Systematic analysis of the mechanism of hydroxysafflor yellow A for treating ischemic stroke based on network pharmacology technology.

In this study, we analyzed the mechanism of hydroxysafflor yellow A (HSYA) for treating ischemic stroke (IS) based on network pharmacology tools, and verified the kernel targets via animal experiments. The targets of HSYA were collected via PharmMapper server and the IS-related targets were searched using Genecards, Online Mendelian Inheritance in Man, Therapeutic Target, and Disgenet databases. The targets identified from the above two steps were overlapped to acquire candidate targets involved in the effects of HSYA for treating IS. Subsequently, the Database for Annotation, Visualization, and Integrated Discovery was used for gene ontology analysis and the Kyoto encyclopedia of genes and genomes pathway analysis. Cytoscape 3.7.1 was applied to establish the component-target-pathway network. Potential core targets were obtained by protein-protein interaction analysis. Furthermore, Autodock Vina was used to identify core genes, and animal experiments was used to verify the expression level of core genes. On the basis of the modified neurologic severity score and the results of 2,3,5-Triphenyltetrazolium chloride and Hematoxylin-eosin staining, we confirmed that HSYA reduced the infarct volume in rats and protected neuronal cells in the hippocampal region after IS. Western blot and immunohistochemical staining showed that HSYA increased the expression of epidermal growth factor receptor, hypoxia inducible factor 1 alpha, and endothelial nitric oxide synthase (P < 0.05). The effects of HSYA on IS are mediated through several targets and pathways related to the regulation of oxidative stress and the renewal of cell and blood vessels while improving post-ischemic brain impairment.

A network pharmacology approach to investigate the mechanism of Shuxuening injection in the treatment of ischemic stroke.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuxuening injection (SXNI), a popular herbal medicine, is an extract of Ginkgo biloba leaves (GBE), and is used to treat ischemic stroke (IS) in China. However, its specific active ingredients and molecular mechanisms in IS remain unclear. AIM OF THE STUDY: The purpose of the research is to identify the main active ingredients in GBE and explore its molecular mechanisms in the treatment of IS. MATERIALS AND METHODS: The main active components of GBE were discerned through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis (TCMSP), Traditional Chinese Medicine Integrated Database (TCMID), Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) database, and absorption, distribution, metabolism and excretion (ADME) analysis. The targets related to IS were obtained using Genecards, Online Mendelian Inheritance in Man (OMIM), Therapeutic Target Database (TTD), and Disgenet. We discovered an intersection of genes. Subsequently, protein-protein interaction (PPI) networks were constructed with Cytoscape 3.7.1 and the String database. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to analyze the intersection of targets via the Database for Annotation, Visualization, and Integrated Discovery (DAVID) 6.8. Built on the above analysis, we made a Compound-Target-Pathway (C-T-P) network. Autodock Vina was used for molecular docking analysis. Maestro 11.9 was used to calculate the root-mean-square deviation (RMSD). Animal experiments were performed to verify the core targets. Triphenyl tetrazolium chloride (TTC) staining was used to calculate the infarct volume in rats. Hematoxylin-eosin (HE) staining was employed to observe the morphology of hippocampal neuron cells. RT-qPCR was applied to detect relative mRNA levels, and protein expression was determined using Western blotting. RESULTS: Molecular docking showed that PTGS2, NOS3 and CASP3 docked with small molecule compounds. According to RT-qPCR and Western blotting, mRNA and protein expression of PTGS2 and CASP3 were up-regulated (P < 0.05), and mRNA and protein levels of NOS3 were down-regulated (P < 0.05). CONCLUSIONS: SXNI can treat IS through multiple targets and routes, and reduce the apoptosis of neuron cells in brain tissue by inhibiting inflammation and regulating the level of oxidative stress, thereby protecting rats brain tissue.

Preliminary investigation of the effects of an FCF inhibitor interference with Septin in the early stage embryos in mice.

