Zengyong Wang

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 17β-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 &mgr;g/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 &agr;-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 &agr;-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 &agr;-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 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 (Up4A), an endothelium‐derived contraction factor, induces vascular SM contraction. Its effect on gastric SM contractions, however, is unknown. We addressed the hypothesis that Up4A 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. Up4A induced transient contractile responses in both CM and LM, which were similar to those induced by ATP and UTP. Up4A failed to induce contraction of either LM or CM in the absence of extracellular Ca2+ or in the presence of nimodipine, an inhibitor of voltage‐gated Ca2+ 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. IP5I (a P2X receptor antagonist) and α,β‐methylene‐ATP (a P2X receptor agonist) had no effect on Up4A‐induced contractions of either LM or CM, and α,β‐methylene‐ATP alone failed to induce a contractile response in either tissue. Suramin (a P2Y receptor antagonist), on the other hand, significantly inhibited Up4A‐induced contraction of CM, but not LM. Up4A‐induced contraction of CM, but not LM, was also inhibited by pretreatment with Y‐27632, an inhibitor of Rho‐associated kinase. We conclude that Up4A induces extracellular Ca2+‐dependent contractions of rat gastric LM and CM, and Up4A‐induced contraction of CM is mediated by suramin‐sensitive P2Y receptors and subsequent activation of the Rho‐associated kinase pathway.

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,β-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.

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. J. Cell. Physiol. 230: 885–895, 2015.

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.

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.

Cimicifugamide from Cimicifuga rhizomes functions as a nonselective β-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 β2-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 β2-AR agonist from CR and cimicifugamide could activate β1, 2-ARs more significantly than β3-AR in β-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.

Mahuannin B an adenylate cyclase inhibitor attenuates hyperhidrosis via suppressing β2-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 β2-adrenoceptor (β2-AR) activity luciferase reporter assay system was used to screen active inhibitors; molecular docking and a series of biological assays centered on β2-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 β2-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.