丹参酮ⅡA调控子宫炎性微环境改善着床障碍大鼠着床窗期子宫内膜容受性的研究

TanshinoneⅡA regulates intrauterine inflammatory microenvironment to improve endometrial receptivity at implantation window in rats with implantation disorder

目的观察丹参酮ⅡA对着床障碍模型大鼠子宫炎症微环境和子宫内膜容受性的影响,并探讨其潜在的作用机制。方法选择动情周期规律的健康6~7周龄SPF级SD大鼠共40只为研究对象,雌雄比为3∶1,进行雌雄鼠合笼后将妊娠成功的20只大鼠按照随机数字表随机分为4组:正常对照组、模型组、丹参酮ⅡA低剂量组(10 mg·kg^(-1)·d^(-1))、丹参酮ⅡA高剂量组(20 mg·kg^(-1)·d^(-1)),每组5只。妊娠第1天,丹参酮ⅡA低剂量组、高剂量组大鼠分别进行相应剂量的丹参酮ⅡA灌胃,连续7 d;正常对照组和模型组给予等体积生理盐水。除正常对照组外,其余各组在妊娠第4天行颈背部皮下注射米非司酮(5 mg/kg),构建胚胎着床障碍模型。各组大鼠均于妊娠第8天行颈椎脱臼法处死,取材后,采用扫描电镜观察子宫内膜胞饮突数量;Western blot检测子宫内膜植入位点组织中白血病抑制因子(LIF)、血管紧张素2(Ang-2)、血管内皮生长因子(VEGF)、Toll样受体4(TLR4)、髓样分化因子88(MyD88)、核因子-κB p65磷酸化蛋白[NF-κB(p-p65)]表达情况;免疫组化法检测大鼠子宫内膜植入位点组织中转化生长因子-β1(TGF-β1)及p65蛋白表达水平;ELISA检测子宫内膜植入位点组织脂氧素A4(LXA4)、白介素(IL)-1α、IL-1β、IL-6、IL-4表达水平。结果扫描电镜下观察发现,模型组大鼠子宫内膜表面偶见较少数量的胞饮突,而丹参酮ⅡA低剂量组、高剂量组的子宫内膜胞饮突数量明显增加。Western blot检测显示,模型组中LIF、Ang-2和VEGF表达均显著低于正常对照组(P<0.01);丹参酮ⅡA低剂量组和高剂量组LIF、Ang-2和VEGF表达均显著高于模型组(P<0.05);模型组中TLR4、MyD88、p-p65表达水平均显著高于正常对照组(P<0.01);丹参酮ⅡA低剂量组和高剂量组中TLR4、MyD88、p-p65表达水平均显著低于模型组(P均<0.05)。免疫组化染色法显示,模型组中TGF-β1表达水平显著高于正常对照组(P<0.01);丹参酮ⅡA低剂量组和高剂量组中的TGF-β1表达水平均显著低于模型组(P<0.05)。ELISA检测结果显示,与正常对照组相比,模型组中LXA4、IL-1α、IL-1β和IL-6水平显著增加(P<0.01),IL-4水平显著降低(P<0.05);与模型组相比,丹参酮ⅡA低剂量组和高剂量组中LXA4、IL-1α、IL-1β和IL-6水平均显著降低(P均<0.05),IL-4水平显著升高(P<0.05)。结论丹参酮ⅡA可能通过抑制TLR4/MyD88/NF-κB信号通路进一步改善子宫炎性微环境,进而改善着床障碍模型大鼠着床窗期子宫内膜容受性。

Objective:To investigate the effects of tanshinoneⅡA on intrauterine inflammatory microenvironment and endometrial receptivity in implantation disorder model rats,and to explore the underlying mechanisms involved.Methods:A total of 40 healthy SPF grade SD female rats aged 6 to 7 weeks with regular estrous cycles were selected.After caging with male rats,the successful pregnancy rats were randomly divided into 4 groups:normal control group,model group,tanshinoneⅡA low-dose group(10mg·kg^(-1)·day^(-1))and tanshinoneⅡA high-dose group(20 mg·kg^(-1)·day^(-1)),5 rats for each group.On the first day of pregnancy,the rats in the low-dose and high-dose tanshinoneⅡA groups were given corresponding doses of tanshinoneⅡA by gavage for 7 days.Normal control group and model group were given equal volume of normal saline.Except the normal control group,the rats in the other group were injected subcutaneously with mifepristone(5 mg/kg)at the back of the neck to construct implantation disorder model.The rats in each group were killed by cervical dislocation on the 8^(th) day of pregnancy to collected the tissues.The number of pinopodes in endometrium was observed by scanning electron microscope.The protein expressions of leukemia inhibitory factor(LIF),human angiotensin-2(Ang-2),vascular endothelial growth factor(VEGF),toll like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),nuclear factor-κB p65 phosphorylated protein[NF-κB(p-p65)]in the tissues at endometrial implantation site were detected by Western blot.The expression levels of transforming growth factor-β(TGF-β1)and p65 protein in the tissues at endometrial implantation site were detected by immunohistochemistry.The expression levels of lipoxin A4(LXA4),IL-1α,IL-1β,IL-6 and IL-4 in the tissues at endometrial implantation site were detected by ELISA.Results:Under scanning electron microscope,it was found that a small number of pinopodes were occasionally seen on the surface of endometrium of rats in the model group,but the number of pinopodes significantly increased in both low and high doses of tanshinoneⅡA groups.Western blot showed that the expressions of LIF,Ang-2 and VEGF in the model group were significantly lower than those in the normal control group(P<0.01),while the expressions of LIF,Ang-2 and VEGF in low/high dose tanshinoneⅡA groups were significantly higher than that in the model group(P<0.05).The expression levels of TLR4,MyD88 and p-p65 in the model group were significantly higher than those in the normal control group(P<0.01),and the expression levels of TLR4,MyD88 and p-p65 in the low-dose group and high-dose group of tanshinoneⅡA were significantly lower than those in the model group(P<0.05).Immunohistochemical staining showed that the expression level of TGF-β1 in model group was significantly higher than that of the normal control group(P<0.01).The expression levels of TGF-β1 in low-dose and high-dose tanshinoneⅡA groups were significantly lower than that of the model group(P<0.05).ELISA test results showed that the levels of LXA4 and IL-1α,IL-1βand IL-6 in the model group were significantly increased compared with the normal control group(P<0.01),while IL-4 levels decreased significantly(P<0.05).Compared with the model group,the levels of LXA4,IL-1α,IL-1βand IL-6 in the low-dose and high-dose tanshinoneⅡA groups were significantly decreased(P<0.05),while IL-4 levels increased significantly(P<0.05).Conclusions:TanshinoneⅡA can improve the intrauterine inflammatory microenvironment by inhibiting TLR4/MyD88/NF-κB signaling pathway,to further improve the endometrial receptivity at implantation window of rats with implantation disorder.