The objective of the present study was to investigate the effects of for chlorfenuron (FCF) interference with the septin protein on early stage embryos in mice. The 1­cell embryos were collected and divided into an FCF interference group and a control group. The FCF interference group was cultured in FCF media and the control group was cultured in dimethyl sulphoxide media at 37˚C with 5% CO2 until the desired phase was achieved. Septin2 protein expression was detected using immunofluorescence and western blot analysis. Blastocyst α­tubulin was stained by immunofluorescence to observe the alterations in spindles and microtubules. The rate of early embryo development into blastocysts was significantly reduced following FCF treatment (P<0.05). In the control group, septin2 was observed with a confocal microscope; septin2 was expressed in embryos at all stages and mainly in the blastomeres from the 2­cell stage onwards, with the expression concentrated in the nuclei of the blastomeres as identified by strong fluorescence. In the FCF interference group, septin2 was weakly expressed in the nuclei of blastomeres at the 2­ and 4­cell stages, and in the granulated blastomeres at the 4­ and 8­cell stages. Expression was barely observed in and following the morula. Granulation was observed starting from the 4­ and 8­cell stages. Compared with the control group, the FCF interference group exhibited irregular microtubules, abnormal spindle morphology and disordered chromosome arrangement in the blastocysts. The septin2 protein was expressed throughout the early stage embryo from the 2­cell stage to the blastocyst and localized in the nuclei of blastomeres. When the septin protein experienced interference by the FCF inhibitor, septin2 protein expression was reduced, which simultaneously resulted in abnormal embryonic development, uneven cytoplasmic division, various sizes and a reduced number of blastomeres, granulation in the blastomeres, disordered blastocyst microtubule distribution, spindle shape alterations and an abnormality of chromosome arrangement.

[Research progress in obesity-induced low fertility in males].

The prevalence of obesity in men of reproductive age is globally increasing. Obesity alters the ratio of testosterone to estradiol and the homeostasis of leptin and other hormone levels by interfering with the hypothalamus-pituitary-gonadal axis. In addition, it may change epigenetic modifications and intergenerational transmission, which would affect the health of the offspring. Both of the pathways reduce male fertility, which may be associated with the obesity-induced change in the levels of some hormones and consequently the alteration of epigenetic modifications. This review focuses on the adverse effects of obesity on male fertility by influencing endocrine hormones and epigenetic modifications, and further discusses the effects of endocrine hormones on male fertility by epigenetic modification, aiming to provide some basic data for the prevention and treatment of obesity-related male fertility in clinical practice.

Epigenetic dynamics and interplay during spermatogenesis and embryogenesis: implications for male fertility and offspring health.

Mapping epigenetic modifications and identifying their roles in the regulation of spermatogenesis and embryogenesis are essential for gaining fundamental medical understandings and for clinical applications. More and more evidence has shown that specific epigenetic modifications are established during spermatogenesis, which will be transferred into oocyte via fertilisation, and play an important role in the early embryo development. Defects in epigenetic patterns may increase the risk of abnormal spermatogenesis, fertilisation failure, early embryogenesis abnormality and several other complications during pregnancy. This review mainly discusses the relationship between altered epigenetic profiles and reproductive diseases, highlighting how epigenetic defects affect the quality of sperm and embryo.

[Effects of obesity on global genome DNA methylation and gene imprinting in mouse spermatozoa].

