| Sterility has long been one of the research hotspots and difficulties in andrology. It is reported that oligoasthenospermia are responsible for three quarters of all the cases."Kidney deficiency, coupled with damp heat, blood stasis, toxin and bacteria" are identified by Professor Wang Qi through clinical practice as the pathogenesis of oligoasthenospermia. This is followed by the development of HuangJingZanyu Capsule, the first spermatogenic prescription of its kind in China for the treatment of serility,whose positive therapeutic effect is well recieved clinically.Huangjing Zanyu Capsule consists of20herbs in total, including huangjing, zhishouwu, tusizi, gouqizi, danshen and pugongying, etc. Since the ingredients are complex and the pharmacological functions are multi-targeted and the overall therapeutic mechanism hasn’t been fully understood, therefore, effective research ideas and approaches are needed to investigate the active components, its therapeutic effect and mechanisms to ensure safety and and preparation of the decoction.This thesis is divided into two sections-literature review and experimental study.Literature review systematically summarized the current research on sterility from the perspective of TCM (Traditonal Chinese Medicine) in recent3years. It discussed the mechanism of relevant formulas, etiology and pathogenesis and syndrome differentiation by literature retrieval, experimental study and clinical study. The regulating functions of testis in sperm production and its associated factors are discussed from the perspective of modern medicine. Hormonal regulation, immune regulation, pharmacological regulation and gene regulation in its production of sperm were explored, particularly on the regulatory mechanism of apoptosis on sperm production. It also reviewed the clinical and experimental research of Huangjing Zanyu Capsule in recent years, which paved way for further research.This experimental study contains eight parts.Objective:It intends to establish the fingerprint spectrum of HPLC and MS which are in vitro chemical components of Huangjing Zanyu Capsule and the dynamic fingerprint spectrum of UPLC-MS through chemical composition in vitro and in vivo serum transitional components analysis; build oligoasthenospermia rat model and discuss changes of sperm quality, endocrine hormone, testis pathological morphology, changes in the level of apoptosis-related factors Caspase-3, Bax, protein Bcl-2,mRNA expression as well as gene expression of the testis; provide foundation for future reaseach on active component analysis and molecular mechanism of Huangjing Zanyu Capsule. Methods:Fingerprint technology and a variety of detection methods are used to analyze the ingredients of Huangjing Zanyu Capsule in vitro studies and drug-induced substance in serum was also examined. Oligoasthenospermia model was also created by cyclophosphamide; semen analysis, Elisa, light microscopy, electron microscopy, immunohistochemistry, Western Blot, gene chip technology and other methods were also involved. Subjects were devided into the normal group, model group, Huangjing Zanyuhigh-medium-low dose group, Wuziyanzong pill control group andl-carnitine control group. Serum sex hormone (FSH, LH, T, E2, PRL) examination, testicular tissue morphology, ultrastructure and the apoptosis-related factors Bcl-2, Bax and Caspase3expression were also examined in each group. We Also screened and analyzed differentially expressed genesmicroarray of testicular tissue and detected the effect that deoxyschizandrin-an active ingredient extracted from Huangjing Zanyu Capsule-has on Leydig cells vitality through MTT method.Results:1. Established Huangjing Zanyu Capsule in vitro database which contains more than1000kinds of chemical composition, combined with high resolution mass spectrometry and standard contrast analysis, for the first time found betaine, represented by Gouqi; icariinA, icariinB, icariinC, anhydroicaritin-3-O-α-L-rhamnose-7-O-β-D-glucopyranoside, Wushan epimedium flavone glycosides,2"-O-Rhamnose epimedium flavone glycosidesⅡ, Rhamnose dehydration epimedium,3-O-alpha-L-pyran rat sugar-based li-(1→2)-alpha-L-pyran buckthorn glycoside and treasure grandiflorum glycosides I epimedi um flavone glycosides, represented by Yinyanghuo; Fructus schisandrae shell element, Mr M D, Mr M J, xin xin, Mr M O O watch Mr M, Mr Sheen, before the fruit of Chinese magnoliavine element, fructus schisandrae butyl ester sheen, Mr M C, m G, pseudo-gamma fructus schisandrae, fructus schisandraeC, represented by Wuweizi; Jade leaf golden flower acid, represented by Roucongrong; Swertia food glycosides and also saponins D, represented by Xuduan; Tanshinone Ⅱ a, tanshinone Ⅱ a, tanshinone Cain and isoimperatorin, represented by Danshen; Anise lactone and parsley phenol-7-methyl ether, represented by Danggui; Loganin, represented by Baijiangcao; Celery-7-O-glycosidase, represented by Cheqianzi and Pugongying. It demenstrated its scientific ground through the analysis of the chemical components of Huangjing Zanyu Capsule, which can cure oligoasthenospermia by targeting its pathogenesis-kidney deficiency combined with damp heat, blood stasis, toxin and bacteria. 