[Analysis of a child with 46,XY Disorder of sex development due to a novel variant of NR5A1 gene].

OBJECTIVE: To analyze the clinical features and genetic basis of a child with Disorder of sex development (DSD). METHODS: A child who was admitted to the Linyi People's Hospital for primary amenorrhoea on July 29, 2019 was selected as the study subject. Clinical data of the child was collected. Chromosomal karyotyping and quantitative real-time PCR were used to detect Y chromosome microdeletions and other chromosomal aberrations. Next-generation sequencing was carried out for the child and her parents. Candidate variant was verified by Sanger sequencing and bioinformatic analysis. RESULTS: The child, a 13-year-old girl, has featured primary amenorrhoea and onset of secondary sex characteristics of males. Ultrasound exam had detected no uterus and definite ovarian structure, but narrow band vaginal hypoecho and curved cavernoid structure. The child was found to have a 46,XY karyotype without an AZF deletion. DNA sequencing revealed that she has harbored a maternally derived c.323delA (p.Q108Rfs*188) variant in the nuclear receptor subfamily 5 group A member 1 (NR5A1) gene, which may result in a truncated protein. The variant was classified as pathogenic (PVS1+PM2_Supporting+PP4) based on the guidelines from the American College of Medical Genetics and Genomics. CONCLUSION: The NR5A1: c.323delA variant probably underlay the pathogenesis of 46,XY DSD in this child. The discovery of the novel variant has enriched the mutational spectrum of the NR5A1 gene and provided a basis for clinical diagnosis, treatment and prenatal diagnosis.

Stellera chamaejasme L. extracts in the treatment of glioblastoma cell lines: Biological verification based on a network pharmacology approach.

Background: Stellera chamaejasme L (RXLD) has been demonstrated with good clinical effects and medicinal value in the treatment of cancer in vivo and in vitro. Specifically, RXLD can eliminate aggregation accumulation, which is depicted as a vital characteristic feature of intracranial tumors. The potential pharmacological mechanisms of anti-glioblastoma (GBM) have not been adequately identified. Methods: The 3D structures of the chemical ingredients in RXLD were imported into the PharmMapper database to construct the pharmacophore models. The gene targets of GBM were obtained from databases. The pharmacophore-targets network and the protein-protein interactions (PPI) were constructed using the String database and were visualized by using Cytoscape. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were conducted using Bioconductor software. Cytoscape visualized the relationship of pathways and candidate genes to screen for key target genes. Software packages PyMOL, AutoDock, and Vina acquired the molecular docking results. In vitro experiments were undertaken to characterize RXLD extracts' effects on A172 cell line proliferation, viability, apoptosis, cell cycle, cell wound healing, cell migration, reactive oxygen species generation, and mitochondrial membrane potential. The expression of core genes in the related pathways was detected by Western blotting. Results: We identified 216 potential targets associated with GBM. The core components in RXLD were neochamaejasmin A, wikstrol A, isochamaejasmin, chamaejasmine, and subtoxin A. The undertaken GO enrichment analysis revealed that oxidative stress, cell proliferation, cell cycle, cell invasion, and cell migration were involved in the biological processes. The KEGG enrichment analysis revealed that the crucial pathway was MAPK pathway, while HRAS, PRKCB, MAPK9, CCND1, and TP53 were distributed in core locations. A total of seven RXLD pharmacophores demonstrated strong spontaneous docking activities with MAPK9. In vitro assays indicated that RXLD can induce apoptosis, block the cell cycle in the G2/M and S phases, inhibit cell migration via the Wnt/ß-catenin pathway, and inhibited p62/Nrf2 pathway. Conclusions: We speculate that the RAS/MAPK pathway might be an upstream pathway through which the RXLD exerts its anti-GBM effects and might be able to regulate further the Wnt/ß-catenin, the oxidative stress, and the ferroptosis pathways.

Ursolic Acid, a Natural Nutraceutical Agent, Targets Caspase3 and Alleviates Inflammation-Associated Downstream Signal Transduction.

