丸组织凋亡阳性细胞率明显低于模型组(P<0.01)。⑤睾丸组织波形蛋白表达的变化:空白组睾丸组织波形蛋白主要分布于睾丸支持细胞的核周围,并且从基底区域向近腔小室延伸。模型组睾丸组织波形蛋白信号和分布范围与空白组比较明显减弱,波形蛋白表达量明显降低(P<0.001)。低剂量组和高剂量组的睾丸组织波形蛋白的主要分布从睾丸支持细胞的核周围区域向内腔延伸,并且波形蛋白在两组间分布也趋于正常,波形蛋白表达量明显高于模型组(P<0.01)。⑥睾丸组织ERK信号通路的变化:模型组大鼠睾丸组织p-ERK蛋白表达量明显高于空白组(P<0.001);低剂量组和高剂量组大鼠的睾丸组织p-ERK蛋白表达量明显低于模型组(P<0.001)。结论1.润精汤是曾庆琪教授基于“精室理论”,根据精室的功能特点,总结出“通补并用”为核心的治疗法则,创制而成的治疗少弱精子症的经验方,药物组成有菟丝子、黄精、山药、枸杞、仙灵脾、水蛭、刺五加、红景天、陈皮、川牛膝,诸药具有“肝脾肾三脏兼顾、气血阴阳平调、补而不腻、通而不损”的特点,全方以补肾健脾为主,兼有补血养肝、活血通络、行气利湿的作用,本方治法较单补肾填精法或补肾活血法更为全面,更加符合当今大多数男性不育症的临床实际情况,尤其是对江南地区的患者更为适宜。2.润精汤和还少胶囊均能提高精液质量(精液量、精子浓度、精子总数、精子前向运动百分率、精子总活动力)、调节性激素水平和改善中医临床症状,从而提高患者配偶妊娠率和临床有效率,且润精汤改善更为明显。所以说润精汤是安全和有效的,且未见明显不良反应,有一定的临床推广价值。3.润精汤可以改善奥硝唑诱导的少弱精子症大鼠的精液质量、血清性激素和睾丸组织病理形态学变化,初步猜测可能与润精汤调控下丘脑-垂体-睾丸性腺轴,调节生殖内分泌激素的水平,改善睾丸微循环和精子生成的局部微环境有关;此外润精汤可上调睾丸波形蛋白的表达和下降p-ERK的表达,提高生殖细胞结构的稳定性和抑制大鼠生殖细胞的凋亡,促进生殖细胞增殖等,其作用机理可能与抗氧化应激有关。更多还原

【Abstract】 ObjectiveThis article first analyzes and summarizes Professor Zeng Qingqi’s academic thoughts and clinical experience in treating male infertility.Based on the previous theory,this article initially explores the clinical effectiveness and safety of Runjing Decoction for patients with idiopathic oligospermia.Based on the male rat model of oligozoospermia induced by Ornidazole,the sperm quality,sex hormone levels,pathological changes of testicular tissue,and testicular tissue cells of oligozoospermia model rats were observed by Runjing Decoction.Apoptosis,testicular vimentin expression,and changes in the expression of ERK signaling pathways,preliminary studies on the target and mechanism of Runjing Decoction in the treatment of oligozoospermia,provide scientific basis for the clinical application of Runjing Decoction.Methods:1.Experience summary:Analyze and summarize Professor Zeng Qingqi’s academic thoughts and clinical experience in treating male infertility,from the etiology and pathogenesis,clinical differentiation,diagnosis and treatment,prescription compatibility,eugenic pregnancy and preventive care Discussed in detail.2.Clinical study:72 patients with idiopathic oligozoospermia who met the diagnostic criteria and inclusion criteria were randomly divided into a test group(Runjing Decoction)and a control group(Huaishao Capsule).Months,the semen volume,semen concentration,total sperm count,forward sperm motility(PR),total sperm motility(PR+NP),sex hormone levels,TCM symptom scores,and adverse reactions were recorded before and after treatment in both groups.To evaluate the clinical effectiveness and safety of Runjing Decoction.3.Experimental study:32 male rats are divided into four groups using a simple random sampling method,with 8 rats in each group,which are blank group,model group,low dose group and high dose group.The blank group is orally administered with saline every day;the model group is orally administered with ornidazole ORN(400 mg/kg·d);the low-dose group is administered with Runjing Decoction(14g/kg)+ornidazole ORN 400 mg/(kg·d)Gavage;high-dose group is administered with Runjing Decoction(56g/kg)+Ornidazole ORN400 mg/(kg·d)for 4 weeks.After the experiment,rat serum sex hormone indicators(FSH,LH,T),rat semen quality(concentration,proportion of sperm forward movement,total motility,and sperm deformity rate)were measured.Western Blot and immunohistochemical methods are used to determine The expression of vimentin in rat testis and the expression of ERK signal pathway in testis are tested by TUNEL method.Results:1.Clinical research:①Both the test group(Runjing Decoction)and the control group(Huaishao Capsule)can improve sperm quality(semen volume,semen concentration,total sperm count,forward motion ratio of sperm,total sperm motility),and regulating sex hormones Level,increase the spouse’s pregnancy rate,improve the clinical symptoms and clinical effectiveness of Chinese medicine,and the test group(Run Jing Decoction)improved more significantly.②No obvious side effects and adverse reactions were observed in Runjing Decoction.2.Experimental research:① sperm quality:the sperm concentration,total sperm motility,and the percentage of forward sperm motility of the model group rats are lower than those of the blank group(P<0.05),and the sperm deformity rate is higher than that of the blank group(P<0.05);low The total activity of the low-dose and high-dose groups are higher than that of the model group(P<0.05),and the sperm deformity rate is lower than that of the model group(P<0.05).The concentration and percentage of sperm forward movement in the low-dose group are slightly higher than those in the model group.(P>0.05);the concentration and percentage of sperm forward movement in the high-dose group are higher than those in the model group(P<0.05).②Sex hormones:There is no significant change in luteinizing hormone(LH)in each group(P>0.05);follicle stimulating hormone(FSH)in the model group was higher than that in the blank group(P<0.05),and the low-dose and high-dose groups were lower than the model group(P<0.05);testosterone(T)in the model group was lower than that in the blank group(P<0.05),and the low-dose and high-dose groups are higher than the model group(P<0.05).