2.Seven Serum effective active components of Huangjing Zanyu Capsule was found, including sweroside(Xuduan), gomisinD(Wuweizi), isoimperatorin(Danshen), gomisinC(Wuweizi), gomisinG(Wuweizi), schizandrin(Wuweizi), Pseudo-y-schizandrin(Wuweizi) and this provided a basis for the index selection of the quality control of Huangjing Zanyu Capsule.3. Proved that cyclophosphamide successfully copied oligoasthenospermiarats model by observing the general condition, testis, epididymis index, sperm routine and testicular pathological morphology observation of the rats.4.Cyclophosphamide leads to a decline in the vitality of sperm and a decrease in the amount of sperm and Huangjing Zanyu Capsule can significantly improve the sperm motility of rats (P<0.05) and increase sperm density (P<0.05), therefore improve the overall qualityof the sperm of the model rats.5.Model rats appeared to be hypothalamus-pituitary-gonadal axis dysfunction and Huangjing Zanyu Capsule can restore the balance of sex hormone level and improve the function of testicular sperm production.6.Screened differently expressed genes1241in total between normal group and model groupwhich contains739up-regulated genes and502down-regulated genes; Screened the differently expressed genes774in total between Huangjing Zanyu group and model group which contains420up-regulated genes and354down-regulated genes.Differently expressed genes are mainly distributed in eight signaling pathways such as cell apoptosis pathways, cellular adhesion molecular signaling pathways, neurotrophic factor signaling pathways, olfactory transduction pathways, biosynthetic pathway of steroid hormones, metabolic pathways, metabolism pathways of retinol and arachidonic acid,etc. It is also discussed that Huangjing Zanyu Capsule can make the down-regulated genes-zfp37and StAR in the model group to rise up and can make the up-regulated gene-Bax to go down. Six new key genes-Adam7, Lcn5, Slc7a8, Hoxa10, Slc4a9and Vom2r57were found through the mas3.0system analysis and this has laid foundation for the further experiment.7. In the testicular tissue of the model group rats induced by cyclophosphamide, protein Bcl-2and mRNA have a down-regulated expression and protein Bax, Caspase3and mRNA have an up-regulated expression, indicate that CP can cause the dysfunction of testicular sperm production through apoptosis, which further leads to oligoasthenospermia. Huangjing Zanyu Capsule can trigger a distinct up-regulated expression of gene Bcl-2’s protein and mRNA and a down-regulated expression of Bax and Caspase3’s protein and mRNA. 8. A10umol/L concentration of deoxyschizandrin which extracted from Huangjing Zanyu Capsule can significantly promote the proliferation of leydig cells (P<0.05), while the effect will be inhibited when the concentration is50umol/L or higher.Conclusion:Found32chemical components in vitro Huangjing Zanyu Capsule for the first time and7serum effective active components are included. Preliminarily analyzed the scientificity of the core pathogenesis that explains the therapeutic effect of Huangjing Zanyu Capsule in treating sterility. Huangjing Zanyu Capsule can significantly improve the sperm motility and quantity, improve the morphology and ultrastructure of testicular tissue, and promote gonadal axis functional recovery. Screened the differently expressed genes1241in total between normal group and model groupwhich contains739up-regulated genes and502down-regulated genes; Screened the differently expressed genes774in total between Huangjing Zanyu group and model group which contains420up-regulated genes and354down-regulated genes. These differently expressed genes can play a role in the regulation of testicular sperm production function through cell cycle, metabolic pathway, signal transduction and other6pathways in order to improve the sperm quality of the model rats. Huangjing Zanyu Capsule can control the apoptosis of spermatogenic cells through enhancing the expression of anti-apoptosis gene Bcl-2’s protein and mRNA and inhibiting the expression of apoptosis gene Bax’ protein and mRNA, in order to reduce the activity of Caspase-3which is known as a apoptotic factor and control the apoptosis of sperm production cells to promote the production of sperm and improve the quality of semen. Such mechanism is likely to be associated with an active ingredient deoxyschizandrin extracted from Huangjing Zanyu Capsule.
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