| 1 ObjectiveBy observing the expression of peripheral blood Tim-3,Gal-9 to the patients with recurrent spontaneous abortion and spleen-kidney deficiency syndrome,also to the normal pregnancy patients,for exploring the relationship which between patients with recurrent abortion peripheral blood related protein expression and recurrent abortion from the perspective of immune regulation;Through detecting the protein expression of Tim-3 and Gal-9 in decidual tissues of normal pregnant mice and recurrent aborted mice,as well as the proliferation of lymphocytes in spleen tissues,to explore the mechanism of tonifying kidney and fetus medicinal granules,through regulating the Tim-3-Gal-9 access to interve the recurrent aborted mice,so as to provide further experimental basis for the treatment of recurrent abortion,with herbs medicine on clinical tonifying kidney and strengthening spleen.2 MethodsCollect 20 patients with recurrent abortion who were hospitalized in the gynecological clinic,of Anhui Hospital of traditional Chinese medicine,as the experimental group,during the period from Jan.1,2019 to Dec.31,2019,also collect 20 patients with first-trimester pregnancy in the same period as the control group.Detect respectively to the expression of the mRNA and protein of peripheral blood Tim-3,Gal-9 from two groups by applying Real-time PCR and Western blot methods,also statistically analyze the test dataExperiment on 40 female CBA/J mice,15 DBA/2 male mice,and 5 BALB/c male mice,after adaptive raising for one week,mating in a cage at a ratio of 2:1 every night.It established the recurrent abortion(RSA)model,while the female CBA/J mice x male DBA/2 mice were caged.It established the normal pregnancy model,while female CBA/J mice x BALB/c mice were caged.It’s considered as the zero day of gestation when the detection of vaginal suppository in female mice or spermatozoa in the vagina.After pregnancy,the mice were randomly divided into four groups,with 10 mice in each group,respectively named as normal pregnancy group,the recurrent abortion model group,the low-dose herb medicine group,the high-dose herb medicine group.The intragastric intervention from the first day of gestation,the normal pregnancy group and the recurrent abortion model group were given the same amount of distilled water by gavage;for the low-dose and high-dose group,were respectively given tonifying kidney and fetus medicinal granules which were diluted with distilled water by gavage(the doses were 11.7g/kg.d、23.4g/kg.d respectively)Keeping above gavage for 15 consecutive days to each group.After 24h of final gavage,then weighed them,took their eyeballs for blood collection,and killed the mice,extracted their uterine decidua,calculated the organ weight index,dissected the uterus to observe the uterus and embryo development status of each mice group,and calculated the abortion rate,as well as embryo loss rate.Detected the proliferation of lymphocytes in spleen tissues of mice by MTT method,respectively detected the protein expression of the mRNA and protein of Tim-3 and Gal-9 from decidual tissues of mice by Real-time PCR and Western blot.3 Results 3.1 Clinical research results3.1.1 Two groups in general conditions.There is no significant difference in age and gestational age between the two groups(P>0.05)3.1.2 Analysis of the Real-time PCR test and the Western blot test resultsAnalysis of the Real-time PCR test and the Western blot test results:Compared with the control group,the expression of the mRNA and protein of Tim-3 in the experimental group was significantly higher than that in the control group(P<0.01).The expression of Gal-9 in peripheral blood between two groups of patients.Gal-9 was expressed in peripheral blood lymphocytes in both groups,moreover,the expression of the mRNA and protein of Gal-9 in the experimental group was lower than the control group,with obviously significant difference(P<0.01).3.2 Experimental research results3.2.1 The uterus and embryo development of pregnant mice in each groupIn the normal pregnancy group,the uterus morphology of pregnant rats showed regular concave convex shape,the embryo showed bead like protrusion,the appearance color was light red,no blood stasis and bleeding spots were found in the uterine cavity;in the recurrent abortion model group,the appearance color of uterus was dark red,the bead like embryo protrusion was not obvious,the obvious blood stasis spots were visible;in the low dose group,the uterus color of pregnant rats was dark,the uterus and embryo