The Experimental Researeh Of The Treatment Of Tubal Pregnancy With Tcm That Activating Blood, Dissolving Stasis,Eliminating Mass And Killing Embryo Based On HPLC-MS QC

IntroductionIn the recent years, with the increase of abortion, sexual confusion and infections,surgery in the department of gynecology and obstetries, the rate of ectopic pregnancy is rising.According to the statistics, the incidence of ectopic pregnaneies among the all is above1%[1]. Among them, unmarried, not yet fertility patients also increased significantly. With the technological advances of modern detection and gynecologists to improve the vigilance of the disease, the rate of early diagnosis of tubal pregnancy greatly enhance,and create the conditions for non-surgical treatment. Some patients could avoid physical injuries and economic pressure caused by surge ry, and retain fertility in the maximum extent.Among non-surgical treatment, Chinese medicine is now widely used in clinic. The etiopathogenesis of tubal pregnancy is considered to be syndrome of blood stasis in the lower abdomen by the traditional Chinese medieine. So the Chinese medicine which activating blood, dissolving stasis, eliminating mass and killing embryo has been used to treat tubal pregnancy for a long time. Since the First Affiliated Hospital of Shanxi College of Traditional Chinese Medicine first to use the decoction of ectopic pregnancy Ⅰ and Ⅱ, many hospitals have used them to treat tubal pregnancy based on the addition and subtraction of herb, and have achieved a satisfactory effect. However, the decoction, efficacy of TCM in the treatment of ectopic pregnancy is often fo und in reports, but the mechanism is still in the initial stage, most of the studies focus on hemorheology, there is no specifie,system-level indicators to explain its mechanism.1. The experimental researchobjectiveThe research was to apply HPLC-mass spectrometry joint analytical methods (HPLC-MS) as quality control method of serum pharmacology, to prove that the serum from rats fed with Chinese medicine contain the active ingredients of Chinese medicine. And from the ER,PR,MMPs angle to investigate the functional mechanism of the Chinese medicine which activating blood, dissolving stasis, eliminating mass and killing embryo on treating tubal pregnancy.Research MethodsThrough HPLC-MS to analyze the serum of SD rats fed with the chinese medicine which activating blood, dissolving stasis, eliminating mass and ki lling embryo, and the serum of SD rats fed with saline and Methotrexate as control, detect and analys their difference in qualitatively. The seru m confirmed by quality control that contain active ingredient of previou s decoction was used to interven the gestational trophoblastic cells culture d in vitro, and the cells come from patient with tubal pregnancy that meet th einclusion criteria. And located the group of Methotrexate and group of norma1saline as control. Though real-time fluorescence quantitative (Real-time PCR) to investigate the mechanism of TCM decoction in the factor of ER, PR, MMPs of gestational trophoblastic cells. From the molecular genetic level to explore the possible mechanism of theChinese medicine which activating bloo d, dissolving stasis, elimina-ting mass and killing embryo.Results(1) LC-MS/MS to detect the drug ingredients in serum Citrulline, β β-dimethyl acryloyl Aka Ning, tanshinone Ⅱ A, salvianolic acid B, amygdalin of paeoniflorin were found in TCM group serum. The Metho trexate group and the saline group serum does not contain any of the previou s ingredient.(2)The impact to ER, PR,MMPs after the trophocytes were cultured by medicated serum that contains Chinese medicine①After had been treated with different serum that contained different medicine in vitro,the expression of ER lowered in the group of Chinese medi-cine and group of Methotrexate. There is significant difference between the group of Methotrexate and group of normal saline(P<0.01);There is differe-nce between the group of Chinese medicine and group of normal saline(P<0.05);There is no difference between the group of Methotrexate and group of Chinese medicine(P>0.05).②After had been treated with differentserum that contained different medicine in vitro, the expression of PR lowered in the group of Chinese medic ine and group of Methotrexate. There is significant difference between the gr oup of Methotrexate and group of normal saline(P<0.01);There is significan t difference between the group of Chinese medicine and group of normal salin e(P<0.01);There is no difference between the group of Methotrexate and gro up of Chinese medicine(P>0.05).③After had been treated with different serum that contained different medicine in vitro, the expression of MMPs raised in the group of Chinese medicine and group of Methotrexate. There is significant difference between the group of Methotrexate and group of normal saline(P<0.01);There is no significant difference between the group of Chinese medicine and group of normal saline(P<0.05);There is no difference between the group of Methotr-exate and group of Chinese medicine (P>0.05).2. the Clinical researchObjectiveTo investigate the type of distribution of TCM constitution and the correlated factors of ectopic pregnancy, the healthy non-pregnant women ser-ved as control.It would be a preliminary study to decrease the incidence of ectopic pregnaney through adjusting the TCM constitution of women before pregnancy. MethodsThe patients with ectopic pregnancy treated in the First Affiliated Hos-pital of Guangzhou University of Chinese Medieine during2011.1-2012.3Were collected.122healthy non-pregnant women served as control group. All cases conducted on-site investigations about constitution of TCM questionnaire designed by Professor Wang Qi.ResultCases collect:(1)178patients that the admission diagnosis is ectopic pregnancy were dis-tributed questionnaires. Then re-screening according to discharge diagnosis, excluding the diagnosis unclear or merge with other diseases. Finally,159valid cases were collected. And122healthy non-pregnant women were collected in total.(2)Ectopic pregnaney occurred much in the yang--xu constitution, qi--yu constitution, Xue--Yu constitution.The significant impact factors of ectopic pregnancy were Xue--Yu constitution and Shi--re constitution (B>0, P<0.01).Research Conclusion1. HPLC-MS/MS for detecting the drug ingredients confirmed that Chinese medi-cine direction does contain the above ingredients. These ingredients may be the efficacy basis of the Chinese medicine on tubal pregnancy.2. The traditional Chinese medicine that activating blood, dissolving stasis eliminating mass and killing embryo reduced the expression of ER,PR,suggest-ing that it could antagonize estrogen and progesterone in receptor levels, resulting varying degrees of degeneration and necrosis in the vi1li,decidua, may be it play an important role in the apoptosis of tubal pregnancy.3. The traditional Chinese medicine that activating blood, dissolving stasis eliminating mass and killing embryo raised the expression of MMPs,suggesting that it could accelerated the dissolution of tubal decidua, resulting varying degrees of degeneration and necrosis in the villi, decidua, may be it play an important role in the apoptosis of tubal pregnancy.4. Xue--Yu constitution and Shi--re constitution are relevant factors of ectopic pregnancy. Among them, Xue--Yu constitution is significant different fromnon-pregnant healthy women. intermingled deficiency and excess5. Most of the TCM constitution type of patients with ectopic pregnancy are syndrome of intermingled deficiency and excess.