Transcriptomic Analysis of Wuzi Yanzongwan on Testicular Spermatogenic Function in Semi-castrated Male Mice / 中国实验方剂学杂志

ObjectiveTo screen out the transcriptomes related to the intervention of Wuzi Yanzongwan on the spermatogenic function of semi-castrated male mice， and to explore its potential mechanism in the intervention of the progress of low spermatogenic function. MethodBalb/c mice were randomly divided into sham-operated group， model group， testosterone propionate group（0.2 mg·kg-1·d-1， intramuscular injection） and Wuzi Yanzongwan group（1.56 g·kg-1·d-1， intragastric administration） according to body weight， with 12 mice in each group. The right testicle and epididymis were extracted from the model group and the drug administration group to construct the semi-castrated model of low spermatogenic function， while the fur and the right scrotum of the sham-operated group were only cut and immediately sterilized and sutured. At the end of the intervention， hematoxylin-eosin（HE） staining was used to observe the histopathology of testis， enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of serum testosterone（T）， luteinizing hormone（LH） and follicle stimulating hormone（FSH）. The sperm count and motility of epididymis were measured by automatic sperm detector of small animal. Transcriptomic microarray technology was used to detect the mRNA expression level of testicular tissue in each group， the transcriptome of genes related to the regulation of Wuzi Yanzongwan was screened， and three mRNAs were selected for Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） to verify the transcriptome data. Through the annotation analysis of Gene Ontology（GO） and the signaling pathway analysis of Kyoto Encyclopedia of Genes and Genomes（KEGG）， the related functions of drugs regulating transcriptome were analyzed. ResultCompared with the sham-operated group， the testicular tissue of mice in the model group showed spermatogenic injury， contraction and vacuolization of the seminiferous tubules， reduction of spermatogenic cells at all levels， widening of the interstitial space， obstruction of spermatogonial cell development and other morphological abnormalities， and serum T significantly decreased， LH significantly increased（P<0.01）， and FSH elevated but no statistically significant difference， the count and vitality of epididymal sperm significantly decreased（P<0.01）. There were 882 differentially expressed mRNAs in the testicular tissues， of which 565 were up-regulated and 317 were down-regulated. Cluster analysis showed that these differentially expressed mRNA could effectively distinguish between the sham-operated group and the model group. Compared with the model group， the damage to testicular tissue in the Wuzi Yanzongwan group was reduced， the structure of the seminiferous tubules was intact， vacuolization was reduced， and the number of spermatogenic cells at all levels was significantly increased and arranged tightly. The serum T significantly increased， LH significantly decreased（P<0.01）， and FSH decreased but the difference was not statistically significant. The count and vitality of sperm in the epididymis were significantly increased（P<0.01）. Moreover， Wuzi Yanzongwan could regulate 159 mRNA levels in the testes of semi-castrated mice， of which 32 were up-regulated and 127 were down-regulated， and the data of the transcriptome assay was verified to be reliable by Real-time PCR. GO and KEGG analysis showed that the transcriptome functions regulated by Wuzi Yanzongwan were involved in the whole cell cycle process of sperm development such as sex hormone production of interstitial cells in testis， renewal， differentiation， metabolism， apoptosis and signal transduction of spermatogenic cells， and were closely related to the biological behaviors of signaling pathways such as spermatogenic stem cell function， endoplasmic reticulum protein processing and metabolic program. ConclusionWuzi Yanzongwan can effectively improve the low spermatogenic function of semi-castrated male mice， and its mechanism may be related to the regulation of testicular transcriptional regulatory network， the synthesis of sex hormones in testicular interstitial cells， the function of spermatogenic stem cells， the whole cell cycle process of spermatogenesis， as well as the expression of endoplasmic reticulum protein processing and metabolic program related genes transcription.

Animal Experiments on Male Infertility Treated by Tonifying Kidney / 中国实验方剂学杂志

Experimental research on male infertility is critical to the study of the pathogenesis of male infertility and the evaluation of drug therapy. This paper reviewed animal experiments on male infertility in recent years. The experimental models of male infertility mainly include oligoasthenozoospermia （OA），teratozoospermia，azoospermia， and varicocele animal models. The OA animal models are mostly induced by glycosides of Tripterygium wilfordii （GTW）， adenine，hydrocortisone， and radiation，which are mainly chemical means. The animal models of azoospermia were usually constructed by intraperitoneal injection of bissulfonyl alkylating agent busulfan and immersion of scrotum in 43 ℃ water. There are few studies on animal models of teratozoospermia，and the induction methods by GTW and methyl methanesulfonate（MMS） are common. The animal models of varicocele-caused infertility are usually induced by operation. The ligation of the middle division of the left renal vein between the lateral inferior vena cava and the medial spermatic vein has a significant influence on testicular morphology and epididymal sperm quality. Animal experimental studies have shown that classic prescriptions for tonifying the kidney and promoting spermatogenesis represented by Wuzi Yanzongwan and clinical empirical prescriptions by modern research have played a significant role in the treatment of male infertility. The mechanism of tonifying the kidney in the treatment of male infertility mainly focuses on inhibiting spermatogenic cell apoptosis. The kidney-tonifying method can regulate the apoptosis of spermatogenic cells，which provides a new treatment idea and a reliable scientific basis for traditional Chinese medicine in the field of male reproduction.