SCOPE: Ursolic acid (UA) is a pentacyclicterpenoid carboxylic acid that is present in a wide variety of plant foods. There are many beneficial health effects that are attributed to the properties of UA. However, the specific cellular targets of UA and the mechanism underlying downstream signal transduction processes linked to the anti-inflammation pathway have not been thoroughly elucidated to date. METHODS AND RESULTS: Chemical biology strategies such as target fishing, click reaction synthesis of a UA probe and molecular imaging were used to identify potential target proteins of UA. Cysteinyl aspartate specific proteinase 3 (CASP3) and its downstream signaling pathway were verified as potential targets by molecular docking, intracellular enzyme activity evaluation and accurate pathway analysis. The results indicated that UA acted on CASP3, ERK1 and JNK2 targets, alleviated inflammation-associated downstream multiple signal transduction factors, including ERK1, NF-κB and STAT3, and exhibited anti-inflammation activities. CONCLUSION: As a natural dietary supplement, UA demonstrated anti-inflammation activity via inhibition of CASP3 and shows the potential to improve the therapy effect of several inflammation-associated diseases.

Mahuannin B an adenylate cyclase inhibitor attenuates hyperhidrosis via suppressing ß2-adrenoceptor/cAMP signaling pathway.

BACKGROUND: Based on the traditional application of traditional Chinese Medicines (TCMs), Ephedra Herba (EH) is used to cure cold fever by inducing sweating, whereas Ephedra Radix (ER) is used to treat hyperhidrosis. Although they come from the same plant, Ephedra sinica Stapf, but have play opposing roles in clinical applications. EH is known to contain ephedrine alkaloids, which is the driver of the physiological changes in sweating, heart rate and blood pressure. However, the active pharmacological ingredients (APIs) of ER and the mechanisms by which it restricts sweating remain unknown. PURPOSE: The current work aims to discover the hidroschesis APIs from ER, as well as to establish its action mechanism. METHODS: UPLC-Q/TOF-MS, PCA, and heat map were utilized for identifying the differences between EH and ER. HPLC integrated with a ß2-adrenoceptor (ß2-AR) activity luciferase reporter assay system was used to screen active inhibitors; molecular docking and a series of biological assays centered on ß2-AR-related signaling pathways were evaluated to understand the roles of APIs. RESULTS: The opposite effect on sweating of EH and ER can be attributed to the APIs of amphetamine-type alkaloids and flavonoid derivatives. Mahuannin B is an effective anti-hydrotic agent, inhibiting the production of cAMP via suppression of adenylate cyclase (AC) activity. CONCLUSION: The effects of EH and ER on sweat and ß2-AR-related signaling pathway are opposite due to different alkaloids and flavonoids of APIs in EH and ER. The present work not only sheds light on the hidroschesis action of mahuannin B, but also presents a potential target of AC in the treatment of hyperhidrosis.

Biodistribution of arctigenin-loaded nanoparticles designed for multimodal imaging.

BACKGROUND: Tracking targets of natural products is one of the most challenging issues in fields ranging from pharmacognosy to biomedicine. It is widely recognized that the biocompatible nanoparticle (NP) could function as a "key" that opens the target "lock". RESULTS: We report a functionalized poly-lysine NP technique that can monitor the target protein of arctigenin (ATG) in vivo non-invasively. The NPs were synthesized, and their morphologies and surface chemical properties were characterized by transmission electron microscopy (TEM), laser particle size analysis and atomic force microscopy (AFM). In addition, we studied the localization of ATG at the level of the cell and the whole animal (zebrafish and mice). We demonstrated that fluorescent NPs could be ideal carriers in the development of a feasible method for target identification. The distributions of the target proteins were found to be consistent with the pharmacological action of ATG at the cellular and whole-organism levels. CONCLUSIONS: The results indicated that functionalized poly-lysine NPs could be valuable in the multimodal imaging of arctigenin.

Cimicifugamide from Cimicifuga rhizomes functions as a nonselective ß-AR agonist for cardiac and sudorific effects.

Cimicifuga rhizomes (CR) are used in the treatment of respiratory and cardiovascular diseases in traditional Chinese medicine, but their key effective components and mechanism of action have not yet been reported. In this study, the cardiac, antipyretic and sudorific effects of CR were evaluated using the toad heart failure in vitro model and mice fever and sweating in vivo models. Moreover, the UPLC/Q-TOF-MS-integrated ß2-AR luciferase reporter gene assay system was used to screen the bioactive ingredients from CR extract, and the activity of this ingredient were verified using the above-mentioned in vitro and vivo models. Our results showed that CR had anti-heart failure, antipyretic and sweating effects, which could be antagonized by propranolol. On the other hand, cimicifugamide was screened as ß2-AR agonist from CR and cimicifugamide could activate ß1, 2-ARs more significantly than ß3-AR in ß-ARs selectivity assessment. The results not only revealed the key effective components and mechanism of CR in traditional use but also supplied a characteristic complementary ingredient for quality control of CR.

