补肾活血方“导法”调控5/12-LOX/LXA4/FPR2改善子宫内膜容受性不良大鼠的助孕机制

Bushen Huoxue Recipe(补肾活血方)with"Enema Method"Regulates 5/12-LOX/LXA4/FPR2 to Improve Pregnancy Supporting Mechanism of Rats with Endometrial Receptivity

目的观察补肾活血方“导法”调控肾虚血瘀子宫内膜容受性不良大鼠种植窗期LXA4相关信号轴的助孕机制。方法通过分子对接预测补肾活血方有效组分与脂氧素A4(LipoxinA4,LXA4)合成酶及受体的结合程度;通过羟基脲灌胃+肾上腺素皮下注射构建肾虚血瘀内膜容受性不良复合模型大鼠;随机分为模型组、空白组、补肾活血导法低、中、高组、阳性组6组;各组造模同时予以相应干预方法,连续10 d,并于动情期按照2∶1雌雄合笼,次日晨检发现阴栓脱落或阴道涂片发现阴栓记为1.0 dpc,于6.0 dpc麻醉处死剖出子宫组织;记录每组子宫内膜、胞饮突形态并评分,观察子宫内膜上胰岛素样生长因子结合蛋白1(insulin-like growth factor-binding protein-1,IGFBP-1)、血管内皮生长因子(Vascular Endothelial Growth Factor,VEGF)蛋白分布表达和同源盒基因A10(HomeoboxA10,HOXA10)mRNA表达,测定子宫组织匀浆中炎症介质LXA4、合成酶5/12/15-脂氧合酶(Lipoxygenase,LOX)、受体甲酰肽受体2(Formyl Peptide Receptor-2,FPR2)和IGFBP-1、VEGF的蛋白含量。结果与空白组相比,模型组子宫内膜被覆上皮菲薄、腺体较少,与模型组对比;补肾活血方导法低、中、高剂量组、阳性组可下调肾虚血瘀PER模型大鼠种植窗期5-LOX、12-LOX、LXA4及FPR2的表达(P<0.05),提高了子宫内膜容受性标记物IGFBP-1、VEGF蛋白和HOXA10 mRNA表达。结论补肾活血方导法通过调控大鼠种植窗期子宫5/12-LOX/LXA4/FPR2信号通路纠正炎性微环境的平衡,进而改善子宫内膜容受性,促进胚-膜交互提高种植及妊娠成功率。

Objective To observe the effect of Bushen Huoxue Recipe(补肾活血方)with enema method on LXA4 signaling pathway in the implantation window of rats with endometrial receptivity disorder due to kidney deficiency and blood stasis.Methods The binding degree of effective components of Bushen Huoxue Recipe to LXA4 synthase and receptor were predicted by molecular docking.A composite model rat with poor intimal receptivity of kidney deficiency and blood stasis was constructed by hydroxyurea gavage+adrenaline subcutaneous injection.The rats were randomly divided into six groups:model group,blank group,low,medium,and high dose groups by Bushen Huoxue Recipe with enema method.Each group was treated with corresponding interventions at the same time for 10 days,and the rats were caged according to 2:1 hermaphroditism in estrous phase.The vulval plugs or sperms of rats were found to be recorded at 1.0 dpc in the following morning,and the uterine tissues were dissected out by anesthetizing and sacrificed at 6.0 dpc.The endometrium and pinocytosis morphology were observed and scored.We observed insulin-like growth factor binding protein-1(IGFBP-1),vascular endothelial growth factor(VEGF)protein distribution and homeobox gene A10(HOXA10)mRNA expression in the endometrium.Protein levels of inflammatory mediator LXA4,synthase 5/12/15 lipoxygenase(LOX),receptor formyl peptide receptor-2(FPR2),IGFBP-1 and VEGF were measured in uterine homogenates.Result Compared with the blank group,the model group had a thin endometrium coating and fewer glands.Compared with the model group,Bushen Huoxue Recipe low,medium and high dose groups and control group could down-regulate the expressions of 5-LOX,12-LOX,LXA4 and FPR2(P<0.05)and improve the mRNA expression of endometrial receptivity markers IGFBP-1,VEGF protein and HOXA10 at implantation window stage in per model rats with kidney deficiency and blood stasis.Conclusion By regulating the uterine 5/12-LOX/LXA4/FPR2 signaling pathway at the implantation window in rats,Bushen Huoxue Recipe with with enema method corrects the balance of inflammatory microenvironment,which improves endometrial receptivity and promotes embryo membrane interaction to improve the implantation and pregnancy success rate.