③Pathological changes of testicular tissue:The seminiferous seminiferous epithelium in the testicular tissue of the blank group had regular morphology,regular arrangement and complete structure.The spermatogenic tubule lumen showed active division at all levels,regular morphology,regular arrangement and structure.Intact spermatogenic cells and a large number of normal and mature sperm.The seminiferous seminiferous epithelium in the testis of the model group rats had disordered morphology,irregular arrangement and loose structure,and the spermatogenic cells at all levels in the seminiferous tubules were disorderly arranged,and there was a large reduction,even shedding,sperm and spermatogenesis.The number of cells was significantly reduced,and the morphology was significantly abnormal.A large number of vacuoles were seen in the partial degeneration of the seminiferous epithelium.The regularity,arrangement and structural integrity of seminiferous seminiferous epithelium in the testicular tissue of rats in the low-dose group were improved compared with the model group.The number and morphology of normal spermatogenic cells in the seminiferous tubule lumen were improved.The number of normal and mature spermatozoa also increased compared with the model group,and the number of shed spermatogenic cells decreased.The pathological morphology of testis in the high-dose group was further improved in the lower-dose group,which was closer to the blank group.④Changes of apoptosis in testis tissue:a large number of germ cell apoptosis can be seen in the seminiferous tubules of the model group,and the rate of positive cells in rat testis tissue is significantly higher than that in the blank group(P<0.01).Low-dose and high-dose groups can undergo a small amount of germ cell apoptosis in the seminiferous tubules.The rate of apoptotic positive cells in the testis of rats in the low-dose group is significantly lower than that in the model group(P<0.05).The rate of apoptotic positive cells was significantly lower than that in the model group(P<0.01).⑤ Testicular tissue vimentin expression:The blank testicular tissue vimentin is mainly distributed around the nucleus of testicular sertoli cells and extends from the basal area to the proximal cavity.The vimentin signal and distribution range of the testis tissue in the model group is significantly weakened compared with the blank group,and the expression of vimentin is significantly reduced(P<0.001).The main distribution of vimentin in the testis tissue of the low-dose group and the high-dose group extended from the perinuclear region of the testicular sertoli cells to the inner cavity,and the distribution of vimentin also became normal between the two groups,and the expression of vimentin is significantly higher than that of the model group.(P<0.01).⑥ Changes in testicular tissue ERK signaling pathway:p-ERK protein expression in testis tissue of model group is significantly higher than that in blank group(P<0.001);p-ERK protein expression in testis tissue of rats in low-dose and high-dose groups Significantly lower than the model group(P<0.001).Conclusion:1.Runjing Decoction is an empirical formula for the treatment of idiopathic oligospermia developed by the former teacher based on the theory of refined chambers.The drug composition includes Cuscuta,Polygonatum,Yam,Wolfberry,Fairy Spleen,Leech,Acanthopanax,Rhodiola,Chenpi,and Achyranthes bidentata,all the medicines have the characteristics of "taking care of the liver,spleen and kidney,balance of qi,blood,yin and yang,tonic without greasiness,and pass without damage".It has the effects of nourishing blood and nourishing the liver,promoting blood circulation,collaterals,and regulating qi and dampness.Patients in the Jiangnan area are more suitable.2.Runjing decoction and Huaishao Capsule could improve the quality of semen(total number of sperm,sperm concentration,sperm,sperm activity,sperm forward movement percentage),adjust the sex hormone levels and improve the clinical symptoms of traditional Chinese medicine,thus improve the patients’ spouses and clinical pregnancy rate efficiently,and Runjing decoction improvement is more apparent.So Runjing decoction is safe and effective,and no obvious adverse reaction,has certain clinical value.3.Runjing Decoction can improve the sperm quality,serum sex hormones,and histopathological changes of testicular tissue in ornidazole-induced oligozoospermia rats.Preliminary speculation may be that Runjing Decoction may regulate the function of hypothalamus-pituitary-testicular gonadal axis,Regulate the level of reproductive endocrine hormones,improve testicular microcirculation and the local microenvironment of spermatogenesis;In addition,Runjing Decoction can up-regulate the expression of vimentin and decrease the expression of p-ERK,improve the stability of germ cell structure and inhibit the Apoptosis of rat germ cells and promotion of germ cell proliferation may be related to antioxidant stress.更多还原