Comparison and evaluation of antimuscarinic and anti-inflammatory effects of five Bulbus fritillariae species based on UPLC-Q/TOF integrated dual-luciferase reporter assay, PCA and ANN analysis.

Many species of Bulbus fritillariae are used as traditional medicines for thousands of years; however, their application is not standardized. To clarify the differences and homologies, the antimuscarinic and anti-inflammatory effects of five BM species were firstly tested and compared at cellular level. With an integrated strategy combining UPLC-Q/TOF MS, PCA and ANN analysis, the active ingredients among 28 different chemical markers were predicted and identified. SB and QB extracts showed the best antimuscarinic effects and several steroidal alkaloids, such as solanidine, contributed to this effects. However, ZB was superior to reduce the inflammatory response. Another five components were responsible by decreasing the expression of NF-κB, including puqiedine, zhepeiresinol, 2-monopalmitin, N-demethylpuqietinone, and isoverticine. More novelty, a new cluster of five BM species based on active ingredients as potential quality markers was depicted to illustrate their functions. These results of the study could make a reference for the medicinal application of BM species in clinic; and the integrated strategy provided an effective method to obtain the quality markers from medical herbs, which was helpful for the quality control of traditional medicinal products.

Screening and identification of Caulis Sinomenii bioactive ingredients with dual-target NF-κB inhibition and ß2- AR agonizing activities.

Caulis Sinomenii (CS) is a valuable traditional medicine in China. Its extract can act as an anti-inflammatory agent and a vascular smooth muscle relaxant. However, the underlying mechanisms remain unknown. In this study, we developed a simple dual-target method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry combined with a dual-target bioactive screening assay for anti-inflammatory and antispasmodic activities to characterize the chemical structure of various bioactive compounds of CS rapidly. Seven potential NF-κB inhibitors were identified, including laudanosoline-1-O-xylopyranose, 6-O-methyl-laudanosoline-1-O-glucopyranoside, menisperine, sinomenine, laurifoline, magnoflorine and norsinoacutin. Furthermore, IL-6 and IL-8 assays confirmed the anti-inflammatory effects of these potential NF-κB inhibitors, in which laudanosoline-1-O-d-xylopyranose and menisperine were revealed as novel NF-κB inhibitors. Among the seven identified alkaloids, three potential ß2 -adrenergic receptor agonists, including sinomenine, magnoflorine and laurifoline, were characterized using a luciferase reporter system to measure for the activity of ß2 -adrenergic receptor agonists. Finally, sinomenine, magnoflorine and laurifoline were identified not only as potential NF-κB inhibitors but also as potential ß2 -adrenegic receptor agonists, which is the first time this has been reported. Molecular dynamic simulation and docking results suggest that the three dual-bioactive constituents could not only inhibit Pseudomonas aeruginosa PAK strain-induced inflammatory responses via a negative regulation of the Braf protein that participates in MAPK signaling pathway but also activate the ß2 -adrenegic receptor. These results suggest that CS extract has dual signaling activities with potential clinical application as a novel drug for asthma.

Looking for agonists of ß2 adrenergic receptor from Fuzi and Chuanwu by virtual screening and dual-luciferase reporter assay.

More and more studies demonstrated that ß2 adrenergic receptor (ß2-AR) plays a crucial role for the treatment of heart failure. Chuanwu and Fuzi have been used over thousands of years in China for the treatment of heart failure. Considering the effects of these herbs are very similar to ß2-AR agonists, we presume whether ß2-AR agonists can be found from Fuzi and Chuanwu. Fuzi and Chuanwu decoction were used to receive the luciferase reporter activity assay to verify the hypothesis, and the result is positive and encouraging. For it is very difficult to get all of the monomer compounds of Fuzi and Chuanwu, virtual screening was used to find potential ß2-AR agonists and a cell-based ß2-AR agonist functional evaluation model, combined with a luciferase reporter assay system, was used to confirm the final result. In this research, 45 compounds were identified as ß2-AR agonists, and four compounds were verified and the rest need further experiment.

The GPER agonist G-1 induces mitotic arrest and apoptosis in human vascular smooth muscle cells independent of GPER.

