Study Of The Molecular Mechanism Of LPS Induced Embryo Implantation Failure And The Protective Effects Of Quercetin

The aim of this experiment was to study the mechanism of the embryo implantationfailure induced by LPS and the protective effects of Quercetin. In this experiment, In vivoexperiment combined with In vitro experiment was adpoted to investigate the possiblemechanisms through two parts of experiments. The effects of Que on the expression ofBcl-2/Bax apoptosis proteins in endometrial cells of mice induced with LPS were studiedalong with LPS mediated TLR4/NF-κB signaling pathway and the effects of Quercetin onendometrial cells of mice.In the in vivo experiment, twenty five Kunming mice were randomly divided into fivegroups at day of4pregnancy, with5mice in each group. The mice were treated with LPSby tail intravenous injection at day4of pregnancy, and were given different doses of Queby intragastric administration consecutively at days5~6of pregnancy. On day7ofgestation, the mice were sacrificed. Histopathological changes of the uterus tissue wereobserved, immunohistochemical technique was applied to detect the expression ofBcl-2/Bax apoptosis proteins in endometrial cells of mice. LPS could diminish theexpression of Bcl-2, enhance the expression of Bax, and diminish the ratio of Bcl-2/Bax.The expression of Bcl-2of the moderate dose Que was significantly increased than that inthe model group, and the expression of Bax was on the contrary. Results suggest that Quecan restrain endometrial cell apoptosis through regulating the expression of Bcl-2/Baxapoptosis protein, then alleviate the embryo implantation failure induced by LPS, thusshows anti-abortive effect.In the in vitro experiment, the primary endometrial cells of mice were cultured byuterus tissue mass cultivation at day4.5of pregnancy. When the amount of the cells growsbe above80%, they were treated with LPS for4h,8h,12h,24h and48h. ELISA wascarried out to test the changes of IL-10and TNF-α in culture supernatants, then theWestern blotting was performed to detect the expression of TLR4and NF-κB proteins afterthe proteins in cytoplasm and karyon were collected respectively. After supplement withdifferent concentrations of Que (10,50,100μmol/L) for12h using the optimumconcentration of LPS screened concentration, the changes of IL-10and TNF-α were measured with ELISA in culture supernatant, the cells were stained by Hematoxylin-Eosin.Immunofluorescence staining technique was used to detect the transmembrane expressionof NF-κB, the Western blotting was used to detect the expression of TLR4protein incytoplasm and NF-κB protein in karyon. The Western blotting was used to detect theexpression of Bcl-2/Bax apoptosis proteins after the hole proteins were collected. After thecells were induced by LPS, the results of ELISA showed LPS had diminished theexpression of IL-10but enhanced the expression of TNF-α in cells, causing inflammation.And the immune balance of Th1/Th2biased towards Th1that was unfavorable topregnancy. The results of immunofluorescence staining technique showed the phenomenonof the transmembrane expression of NF-κB protein from cytoplasm to karyon. Theexpressions of TLR4protein in cytoplasm and NF-κB protein in karyon were positivelycorrelated with LPS for time, and the results showed the synchronization consistencybetween them. After supplement with Que, the results of ELISA showed Que had enhancedthe expression of IL-10but diminished the expression of TNF-α in cells, restraininginflammation. So the immune balance of Th1/Th2tended to Th2which was beneficial topregnancy. The concentrations of10μmol/L of Que had diminished the expression ofTLR4protein in cytoplasm and NF-κB protein in karyon significantly and alleviated theinflammatory injury in endometrial cells induced with LPS. Que had increased theexpression of Bcl-2but reduced the expression of Bax obviously to restrain apoptosis inendometrial cell.Conclusion: LPS can cause inflammatory injury to the endometrial cells and excessiveapoptosis in the endometrial cells, so the immune balance of Th1/Th2biased towards Th1that was unfavorable to pregnancy. The results suggest that LPS had activated theTLR4/NF-κB signaling pathway effectively in endometrial cells of mice. Que caneffectively protect the endometrial cells against the inflammatory injury and excessiveapoptosis induced by LPS, and the immune balance of Th1/Th2biased towards Th2thatwas beneficial to pregnancy. The results suggest Que could evidently block theTLR4/NF-κB signaling pathway mediated by LPS.