Anti-testicular Inflammation Mechanism of Wuzi Yanzongwan Based on Network Pharmacology / 中国实验方剂学杂志

ObjectiveTo explore the anti-testicular inflammation mechanism of Wuzi Yanzongwan through network pharmacological analysis and experimental verification. MethodThe active ingredients of Wuzi Yanzongwan were searched in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）， and their targets were predicted via SwissTargetPredicition. GeneCards and Online Mendelian Inheritance in Man （OMIM） were used to obtain the known targets for the treatment of testicular inflammation. STRING 11.0 was employed to construct the protein-protein interaction （PPI） network， and Cytoscape 3.7.1 to construct the “herbal medicine-active ingredient-target” network of Wuzi Yanzongwan and “active ingredient-disease target” network. Metascape was used for gene ontology （GO） annotation and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment of signaling pathways. ResultA total of 72 active ingredients and 624 potential targets of Wuzi Yanzongwan were screened out， and 196 key targets were identified for the treatment of testicular inflammation by Wuzi Yanzongwan. The results of GO annotation showed that the anti-testicular inflammation targets of Wuzi Yanzongwan were involved in the biological processes such as the response to molecule of bacterial origin， the positive regulation of response to external stimulus， and the response to extracellular stimulus. KEGG pathway enrichment revealed that the major pathways associated with the treatment （P<0.01） included the pathways in cancer， epidermal growth factor receptor （EGFR） tyrosine kinase inhibitor resistance， transcriptional misregulation in cancer， calcium signaling pathway， apelin signaling pathway， and NOD-like receptor signaling pathway. Wuzi Yanzongwan may exert the anti-testicular inflammation effect by inhibiting the activation of the NLR family pyrin domain-containing 3 （NLRP3） inflammasome pathway to alleviate the inflammatory response， which verifies the prediction results based on network pharmacology to a certain extent. ConclusionThe inhibitory effect of Wuzi Yanzongwan on testicular inflammation is multi-target， multi-pathway， and multi-mechanism. The findings of this study provide evidence support for the clinical application of Wuzi Yanzongwan.

Efficacy and Mechanism of Yishen Shengjing Prescription in Treatment of Oligoasthenospermia / 中国实验方剂学杂志

ObjectiveWe aimed to investigate the efficacy and mechanism of Yishen Shengjing Prescription （YSP） in the treatment of oligoasthenospermia in rats. MethodThe oligoasthenospermia rat model was established by injection with cyclophosphamide （35 mg·kg-1·d-1） for 5 consecutive days. Rats were randomly assigned into control group （without treating with cyclophosphamide）， model group， low- （YSP-L）， medium- （YSP-M）， and high- （YSP-H） dose （2.91， 5.83， and 11.66 g·kg-1， respectively） groups， Wuzi Yanzongwan （WYW， 1.03 g·kg-1） group， and L-carnitine （0.17 g·kg-1） group， with 8 rats in each group. After 28 days of drug intervention， the body weight， testicular weight， and testicular index of rats were recorded. The sperm quality in epididymis was detected by computer-assisted sperm analysis （CASA） system. Hematoxylin-eosin （HE） staining was employed for observation of testicular tissue morphology. The levels of malondialdehyde （MDA） and superoxide dismutase （SOD） in testicular tissue were detected by colorimetry. The levels of follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and testosterone （T） in serum were determined by enzyme-linked immunosorbent assay（ELISA）. TdT-mediated dUTP nick-end labeling （TUNEL） was employed to detect the apoptosis of testicular cells. The protein levels of B cell lymphoma-2（Bcl-2）， Bax， and cleaved Caspase-3 in testicular tissue were detected by Western blot. ResultCompared with the control group， the model group showed decreased body weight， testicular weight and index， sperm concentration and motility （P<0.01） and increased testicular pathological score （P<0.01）. Compared with the model group， the YSP-M， YSP-H， WYW， and L-carnitine groups showed increased body weight， testicular weight， testicular index， sperm concentration and motility and decreased testicular pathological score. After modeling， the SOD level decreased （P<0.01） while the MDA content increased （P<0.01） in the testicular tissue. YSP-H， WYW， and L-carnitine reversed the SOD and MDA level changes caused by modeling. Compared with the control group， the model group exhibited declined T level （P<0.01） and increased FSH and LH levels （P<0.01）. Compared with the model group， YSP， WYW， and L-carnitine increased the T level （P<0.01） and decreased the LH level （P<0.05， P<0.01）. The apoptosis rate of spermatogenic cells in the model group was higher than that in the control group （P<0.01）， whereas YSP-M， WYW， and L-carnitine reversed such changes （P<0.01）. The model group rats showed decreased expression of Bcl-2（P<0.05） and increased expression of Bax and cleaved Caspase-3 （P<0.05， P<0.01）. Compared the model group， YSP-M， YSP-H， WYW， and L-carnitine up-regulated the Bcl-2 expression and down-regulated the cleaved Caspase-3 expression （P<0.05， P<0.01）. ConclusionYSP improved the sperm quality of oligoasthenospermia model rats by regulating the antioxidant system and sex hormone levels and inhibiting the apoptosis of spermatogenic cells.