The G protein-coupled estrogen receptor (GPER) has been implicated in the regulation of smooth muscle cell (SMC) proliferation. The GPER selective agonist G-1 has been a useful tool for exploring the biological roles of GPER in a variety of experimental settings, including SMC proliferation. The present study, originally designed to investigate cellular and signaling mechanisms underlying the regulatory role of GPER in vascular SMC proliferation using G-1, unexpectedly revealed off-target effects of G-1. G-1(1-10 µM) inhibited bromodeoxyuridine (BrdU) incorporation of human SMCs and caused G2/M cell accumulation. G-1 treatment also increased mitotic index concurrent with a decrease in phosphorylation of Cdk1 (Tyr 15) and an increase in phosphorylation of the mitotic checkpoint protein BuBR1. Furthermore, G-1 caused microtubule disruption, mitotic spindle damage, and tubulin depolymerization. G-1 induced cell apoptosis as indicated by the appearance of TUNEL-positive and annexin V-positive cells with enhanced cleavage of caspases 3 and 9. However, neither the GPER antagonist G-15 nor the MAPK kinase inhibitor PD98059 prevented these G-1 effects. Down-regulation of GPER or p44/42 MAPK with siRNA transfection also did not affect the G-1-induced apoptosis. We conclude that G-1 inhibits proliferation of SMCs through mechanisms involving mitotic arrest and apoptosis, independent of GPER and the MAPK pathway.

Arctigenin exhibits relaxation effect on bronchus by affecting transmembrane flow of calcium.

Arctigenin, a lignan extract from Arctium lappa (L.), exhibits anti-inflammation, antioxidation, vasodilator effects, etc. However, the effects of arctigenin on bronchus relaxation are not well investigated. This study aimed to investigate how arctigenin regulates bronchus tone and calcium ion (Ca(2+)) flow. Trachea strips of guinea pigs were prepared for testing the relaxation effect of arctigenin to acetylcholine, histamine, KCl, and CaCl2, respectively. Furthermore, L-type calcium channel currents were detected by patch-clamp, and intracellular Ca(2+) concentration was detected by confocal microscopy. The results showed that arctigenin exhibited relaxation effect on tracheae to different constrictors, and this was related to decreasing cytoplasmic Ca(2+) concentration by inhibiting Ca(2+) influx partly through L-type calcium channel as well as promoting Ca(2+) efflux. In summary, this study provides new insight into the mechanisms by which arctigenin exhibits relaxation effect on bronchus and suggests its potential use for airway disease therapy.

Uridine adenosine tetraphosphate induces contraction of circular and longitudinal gastric smooth muscle by distinct signaling pathways.

Extracellular nucleotides uridine-5'-triphosphate (UTP) and adenosine-5'-triphosphate (ATP) induce contraction of gastric smooth muscle (SM). The dinucleotide uridine adenosine tetraphosphate (Up4 A), an endothelium-derived contraction factor, induces vascular SM contraction. Its effect on gastric SM contractions, however, is unknown. We addressed the hypothesis that Up4 A induces gastric SM contraction via a mechanism that may differ between circular and longitudinal muscle (CM and LM, respectively). CM and LM were isolated from rat gastric fundus for the measurement of isometric tension. Up4 A induced transient contractile responses in both CM and LM, which were similar to those induced by ATP and UTP. Up4 A failed to induce contraction of either LM or CM in the absence of extracellular Ca(2+) or in the presence of nimodipine, an inhibitor of voltage-gated Ca(2+) channels. P2X1, 2, 4, 5 and 7 and P2Y1, 2, 4 and 6 receptor expression was detected in gastric SM by reverse transcription-polymerase chain reaction. IP5 I (a P2X receptor antagonist) and α,ß-methylene-ATP (a P2X receptor agonist) had no effect on Up4 A-induced contractions of either LM or CM, and α,ß-methylene-ATP alone failed to induce a contractile response in either tissue. Suramin (a P2Y receptor antagonist), on the other hand, significantly inhibited Up4 A-induced contraction of CM, but not LM. Up4 A-induced contraction of CM, but not LM, was also inhibited by pretreatment with Y-27632, an inhibitor of Rho-associated kinase. We conclude that Up4 A induces extracellular Ca(2+) -dependent contractions of rat gastric LM and CM, and Up4 A-induced contraction of CM is mediated by suramin-sensitive P2Y receptors and subsequent activation of the Rho-associated kinase pathway.

Differential expression of G-protein-coupled estrogen receptor-30 in human myometrial and uterine leiomyoma smooth muscle.