OBJECTIVE: To investigate the influence of high fat diet-induced obesity (HFDIO) on the differentially methylated region (DMR) of the imprinted gene and global genome methylation of sperm DNA. METHODS: We performed bisulfite sequencing on the DMR of the imprinted gene and global genome methylation of sperm DNA in the mouse model of HFDIO. RESULTS: No statistically significant differences were found between the HFDIO model and normal control mice in MEG3-IG (93.73 vs 97.26%, P = 0.252), H19 (98.00 vs 97.83%, P = 0.920), IGF2 (97.34 vs 96.25%, P =0.166), IGF2R (1.43 vs 1.11%, P = 0.695), PEG3 (0.19 vs 0.38%, P = 0.537), MEST (0.23 vs 0.68%, P = 0.315), NNAT (0.31 vs 0.00%, P = 0.134), or SNRPN (1.88 vs 3.13%, P = 0.628). A total of 8 942 DMRs were detected across the sperm genome (P <0.05). Gene functional enrichment analysis indicated that the enriched terms with the largest numbers of genes were the metabolic process (n = 1 482), RNA synthesis (n = 779), and transcription (n = 767). CONCLUSIONS: The methylation level underwent no significant change in the DMRs of the imprinted genes from the mice with HFDIO, but the CG methylation of the genes involved in the metabolic process, RNA synthesis and transcription were significantly altered.

[Association of single nucleotide polymorphisms(SNPs) in Pygo2 coding gene with idiopathic oligospermia and azoospermia].

Male infertility is often associated with a decreased sperm count. Pygo2 gene is expressed in the elongating spermatid when chromatin remodeling occurs, thus it is possible that impairment of Pygo2 function could lead to spermatogenic arrest, reduction of sperm count and subsequent infertility. The aim of this study was to detect mutations in Pygo2 that lead to idiopathic oligospermia and azoospermia in human. DNA was isolated from venous blood from 77 fertile and 195 idiopathic oligospermic or azoospermic men. PCR-sequencing analysis was performed for the 3 coding regions of Pygo2. Non-synonymous single nucleotide polymorphisms (SNPs) were detected and analyzed using SIFT, Polyphen-2 and Mutation Taster software to determine possible changes in protein structure that could affect phenotype. Of the 195 patients analyzed, sufficient gene sequencing was accomplished for 178 men (30 mild or moderate oligospermic, 57 severe oligospermic and 91 azoospermic men). Three previously reported non-synonymous SNPs were identified in azoospermic and severe oligospermic patients and not in mild and moderate oligozoopermic or normozoospermic men. SNP rs61758740 (M141I) causes the replacement of a hydrophobic amino acid with another hydrophobic amino acid, rs61758741 (K261E) causes the replacement of a basic amino acid with an acidic amino acid and rs141722381 (N261I) causes the replacement of a hydrophilic amino acid with another hydrophobic amino acid. The data predicted by three different software programs showed that SNP rs141722381 results in the damage of tertiary protein structure and thus could be involved in relevant diseases. The study demonstrates that SNPs in the coding region of Pygo2 gene may be one of the causative factors in idiopathic oligospermia and azoospermia, resulting in male infertility.

Sperm selection based on electrostatic charge.

Charge is a fundamental property of all forms of matter that exhibit attraction or repulsion in the presence of another charged particle. This electrokinetic property occurs when the particles exhibiting a net negative or positive charge are subjected to an external electric field that exerts an electrostatic force between them. Sperm surface membranes exhibit varying levels of electrostatic potential that are proportional to the levels of sialic acid residue acquired on the cell surface during maturation. Electrostatic charge-based sperm separation is a recently developed technique that uses an electric field to isolate mature sperm with reduced levels of DNA fragmentation. Two methods for the separation of sperm based on electrostatic charge, the Zeta method and a commercially available electrophoretic method using the SpermSep Cell Sorter 10, are discussed in this chapter including a detailed protocol for sperm separation based on the Zeta method.

[Methylation and acetylation of histones during spermatogenesis].