volume were obvious The uterus of the pregnant rats in the high dose group of Bushen Antai granules was light red,such as bead like protuberance,the embryo developed well,and the bleeding point in the uterine cavity was not obvious.3.2.2 Abortion rate for pregnant mice in each groupCompared with the normal pregnancy group,the abortion rate and embryo loss rate ofRSA model group significantly increased(P<0.01).Compared with the model group,the abortion rate and embryo loss rate in the low-dose group of kidney-tonifying and fetus medicinal granules was lower than the model group,with the difference of statistical significance(P<0.05);the abortion rate and embryo loss rate of RSA in the high-dose group of kidney-tonifying and fetus medicinal granules was obviously lower,with the difference of obviously statistical significance(P<0.01),however,the difference of abortion rate and embryo loss rate between the high and low-dose group of idney-tonifying and fetus medicinal granules,with obviously statistical significance(P<0.01).3.2.3 Proliferation of spleen lymphocytes in pregnant mice in each group.Compared with the normal pregnancy group,the proliferation of spleen lymphocytes inRSA model group was significantly lower,with the difference of statistical significance(P<0.01);Compared with model group,the proliferation of spleen lymphocytes rate of RSA mice in the low and high-dose group of kidney-tonifying and fetus medicinal granules was obviously rising,with the difference of obviously statistical significance(P<0.01),however,the comparison between the high and low-dose group of kidney-tonifying and fetus medicinal granules,with obviously statistical significance(P<0.05).3.2.4 The protein expression of the mRNA and protein of Tim-3 and Gal-9 in decidual tissues of the pregnant mice in each group.Compared with the normal pregnancy group,the mRNA and protein Tim-3 in the model group of decidual tissues significantly increased(P<0.01);compared with the model group,the Tim-3 protein expression in high-dose herb medicine group was significantly reduced(P<0.01),the Tim-3 protein expression in low-dose herb medicine with no statistical significance(P>0.05)Compared with high-dose herb medicine group,the expression level increased in the low-dose group,with the difference of statistical significance(P<0.05).Compared with the normal pregnancy group,the mRNA and protein Gal-9 in the model group of mice decidual tissues significantly reduced(P<0.01);compared with the model group,the Gal-9 protein expression in high-dose herb medicine group significantly increased(P<0.01),the Gal-9 protein expression in low-dose herb medicine with no statistical significance(P>0.05);Compared with high-dose herb medicine group,the expression level reduced in the low-dose group,with the difference of statistical significance(P<0.05).4 Conclusion4.1 The abnormal expression of the mRNA and proteinof of peripheral blood lymphocytes Tim-3 and Gal-9 in patients with recurrent abortion.4.2 The expression of the mRNA and proteinof of peripheral blood lymphocytes Tim-3 in patients with recurrent abortion was obviously higher than the first trimester of normal pregnancy,while the expression of the mRNA and proteinof of Gal-9 was obviously lower than the first trimester of normal pregnancy,it indicated that the abnormal expression of the mRNA and proteinof of peripheral blood lymphocytes Tim-3 and Gal-9 was associated with the incidence of recurrent abortion.4.3 The tonifying kidney and fetus medicinal granules can improve the development of pregnant uterus and embryo in RSA mice.4.4 The tonifying kidney and fetus medicinal granules can reduce the abortion rate and embryo loss rate of RSA mice.4.5 Improving the immune tolerance at the matural-fetal interface,and promoting the proliferation of spleen lymphocytes in pregnant mice,for the kidney-tonifying and fetus medicinal granules,it maybe as one of the immune regulatory mechanisms,in preventing and treating abortion.(1)The kidney-tonifying and fetus medicinal granules,can reduce the risk of miscarriage and maintain pregnancy,which through reducing the Tim-3 protein expression level,after binding with ligand Gal-9,and inhibiting Thl cell function,so as to achieve the maternal fetal interface immune balance in RSA mice,and Th2 domination(2)The kidney-tonifying and fetus medicinal granules,can improve immune tolerance and maintain pregnancy by improving the level of lymphocyte proliferation in spleen tissues. |
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