OBJECTIVE: To determine differential expression of G-protein-coupled receptor 30 (GPR30) in uterine leiomyoma and its matched myometrium. DESIGN: GPR30 expression examined in both tissues and cultured cells. SETTING: Research laboratories. PATIENT(S): Women 35 to 50 years old with uterine leiomyomas. INTERVENTION(S): Hysterectomy. MAIN OUTCOME MEASURE(S): GPR30 expression profile. RESULT(S): Using Western blot and real-time quantitative polymerase chain reaction analyses, we found that GPR30 was highly expressed in uterine leiomyomas compared with their matched myometrium. In only three out of nine patients examined was GPR30 protein detectable by Western blot analysis in myometrial tissues, but at statistically significantly lower levels than in their leiomyomas. Confocal microscopy revealed the nuclear localization of GPR30 in leiomyoma tissues and cultured leiomyoma smooth muscle cells (SMCs). Treatment with 0.1 µM 17ß-estradiol increased mRNA expression of GPR30 in leiomyoma SMCs but decreased expression in myometrial SMCs. Treatment with G-1, a GPR30 agonist, stimulated phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) in both SMC types. PD98059, the MEK inhibitor, completely inhibited G-1-induced phosphorylation of p44/42 in myometrium SMCs, but not in SMCs from leiomyoma. CONCLUSION(S): GPR30 is abundantly expressed in uterine leiomyomas, likely resulting from estrogen stimulation.

Atorvastatin inhibits myocardin expression in vascular smooth muscle cells.

Atorvastatin (ATV), an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, is widely prescribed as a lipid-lowering drug. It also inhibits the RhoA-Rho-associated kinase pathway in vascular smooth muscle (SM) cells and critically inhibits SM function. Myocardin is a coactivator of serum response factor, which upregulates SM contractile proteins. The RhoA-Rho-associated kinase pathway, which directly triggers SM contraction, also increases myocardin gene expression. Therefore, we investigated whether ATV inhibits myocardin gene expression in SM cells. In mice injected with ATV (IP 20 µg/g per day) for 5 days, myocardin gene expression was significantly downregulated in aortic and carotid arterial tissues with decreased expression of myocardin target genes SM α-actin and SM22. Correspondingly, the contractility of aortic rings in mice treated with ATV or the Rho-associated kinase inhibitor Y-27632 was reduced in response to treatment with either KCl or phenylephrine. In cultured mouse and human aortic SM cells, KCl treatment stimulated the expression of myocardin, SM α-actin, and SM22. These stimulatory effects were prevented by ATV treatment. ATV-induced inhibition of myocardin expression was prevented by pretreatment with either mevalonate or geranylgeranylpyrophosphate but not farnesylpyrophosphate. Treatment with Y-27632 mimicked ATV effects on the gene expression of myocardin, SM α-actin, and SM22, further suggesting a role for the RhoA-Rho-associated kinase pathway in ATV effects. Furthermore, ATV treatment inhibited RhoA membrane translocation and activation; these effects were prevented by pretreatment with mevalonate. We conclude that ATV inhibits myocardin gene expression in vivo and in vitro, suggesting a novel mechanism for ATV inhibition of vascular contraction.

Uridine adenosine tetraphosphate induces contraction of airway smooth muscle.

Contraction of airway smooth muscle (ASM) plays an important role in the regulation of air flow and is potentially involved in the pathophysiology of certain respiratory diseases. Extracellular nucleotides regulate ASM contraction via purinergic receptors, but the signaling mechanisms involved are not fully understood. Uridine adenosine tetraphosphate (Up(4)A) contains both pyrimidine and purine moieties, which are known to potentially activate P2X and P2Y receptors. Both P2X and P2Y receptors have been identified in the lung, including airway epithelial cells and ASM. We report here a study of purinergic signaling in the respiratory system, with a focus on the effect of Up(4)A on ASM contraction. Up(4)A induced contraction of rat isolated trachea and extrapulmonary bronchi as well as human intrapulmonary bronchioles. Up(4)A-induced contraction was blocked by di-inosine pentaphosphate, a P2X antagonist, but not by suramin, a nonselective P2 antagonist. Up(4)A-induced contraction was also attenuated by α,ß-methylene-ATP-mediated P2X receptor desensitization. Several P2X receptors were detected at the mRNA level: P2X1, P2X4, P2X6, and P2X7, and to a lesser extent P2X3. Furthermore, the Up(4)A response was inhibited by removal of extracellular Ca(2+) and by the presence of the L-type Ca(2+) channel blocker, nifedipine, or the Rho-associated kinase inhibitor, H1152. We conclude that Up(4)A stimulates ASM contraction, and the underlying signaling mechanism appears to involve P2X (most likely P2X1) receptors, extracellular Ca(2+) entry via L-type Ca(2+) channels, and Ca(2+) sensitization through the RhoA/Rho-associated kinase pathway. This study will add to our understanding of the pathophysiological roles of extracellular nucleotides in the lung.