G Protein-coupled Receptor30Mediated Effects Of Estrogen On The Expression Of TLR4in The Uterus Of Mice

Estrogen has direct or indirect influence and adjustment on local immune of the uterus.Toll-like receptor4(TLR4) as one of toll-like receptor superfamily members, plays animportant function in immune defense and immune regulation in the uterus.Studies havedemonstrated that estrogen secretion affects the expression of TLR4in the uterus.Estrogen’sphysiological effects are mediated via estrogen receptors.Classic estrogen nuclear receptorsinvolving in the uterine local immune regulation of estrogen had been researched. Themembrane receptor, G protein coupled receptor30(G protein-coupled receptor30, GPR30)in recent years mediating estrogen effect on uterine immunity regulation has not been reported.So, we used immunohistochemical method and real-time fluorescence quantification PCRmethod to research the effects of GPR30mediating estrogen on the expresstion of TLR4inthe uterus of the mice. The following results were obtained:1. Distribution and expression rule of TLR4and GPR30in mice uterus during estruscycleGPR30and TLR4differently distributed in uterine endometrium, muscularis andserosalayer of mice during estrus cycle. Mainly in endometrium epithelium, glandularepithelium and stroma cells there were the deepest positive staining and the most positivecells, while in the muscularis and serosa layer were less. In4periods of estrus cycle, GPR30and TLR4had the deepest positive staining and strong positive distribution in endometriumepithelium and glandular epithelium in the estrus, and gradually weakened in metaestrusperiod. There was shallow and weak positive distribution. The distribution trend of GPR30 and TLR4in stroma was same as in endometrium epithelium and glandular epithelium. Inmuscularis and serosa layer, GPR30and TLR4positive immune reaction production had thedeepest staining in the estrus, and they had no obvious difference between other periods.The relative expression of GPR30and TLR4in different period was compared andanalysed, and found that they had the highest relative expression in estrus, significantly higherthan that of other three periods (P <0.01); while had the lowest relative expression indisestrus, significantly lower than that of other three periods (P <0.01). The relativeexpression of GPR30between4stages presented significantly difference (P <0.01), while therelative expression of TLR4in proestrus and metaestrus period had no significant difference(P>0.05). GPR30and TLR4of the uterus in different periods expressed highly inendometrial layer, lowly in muscularis and serosa layer. In endometrial layer in differentperiods, the relative expression change of GPR30in endometrial epithelium, glandularepithelium and stromal was consistent with the relative expression of overall change in theuterus. The relative expression change of TLR4in endometrial epithelium and glandularepithelium was the same as overall change in the uterus; while in endometrial stromal in4periods, the relative expression of TLR4presented significant difference (P <0.01), highest inestrus and lowest in disestrus. In addition, the relative expression of GPR30in endometriumepithelium and glandular epithelium in4periods was always higher than that of endometriumstroma; while the relative expression of TLR4in endometrium stroma in three periods otherthan in the estrus was the highest. The relative expression of GPR30and TLR4wascorrelation analysised and found that the relative expression of GPR30and TLR4in uterus ofmice during estrus cycle showed positive correlation.2. Effcet of G protein-coupled receptor30on the distribution and expression of TLR4inthe uterus of ovariectomized miceThe mice were divided into SHAM group，ovariectomized group (OVX), OVX+G1groups (OVX+0.1nmoL G1、OVX+0.5nmoL G1、OVX+2.5nmoL G1), OVX+G15groups(OVX+0.1nmoL G15、OVX+0.5nmoL G15、OVX+2.5nmoL G15). The relative expressionlevel of TLR4in uterus of mice between groups presented that the OVX group expressedsignificantly higher than other group（sP <0.01）. After giving the agonist G1to ovarian mice,the relative expression of TLR4decreased, significantly lower than OVX group and SHAMgroup (P <0.01). Also, the trend of TLR4expression level in OVX+G1groups decreasedwith the increasing of G1concentration. Between OVX+G1groups, the relative expression ofTLR4in low and medium concentration group was significantly higher than that highconcentration group (P <0.01), and this two groups had no significant difference (P>0.05).After giving the antagonist G15to ovarian mice, the relative expression of TLR4increased with the increasing of G15concentration. The significant difference on relative expression ofTLR4in low, medium and high concentration of OVX+G15groups was consistent withOVX+G1groups. Real-time fluorescent quantitative results showed that, the result offluorescent real-time quantitative PCR showed that the expression level change of TLR4mRNA in uterus of mice between groups was consistent with the relative expression of TLR4.The above results showed that GPR30can mediate estrogen to inhibit the expression ofTLR4in the uterus, and participate in regulating the uterine local immune function.