The Mechanism Study Of Steroidal Saponins On Myometrium Contraction

Objective Total steroidal saponins extracted from the rhizome of Paris polyphyllaSm. var. yunnanensis(TSSPs) have been widely used in China for the treatment ofabnormal uterine bleeding. We previously studied the main active constituents ofTSSPs and their structure-activity relationships with respect to rat myometrialcontractions.Tg (pennogenintetraglycoside) was identified as one of the activeingredients in TSSPs able to induce rat myometrial contractions. However, themechanisms underlying the pharmacological actions on uterine activity have notbeen described clearly.The present study was designed to investigate the role ofmultiple signaling pathways in regulating steroidal saponins-induced myometriumcontraction.Methods Here Tg was screened for effects on contractile activity in isolated uterinestrips from estrogen-primed rats and on MLC20, ERK1/2, CaDphosphorylation andrelated signaling pathways in cultured rat myometrial cells as determined by Westernblot. Intracellular calcium ([Ca2+]i) was monitored under a confocal microscope usingFluo-4AM-loaded myometrial cells.The myometrium contractions induced by Tgorsupressed by various inhibitor were detected by tonotransducer in vitro.Results1. The Relationship between Myometrium Contraction Induced by Tg andPhosphorylation of MLC20Tg dose-dependently stimulated rat myometrial contractions as well as MLC20phosphorylation in vitro, which could be completely suppressed by an inhibitor ofmyosin light chain kinase (MLCK) or inhibitor of CaM.2. Involvment of [Ca2+]iIncrease in Tginduced Uterine Contractions and MLC20PhosphorylationPhosphorylation of MLC20in rat myometrial cells can be mediated by MLCK, whichis predominantly regulated by the concentration of free calcium ions (Ca2+). The alterations in [Ca2+]iin response to Tg were then detected in the Flu-4-AM loadedmyometrial cells.The myometrial cells loaded with Flu-4AM exhibited a transient significant increasein fluorescence after stimulation with2.5mM Tg. In the same experiments, whilecontinuously recording the fluorescence using a time course software, a rapidtransient peak of fluorescence in myometrial cells was observed when challenged withTg, which indicated an increase in [Ca2+]iin rat myometrial cells. Use of Ca2+channelblockers and kinase inhibitors demonstrated that Tg-induced myometrial contractionsare mediated by activation of the phospholipase C(PLC)-inositol triphosphate (IP3)signaling pathway, resulting in increased MLC20phosphorylation. The usage ofRethunium Red(RYR inhibitor) suppressing Tg-induced uterine contraction suggestedthat Ca2+induced Ca2+release(CICR） also took part in Tg-induced uterinecontracton.3. Tho role of rhoA/ROK signaling pathway in Tg-induced myometrium contractionThe ROK inhibitor Y27632was used to determine whether the RhoA/ROK pathwayis involved in Tg-induced MLC20phosphorylation resulting in myometrialcontractions. Y27632could inhibit contractions induced by Tg at5mM and even at2mM, with nearly complete suppression at50mM. In the Western blot analysis,significant inhibition of MLC20phosphorylation induced by500nM Tg wasobserved.4. MAPKs signaling pathways play important roles in Tg-induced myometriumcontractionFurther, MAPKs signaling pathways including ERK/MAPK, p38/MAPK andJNK/SAPK were detected by using specific inhibitors U0126, SB203580andSB600125. All of these inhibitors could partly suppress uterine contractionstimulated by5μM Tg. Tg could dose-and time-dependently stimulated thephosphorylation of ERK1/2, p38and JNK. In ERK/MAPK pathway,phosphorylation of CaD could be induced by Tg and be inhibited by U0126.5. The crosstalk between the relative pathwaysAs there were several pathways included in Tg-induced myometrium contractionprocess, the inherent connections were evaluated. ERK/MAPK pathway activated by Tg could also induce the phosphorylation MLC20together with ERK’sphosphorylation. On the other hand, the phosphorylation effect of ERK1/2could beenhanced by PLC/IP3pathway, which activated MLC20’s phosphorylation after Tgstimulation. But the inhibitors of MLCK and CaM could not influence the level ofp-ERK induced by Tg. GFX, a specific inhibitor of PKC, which could activated byPLC, could diminish the express of p-EKR.ConclusionsIn summary, this study demonstrates that rat myometrial contractility induced by Tgcorrelated with several pathways’ activation, including PLC/IP3, rhoA/ROK, MAPKs.These pathways crossover with each other by signaling molecules so as to form ansignaling pathway web underlying the mechanisms of Tg-induced myometriumcontraction. On the one hand, Tg-induced contraction results from MLC20phosphorylation in myometrial cells while the PLC/IP3and RhoA/ROK signalingtransduction pathways mediate the process. The two signaling pathways act underdifferent mechanisms, one occurs via the changes of [Ca2+]iregulated by PLC/IP3pathway, while the other via Ca2+sensitization of the contractile proteins signaled bythe RhoA/Rho kinase pathway. On the other hand, MAPKs pathways may mediatedTg-induced uterine contraction by increasing p-CaD, which could enhance the abilityof actin to binding with myosin. We speculate that several targets may in the responseto Tg, which may be responsible for the therapeutic effect of TSSPs on AUB. Thesefindings may help facilitate the drug discovery process for AUB therapies.