Protective Effects Of Quercetin On Embryo Implantation Failure And Uterine Impairment Induced By LPS In Mice

Lipopolysaccharide (LPS) is cytoderm’s important structural constitution andpathogenic factor of gram-negative bacteria, which can induce negtive effects inhuman such as embryo abortion, stillbirth, spontaneous abortion etc. when contactedduring early pregnancy. Study shows that Quercetin acts like anti-inflammatory,antioxidant, and can scavenge oxygen free radicals and help regulate immune so asto protect the uterus against the damage induced by LPS. The study builds up themurine model of embryo implantation via LPS, detects the content of IL-10andTNF-α and the expression of mRNA by the methods of ELISA in serum and RT-PCRin uterine tissue, the expression of TLR4/NF-κB protein and mRNA in uterine tissuerespectively by the methods of Western blotting and RT-PCR, in order to researchthe effects of Quercetin on TLR4/NF-κB signaling pathway in uterine tissuemediated by LPS, and probe into its mechanism.The Litter of female Kunming mice never pregnant were exchanged with themales’ after seven days’ feeding. Ones in oestrus detected by vaginal smear wereconhabitate with males one to one overnight, then examined for vaginal plugssignifying at day0of pregnancy. Sixty pregnant mice were randomly divided intosix groups, with10mice in every group, control group and five LPS groups withdiffenrent doses. The mice were treated with PBS by tail intravenous injection incontrol group at pregnancy4-5days, other groups were respectively given LPSconcentration of0.2μg/ml,0.5μg/ml,1.0μg/ml,2.0μg/ml,5.0μg/ml in the sameway. On day6of gestation, the mice were sacrificed. The growth condition of theuterus were observed. The best model of Embryo Implantation Failure wasconfirmed via analyzing the amount of Embryo Implantation dysfunction and theweight of the uterus. The results showed that different doses of LPS blocked EmbryoImplantation and reduced its amount to varing degree and showed apparent shrinkand lessness in uterus and gradural decrease of average of Embryo Implantation,uterus weight and the net weight of uterus with the dose increace of LPS. The datesof LPS groups with dose of0.2μg/ml and0.5μg/ml showed no significantdifference (P>0.05) compared with control group. The amount of EmbryoImplantation LPS group with dose of2.0μg/ml and5.0μg/ml were extreme low. While the average of Embryo Implantation and uterus weight of LPS group withdose of1.0μg/ml show significant difference (P<0.05) compared with control group,hence it was taken as the model of Embryo Implantation dysfunction for thefollow-on experiment.Fifty pregnant mice were randomly divided into five groups, with10mice inevery group, control group, model group and quercetin groups with three differentdoses. At days1-3of pregnancy, the control group and model group were treatedwith physiological saline by gavage, quercetin groups respectively receivedquercetin with concentration of0.25,1.25,2.5mg/ml by gavage. At days4-5ofpregnancy, model group and quercetin groups were given LPS via the tail vein andcontrol group received PBS intravenously. To observe the pathological changes ofuterine by HE. ELISA and RT-PCR were applied to detect the expression of mRNAand the content of IL-10and TNF-α. Western blotting and RT-PCR were used todetect the expression of TLR4/NF-κB protein and mRNA. The uterinehistopathological results showed that quercetin obviously improved the recover ofdamaged uterine tissue induced by LPS, and improvement was obvious in2.5mg/mlquercetin group compared with model group. The ELISA results showed that LPScould reduce the expression of IL-10in mice serum, but increase the expression ofTNF-α. It caused inflammation, and made the immune regulating tend to Th1direction which was unfavorable to pregnancy. But supplement with quercetin inadvance, the content of IL-10increased significantly, TNF-α content was reduced,inflammatory response was controlled, and the results were consistent withRT-PCR’s. The result of Western blotting indicated that LPS can singnificantincrease the expression of TLR4protein from intrauterine cytoplasm and NF-κBfrom intrauterine cytoblast in mouse. After used0.25mg/ml quercetin, theexpression of NF-κB and TLR4protein showed no significant difference comparedwith model group. In the quercetin group of1.25mg/ml, this expression was lowerthan model group, In quercetin group of2.5mg/ml, this expression showedsignificant difference compared with model group. And the expression presented adose-dependent decrease with the dose increase of quercetin. The results of RT-PCRindicated the expression of TLR4mRNA and NF-κB mRNA in uterus induced withLPS significantly enhanced compared with control group (P<0.05) The expression ofTLR4mRNA and NF-κB mRNA from quercetin groups showed a graduallydecreace contrasting with model group (P<0.05), and the expression presented adose-dependent relationship with the dose increase of quercetin.The results of oxidation indexes indicated that the model group activities ofSOD and GSH-Px in serum of mice was reduced by30.10%and38.94%respectively, the levels of MDA and NO was increased by29.96%and42.44%. These indexes returned to normal levels gradually in quercetin groups. The quercetincould inhibit oxidative stress caused by lipopolysaccharide and exhibit antioxidanteffect.Conclusion: LPS can cause impairment of uterine tissues induced by embryoimplantation failure, and made the immune regulating tend to Th1direction whichwas harmful to pregnancy; activated the TLR4/NF-κB signaling pathway effectively,lead to inflammatory reaction. Quercetin can protect the uterine tissues in miceagainst the inflammatory damage and alleviate the embryo implantation dysfunctioninduced by LPS, and tend the immune balance to Th2direction which was beneficialto pregnancy, acting an effect of preventing miscarriages.