Spermatogenesis is a highly complex and unique differentiation process. This process involves development of spermatogonia into spermatocytes, formation of haploid spermatids, and maturation of spermatozoa. It features stage- and testis-specific gene expression, mitotic and meiotic divisions, and the histone-protamine transition. The epigenetic modification plays an important role in meiotic recombination, formation of the synaptonemal complex, sister chromatid cohesion, spermiogenesis during postmeiotic stages, gene expression repression, and heterochromatin formation. The mark of the repressive and/or activating histone methylation and acetylation has a defined composition. It not only ensures proper chromosome pairing and successful bivalent segregation but also mediates highly orchestrated expression of meiosis-specific genes. The incorrect histone methylation and/or acetylation during spermatogenesis will directly affect the establishment and maintenance of epigenetic patterns, resulting in abnormal spermatogenic cells and even male infertility. This article is an effort to review the dynamic changes of methylation and acetylation of histones during spermatogenesis, as well as the regulatory mechanism of the enzymes involved in these processes, which provides some basic information for further study of the epigenetic events during spermatogenesis and the prevention of male infertility.

[The genetic and epigenetic defect from the sperm for intracytoplasmic sperm injection (ICSI)].

Intracytoplasmic sperm injection (ICSI) can be applied to treat male infertility patients of oligospermia, asthenospermia, teratospermia, azoospermia and failure of the common in-vitro fertilization (IVF), which may overcome the sperm deficiency and even obtain sperms directly from percutaneous epididymal sperm aspiration (PESA) and testicular sperm extraction (TESE). As for direct injection of a single sperm into an egg, the ICSI disobeys the biological laws of natural insemination, thus leading to high genetic and epigenetic risk for patients owing to genetic and epigenetic defect of sperm. By reviewing the genetic and epigenetic defects of ICSI sperm, as well as related diseases, this article aims at understanding of the risks resulting from the genetic and epigenetic defects of ICSI sperm at a molecular mechanism level. The results show that the quality control of ICSI sperm via detecting its epigenetic factors, such as methylated DNA and acetylated histone, is essential for reducing the genetic and epigenetic risk from ICSI.

Ultrasound-promoted access to Baylis-Hillman amines.

It is surveyed that the amination of the Baylis-Hillman acetates with primary amines can be dramatically promoted in improved yields and shortened reaction time under ultrasound irradiation than those under conventional stirring. The extensive scope of both amines and acetates are screened to investigate the relationship between substituents and their performance in such transformation.

Ultrasound-promoted synthesis of novel dispirocyclic frameworks from aza-Claisen rearrangements of Baylis-Hillman amines.

A novel tetracyclic frameworks of dispiropyrrolizidines can be obtained in moderate to good yields via the 1,3-dipolar cycloaddition of azomethine ylides with dipolarophiles derived from aza-Claisen rearrangement of Baylis-Hillman amines. The transformations are highly regioselective and stereoselective, affording the desired compounds in reduced time and increased yields under ultrasound irradiation at room temperature. All the products are confirmed by 1H, 13C NMR, IR and MS spectra, while their molecular structures are elucidated by X-ray crystallography of a selected sample.

[Genes involved in spermatogenesis].

During mammalian spermatogenesis, primordial germ cells develop into spermatogonia, giving rise to spermatocytes that undergo two meiotic divisions to become round spermatids. These cells differentiate into spermatozoa during spermiogenesis. Spermatogenesis is a complex process of cell differentiation controlled by many factors, among which gene regulation in the spermatogenic cells plays a pivotal role. Genes important for male gametogenesis involved in highly con-served landmark events such as meiotic recombination, formation of the synaptonemal complex, sister chromatid cohesion, spermiogenesis during postmeiotic stages, and checkpoints and factors required for the meiotic cell cycle. Spermatogenesis is characterized by phase-specific expression of many genes exclusively expressed in the spermatogenic cells. With the development and application of technologies like gene cloning, gene expression and functional characterization, many spermatogenesis-related genes have been found in the past few years, and some of them have been proved to play an important role in spermatogenesis. Here, we review the advances in this field with an emphasis on spermatogenesis-associated genes such as cyclins, proto-oncogenes, azoospermia factor genes, cytoskeleton genes, heat shock genes, nucleoprotein transition genes, centrin genes and apoptosis genes.