Mechanism Of Huayu Xiaozheng Treating Ectopic Pregnancy And Its Effect On Endometrial Receptivity

ObjectiveExperimental part uses tubal gestational trophoblastic cells cultured in vitro as a carrier,based on the extracellular matrix-integrin-cytoskeleton transmembrane signaling system,which centered on the focal adhesion kinase,to explore the influence of Huayu xiaozheng Tang on the invasion and migration ability of tubal gestational trophoblastic cells,and to explore its regulation of the signaling mechanism and target of action in order to further improve the mechanism of the treatment of tubal pregnancy mechanism by Huayu xiaozheng method.Clinical part intends to adopt a prospective randomized controlled trial,concerning the levels of estrogen and progesterone levels,endometrial thickness,and uterine hemodynamics,to evaluate the efficacy of Huayu xiaozheng Tang on the endometrial receptivity of patients with blood stasis syndrome after ectopic pregnancy is cured,so as to provide a new idea of Chinese medicine treatment for the improvement of reproductive fertility of patients after ectopic pregnancy is cured.MethodsExperimental part:（A）In vitro culture and identification of human villous trophoblast cells of tubal pregnancy;（B）Co-culture of drug-containing medium and tubal gestational trophoblast cells,then divided into blank group,Y15group,Y15+medium-dose traditional Chinese medicine group,low and middle,High-dose traditional Chinese medicine group,MTX group;（C）Transwell method was used to detect the effect of each group of drugs on the ability of migration and invasion of tubal gestational trophoblast cells in vitro;（D）Western-blot method was used to detect the effect of each group on the expression of u PA and its receptor,integrin,FAK and its phosphorylation,Paxillin protein and its phosphorylation level.Clinical part:Randomized,controlled method was used to randomize 60cases of blood stasis syndrome patients who were cured after ectopic pregnancy,into the experimental group and the control group.The experimental group used Huayu xiaozheng granules as an intervention measure and took it with boiled water,14g,tid,on the 7th to 14th day of each menstrual cramps,from the 2nd menstrual cycle after the ectopic pregnancy was cured.The control group was a blank group and no drug intervention was given.The duration of the intervention was from the 2nd menstrual cycle to the 4th menstrual cycle after the ectopic pregnancy was cured.After the ectopic pregnancy was cured,at the 6th to 10th days after ovulation of the 1st menstrual cycle and the3rd menstrual cycle of treatment,The following tests and inspection indexes were collected:（A）The chemiluminescent method was used to detect the serum estradiol,progesterone level of the two groups before and after treatment;（B）Abdominal and vaginal utero attachment color Doppler ultrasound was used to monitor and calculate the single score of Salle to assess the endometrial receptivity（including endometrial thickness and morphology,myometrial echo,subendometrial blood flow,early diastolic notch,end diastolic blood flow,uterine arterial beat index PI）;（C）Recorded adverse events.Assessing the effectiveness and safety comprehensively.ResultsExperimental PartTranswell experiment results showed that:（A）In vitro invasion test,compared with the blank group and Y15 group,each group can reduce the number of invasive membranes and enhance the invasion inhibition rate of tubal gestational trophoblastic cells,among which each dose group of traditional Chinese medicine can significantly inhibited cell invasion in a dose-dependent manner,the difference was statistically significant（P<0.05）.Compared with MTX group,the number of invasive membranes of tubal gestational trophoblastic cells was decreased and the inhibition rate was increased in the high-dose traditional Chinese medicine group,and the difference was statistically significant（P<0.05）.（B）In vitro migration ability test,compared with the blank group and Y15 group,each group can reduce the number of migrated trophoblastic membranes and increase the migration inhibition rate of of tubal gestational trophoblastic cells,and each group of traditional Chinese medicine can obviously inhibit cell migration,It was dose-dependent and the difference was statistically significant（P<0.05）.Compared with the MTX group,the number of migrated membranes of tubal gestational trophoblastic cells in the high-dose and middle-dose groups of traditional Chinese medicine decreased,and the migration inhibition rate increased.The difference was statistically significant（P<0.05）.Western Blot experiment results showed that,（A）The expression of u PA,u PA-R expression,compared with the blank group,the expression of u PA and u PA-R in high-dose traditional Chinese medicine group,Y15 group,and in Y15+medium-dose traditional Chinese medicine group decreased（P<0.05）.The expression of u PA was increased（P<0.05）and u PA-R was decreased in MTX group（P>0.05）;Compared with Y15 group,u PA expression was decreased in Y15+medium-dose traditional Chinese medicine group（P<0.05）.The expression in high-dose group increased（P>0.05）;the expression of u PA-R in high-dose group and Y15+medium-dose traditional Chinese medicine group decreased in turn（P<0.05）.（B）The expression of Integrin,Compared with the blank group,each administration group could adjust the expression level of integrin down（P<0.05）.The expression of each dose group of traditional Chinese medicine was dose-dependent and was less than that of MTX Group（P<0.05）;compared with Y15 group,the expression of integrin in high-dose group and Y15+medium-dose traditional Chinese medicine group increased sequentially（P<0.05）.