补肾活血方对抗心磷脂抗体(+)复发性流产患者蜕膜及外周血膜联蛋白A2和Toll样受体4的影响研究

Regulating Effects of Kidney-nourishment and Blood-activation Recipe on Annexin A2 and Toll-like Receptor 4 of Peripheral Blood and Decidua in Patients with Recurrent Spontaneous Abortion and Positive Antiphospholipid Antibody

背景复发性流产(RSA)是妊娠常见疾病之一,抗心磷脂抗体(ACA)与该病的发生密切相关。前期研究发现ACA(+)RSA者蜕膜中Toll样受体4(TLR4)/核因子κB(NF-κB)通路呈活化状态,提示该通路及通路中因子的变化可能是ACA(+)RSA发生的重要机制,故对该通路及其相关因子的干预可能会对ACA(+)RSA的妊娠结局产生一定影响。由于ACA(+)RSA者具有血栓形成倾向,这与中医肾虚血瘀证型相一致,故治以补肾活血法,根据该治法拟定的补肾活血方也在长期、大量临床应用中取得了良好的效果。目的观察补肾活血方对ACA(+)RSA患者蜕膜及外周血中膜联蛋白A2(ANXA2)、TLR4表达情况,进而阐述补肾活血方在ACA(+)RSA患者保胎治疗中的疗效及作用机制。方法选取2015年3月—2016年3月在黑龙江中医药大学附属第一医院门诊及住院部治疗的符合纳入标准的ACA(+)RSA现妊娠失败需行清宫术患者16例,在黑龙江中医药大学附属第一医院门诊行正常人工流产者15例,共收集蜕膜样本31例:ACA(+)RSA者蜕膜为试验组1(n=16)、正常人工流产者蜕膜为对照组1(n=15),其中试验组1中1例因流产组织发现染色体异常故予以排除。选取2015年3月—2016年3月在黑龙江中医药大学附属第一医院门诊及住院部治疗的符合研究标准的要求行保胎治疗的ACA(+)RSA患者11例(试验组2),在黑龙江中医药大学附属第一医院门诊行正常孕检者13例(对照组2),收集其血清样本,其中试验组2中1例自然流产,对照组2中3例自动退出,未计入统计数据。采用RT-qPCR法检测对照组1和试验组1蜕膜中ANXA2mRNA、TLR4 mRNA水平。试验组2给予补肾活血方,对照组2不给予干预,于治疗前和治疗后分别采静脉血,采用ELISA法检测对照组2和试验组2血清孕酮、β人绒毛膜促性腺激素(β-HCG)、ANXA2、TLR4水平。结果对照组1蜕膜组织中ANXA2 mRNA水平高于试验组1,TLR4 mRNA水平低于试验组1(P<0.05)。治疗前对照组2和试验组2血清孕酮、β-HCG、ANXA2、TLR4水平比较,差异有统计学意义(P<0.05);治疗后对照组2和试验组2血清孕酮、β-HCG、ANXA2、TLR4水平比较,差异无统计学意义(P>0.05)。试验组2治疗后血清孕酮、β-HCG、ANXA2水平高于治疗前,血清TLR4水平低于治疗前(P<0.05)。结论 ACA(+)RSA的发生可能与ANXA2低表达、TLR4高表达相关,补肾活血方可以通过上调ANXA2水平、下调TLR4水平改善炎性环境,进而改善妊娠结局。

Background Recurrent spontaneous abortion(RSA)is one of the common diseases in pregnancy,and anti-cardiolipin antibodies(ACA)are closely related to the occurrence of the disease.Previous studies have found that the Tolllike receptor 4(TLR4)/NF-κB pathway in the decidua of ACA(+)RSA is activated,suggesting that the changes in factors in this pathway and pathway may be important mechanisms for the occurrence of ACA(+)RSA,so the pathway and interventions with related factors may have an impact on the pregnancy outcome of ACA(+)RSA.Because ACA(+)RSA has a tendency to thrombosis,which is consistent with the syndrome of kidney deficiency and blood stasis in Chinese medicine,so use the method of reinforcing kidney and activating blood,Kidney-nourishment&Blood-activation Recipe prepared according to the treatment method have also been obtained good effect in long-term and large-scale clinical application.Objective To observe the effect of Kidney-nourishment and Blood-activation Recipe on peripheral blood and decidual Annexin A2(ANXA2),TLR4 in patients with RSA and positive ACA,and then to explain the efficacy and action mechanism of Kidney-nourishment and Blood-activation Recipe in fetus protection for ACA(+)RSA patients.Methods Thirty-one patients admitted to the outpatient and inpatient departments of First Affiliated Hospital,Heilongjiang University of Chinese Medicine from March 2015 to March 2016 were selected.Among them 16 cases had pregnancy failures and needed complete curettage of uterine cavity,and 15 patients performed normal abortion at the outpatient department.A total of 31 decidua were collected and patients were divided into test group 1(n=16)and control group 1(n=15).One case was excluded from the test group 1 because of chromosome abnormality in the abortion tissue.Eleven patients were selected as test group 2 with ACA(+)RSA who asked for a fetus protection andadmitted to the outpatient and inpatient departments of First Affiliated Hospital to Heilongjiang University of Chinese Medicine from March 2015 to March 2016,and 13 patients who received a normal pregnancy test at the outpatient department were selected as control group 2.A total of 24 serum samples were collected.Four cases were excluded from the study,as one case in the test group 2 had spontaneous abortion,and 3 cases in the control group 2 quitted the study.The levels of ANXA2 mRNA and TLR4 mRNA in decidua of patients in the control group 1 and test group 1 were detected with RT-qPCR method.The test group 2 was given Kidney-nourishment and Blood-activation Recipe,and the control group 2 was not given intervention.Their venous blood was taken before and after treatment.The levels of serum progesterone,β-HCG,ANXA2,and TLR4 of control group 2 and test group 2 were detected using the ELISA method.Results The level of ANXA2 mRNA in the abortion decidual tissue of the control group 1 was higher than that of the test group 1 and its TLR4 mRNA level was lower than that of the test group 1(P<0.05).There were significant differences in levels of serum progesterone,β-HCG,ANXA2 and TLR4 between the control group 2 and the test group 2 before the treatment(P<0.05).After the treatment,the differences in serum progesterone,β-HCG、ANXA2 and TLR4 between the control group 2 and the test group 2 was not statistically significant(P >0.05).The levels of serum progesterone,β-HCG,ANXA2 of the test group 2 were all significantly higher than those before treatment,level of serum TLR4 of the test group 2 was significantly lower than that before treatment(P<0.05).Conclusion The occurrence of ACA(+)RSA may be associated with low expression of ANXA2 and high expression of TLR4.Kidney-nourishment and Blood-activation Recipe can improve hypercoagulability and inflammation and in turn improve the pregnancy outcome by up-regulating ANXA2 and down-regulating TLR4.