Effect of Modified Wuzi Yanzongwan Periodic Staging Treatment on Outcome of Assisted Pregnancy in Patients with Poor Ovarian Response / 中国实验方剂学杂志

Objective:To observe the effect of modified Wuzi Yanzongwan periodic staging treatment on the outcome of assisted pregnancy in patients with poor ovarian response （POR） and kidney deficiency syndrome. Method:One hundred and four patients were randomly divided into observation group and control group， with 52 cases in each group. Both groups received gonadotropin releasing hormone （GnRH） antagonist regimen. The patients in control group additionally took Bushen Yutaiwan orally， 5 g/time， 3 times/day. The patients in observation group additionally took modified Wuzi Yanzongwan during pre-ovulation and post-ovulation periods， 1 dose/day. The treatment courses were 3 menstrual cycles （or termination after clinical pregnancy） in both groups. The number of eggs obtained， the number of available embryos， the number of fertilization， the number of high-quality embryos， the number of embryos implanted， the number of cycles cancelled， and the clinical pregnancy were recorded. Human chorionic gonadotropin （HCG） was injected， and then follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， estrogen （E₂）， anti-Müllerian hormone （AMH）， basal antral follicle count （AFC） and endometrial thickness were measured daily. The number of days and dosage of Gn used， scores of kidney deficiency syndrome were recorded before and after treatment， and the adverse reactions during the study period were recorded. Result:The number of eggs captured， rate of harvested eggs， number of available embryos， rate of available embryos， number of high-quality embryos， rate of high-quality embryos， and fertilization rate in observation group were higher than those in control group （<italic>P</italic><0.05 or <italic>P</italic><0.01）. The cycle cancellation rate was lower than that in the control group； the embryo implantation rate and clinical pregnancy rate were superior than those in control group， but the difference was not statistically significant. The FSH level and FSH/LH ratio in observation group were lower than those in control group during HCG day （<italic>P</italic><0.01）， while E₂， AMH， AFC and endometrial thickness were higher than those in control group （<italic>P</italic><0.01）. Simultaneously， the number of days and amount of Gn used in observation group was lower than that in control group （<italic>P</italic><0.01）. Conclusion:The Modified Wuzi Yanzongwan periodic staging treatment combined with GnRH antagonist scheme for patients with POR kidney deficiency syndrome， can regulate the level of endocrine hormones， promote follicular development， improve ovarian reserve， increase the number of eggs obtained， improve egg quality， help improve pregnancy outcomes， and increase the chances of successful pregnancy with assisted reproductive technology. It is worthy of further clinical research.

Network Pharmacology Research of Wuzi Yanzongwan in Treatment of Male Infertility / 中国实验方剂学杂志

Objective:Exploring the material basis and mechanism of Wuzi Yanzongwan in the treatment of male infertility based on network pharmacology. Method:Traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP) was used to screen the active ingredients and targets in Wuzi Yanzongwan. GeneCards, OMIM and PharmGkb databases were used to screen the targets of male infertility. R language software was used to screen common targets of drugs and diseases, Pharmaceutical active ingredients-disease target interaction network was constructed by using Cytoscape software. The common target protein interaction network (PPI) was constructed by STRING platform, the gene ontology(GO) analysis of common target was analyzed by ClueGo plug-in, and Kyoto encyclopedia of genes and genomes(KEGG) pathway was enriched by R language software. Result:A total of 72 active ingredients were obtained from Wuzi Yanzongwan, and 35 possible targets for the treatment of male infertility were obtained. These targets are mainly involved in biological processes such as oxidation and antioxidant activity, and are mainly concentrated in phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt) and hypoxia inducible factor-1(HIF-1) signaling pathways. Conclusion:The network pharmacology confirmed the multi-component, multi-target and multi-pathway action characteristics of Wuzi Yanzongwan, predicted the possible mechanism of Wuzi Yanzongwan in the treatment of male infertility, and provided theoretical basis for further study of its active ingredients and mechanism.