（C）The expression of FAK and p-FAK,Compared with the blank group:The expression of FAK and p-FAK in the high-dose,middle-dose groups of traditional Chinese medicine,Y15 group,and Y15+medium-dose traditional Chinese medicine group decreased（P<0.05）,and the expression in MTX group increased（P<0.05）;Compared with Y15 group,The expression of FAK and p-FAK in the high-dose group was decreased（P<0.05）,and similar to that of the Y15+medium-dose traditional Chinese medicine group（P<0.05）.（D）The expression of p-Paxillin:compared with the blank group,each drug group can down-regulate the expression level of p-Paxillin（P<0.05）,in which the expression of each dose group of traditional Chinese medicine was dose-dependent,and both were less than MTX group（P<0.05）;compared with Y15 group,the expression of p-Paxillin was dose-dependent in each dose group of traditional Chinese medicine（P<0.05）.clinical Part（A）Baseline characteristics analysis:There was no significant difference in age,ectopic pregnancy syndrome differentiation and treatment method,pre-treatment estrogen and progesterone levels,salary scores,and items between the two groups（P>0.05）.The characteristics are basically the same,the grouping is balanced and comparable.（B）Efficacy analysis:Comparison within the group,Estradiol level, progesterone level,Salle score,and intimal thickness of the Huayu xiaozheng group after treatment were higher than those before treatment,and the E₂/P ratio.the value of uterine arterial beat index PI after treatment was lower than before treatment,and the difference was statistically significant （P<0.05）.After treatment,the number of patients who owns multiple layers of endometrial morphology,myometrial echo,early diastolic notch,end diastolic blood flow,and subendometrial blood flow,was increased before treatment,but the difference was not statistically significant（P>0.05）.The progesterone level,Salle score,and endometrial thickness after treatment in the blank group were significantly higher than before treatment（P<0.05）.The estradiol level and E₂/P ratio after treatment were higher than those before treatment.the value of uterine arterial beat index PI after treatment was lower than before treatment.After treatment,the number of cases with multiple layers of endometrial morphology,myometrial echo,end diastolic blood flow,was lower than that before treatment.The number of cases with early diastolic notch and subendometrial blood flow was lower than that before treatment,the difference was not statistically significant（P>0.05）.Comparison between groups,After treatment,estradiol level in Huayu xiaozheng group was higher than that in blank group（P>0.05）.Progesterone level,Salle score and endometrial thickness in Huayu xiaozheng group was higher than that of blank group.E₂/P ratio and PI of uterine artery were lower than that of blank group.The difference was statistically significant（P<0.05）.The number of cases of multiple layers of endometrial morphology and end diastolic blood flow in the Huayu xiaozheng group was more than that in the blank group（P<0.05）.The number of cases with myometrial echo,early diastolic notch and subendometrial blood flow,was more than that in the blank group（P>0.05）.The differences of estradiol level,progesterone level,Salle score,endometrial thickness,and uterine arterial beat index PI between the treatment before and after in the Huayu xiaozheng group were larger than those in the blank group.The difference was statistically significant（P<0.05）.Compared with the efficacy between the two groups,Huayu xiaozheng group was superior to the blank group,and the difference was statistically significant（P<0.05）.（C）Safety analysis:During the observation period,only one case of adverse events occurred in the Huayu Xiaozheng group,which was the symptom of mild diarrhea during the second treatment cycle.It was not considered to be related to medication.No adverse reactions were found in the remaining cases.（D）Follow-up:limited by the observation period,the follow-up observation time of the enrolled patients ranged from 1 to 3 menstrual cycles.During the period of clinical expulsion,2 patients were clinically pregnant,including 1 intrauterine pregnancy and 1 biochemical pregnancy.ConclusionHuayu xiaozheng Tang can inhibit the invasion and migration of tubal trophoblastic gestational cells in a dose-dependent manner.The optimal dose-response was achieved by Huayu xiaozheng Tang of 20mg/m L.Huayu xiaozheng Tang may play a similar or synergistic effect with FAK inhibitors,inhibit the invasion and migration of tubal trophoblastic gestational cells,the mechanism may be via down-regulation of u PA,u PA-R expression associated with degradation of extracellular matrix.At the same time,the expression of FAK,p-FAK,and p-Paxillin were adjusted to inhibit the transmembrane signal transduction system of trophoblast extracellular matrix-integrin-cytoskeleton,and reduce the invasion and destruction of the implantation site of the fallopian tubes.In order to achieve the purpose of treating ectopic pregnancy..Huayu xiaozheng granules may improve the ultrasound Salle scores by increasing the level of progesterone,decreasing the E₂/P ratio,increasing the endometrial thickness,and reducing the resistance of the uterine arterial blood flow,thereby improving the endometrial receptivity of patients with blood stasis after the cure of ectopic pregnancy,and is safe to be used in clinical treatment.