Expression Of Treg Cells And Related Cytokines In Peripheral Blood Of Patients With Spleen And Kidney Deficiency Syndrome And RSA Mice,and Experimental Study On Traditional Chinese Medicine Intercention

1 ObjectiveThe proportion of CD4~+CD25~+Foxp3~+/CD4~+,CD4~+Foxp3~+cells ratio and the levels of IFN-?,TNF-?,IL-2,IL-4,IL-5,IL-6,IL-10,IL-12,IL-17 and IL-23 cytokines in peripheral blood of patients with Recurrent spontaneous abortion and RSA mice models were measured,and the significance of Th1/Th2,Th17/Treg cells in the pathogenesis of RSA;To explore the effect of Bushen Antai Granules on immune regulation in RSA mice The mechanism provides theoretical support for the clinical intervention of Recurrent spontaneous abortion with traditional Chinese medicine for tonifying the kidney and spleen.2 MethodsFrom January 2019 to December 2019,20 patients with Recurrent spontaneous abortion of spleen kidney deficiency type who were treated in the outpatient and inpatient department of gynecology and reproduction center of the First Affiliated Hospital of Anhui University of traditional Chinese medicine were collected as the study group and20 patients with normal early pregnancy at the same period as the control group.Enzyme-linked immunosorbent assay(ELISA)for detecting peripheral blood IFN-?,TNF-?,IL-2,IL-4,IL-5,IL-6,IL-10,IL-12,IL-17,IL-23 expression;CD4~+CD25~+Foxp3~+/CD4~+,CD4~+Foxp3~+cell ratio were detected by flow cytometry.In the experiment,30 Clark classic Recurrent abortion model mice of CBA/J×DBA/2 were randomly divided into a relapsed abortion model group,a traditional Chinese medicine low-dose group,and a traditional Chinese medicine high-dose group,with 10 rats in each group;CBA/J×BALB/c pregnant mice were used as the normal pregnancy group.From the first day when the mice were detected to pregnancy,they were given gavage:normal pregnancy and model group were given gavage with distilled water;Chinese medicine high and low dose groups were 23.4g/kg.d and 11.7g/kg.d,respectively.Diluted with distilled water to administer the stomach for 15 days.24 hours after the last administration,the mice were sacrificed,the uterus was removed,and the development of the embryo was observed under a section to calculate the abortion rate and embryo loss rate.ELISA was used to detect peripheral blood IFN-?,TNF-?,and IL-2,IL-4,IL-5,IL-6,IL-10,IL-12,IL-17,IL-23 expression;CD4~+CD25~+Foxp3~+/CD4~+,CD4~+Foxp3~+cell ratio detected in peripheral blood of mice by flow cytometry3 Results3.1 Clinical research results3.1.1 Comparison of general conditions of two groups of patientsThere was no statistically significant difference in the age and gestational ageage between the two groups(P>0.05).3.1.2 Expression of IL-2,IL-12,IFN-?,TNF-?in peripheral blood of two groups of patientsCompared with the control group,the levels of IL-2,IL-12,IFN-?,and TNF-?in the peripheral blood of the study group were significantly increased,and the difference was statistically significant(P<0.01).3.1.3 Expression of IL-4,IL-5 and IL-10 in peripheral blood of patients in two groups Compared with the control group,the peripheral blood IL-4 levels in the study group were obviously reduced,and the difference was statistically obvious(P<0.05);the levels of IL-5 and IL-10 were significantly reduced,and the difference was statistically significant(P<0.01).3.1.4 Expression of IL-6,IL-17 and IL-23 in peripheral blood of patients in two groups Compared with the control group,the level of IL-6 in the peripheral blood of patients in the study group was obviously increased,and the difference was statistically obvious(P<0.05);the levels of IL-17 and IL-23 were significantly increased,and the difference was statistically significant(P<0.01).3.1.5 Expression of CD4~+CD25~+Foxp3~+/CD4~+,CD4~+Foxp3~+cell ratio in peripheral blood of two groups of patientsCompared with the control group,the peripheral blood CD4~+CD25~+Foxp3~+/CD4~+,CD4~+Foxp3~+cells ratio in the study group were obviously reduced,and the difference was statistically obvious(P<0.05).3.2 Experimental research results3.2.1 Mouse abortion rate and embryo loss rate in each groupCompared with the normal pregnancy group,the abortion rate and embryo loss rate in the model group were significantly increased,and the differences were statistically significant(P<0.01).Compared with the model group,the abortion rate and embryo loss rate in the high-dose group of Chinese medicine were significant.Decreased,the difference was statistically significant(P<0.01);the abortion rate and embryo loss rate in the low-dose group of Chinese medicine were significantly lower than those in the model group,and the difference was statistically obvious(P<0.05);Compared with the low-dose group,The abortion rate and embryo loss rate in the high-dose group were significantly reduced,and the differences were statistically significant(P<0.01).3.2.2 Expression of IL-2,IL-12,IFN-?,TNF-?in peripheral blood of mice in each groupCompared with the normal pregnancy group,the levels of IL-2,IL-12,IFN-?,and TNF-?in the peripheral blood of the model group mice were significantly increased,and the differences were statistically significant(P<0.01);Compared with the model group,the levels of IL-2,IL-12,IFN-?,and TNF-?in peripheral blood of mice in the high-dose group of Chinese medicine were significantly reduced,and the difference was statistically significant(P<0.01);Compared with the model group,the TNF-?in the peripheral blood of mice in the low-dose Chinese medicine group was significantly reduced,the difference was statistically significant(P<0.01),and the levels of IL-2 and IL-12 were obviously reduced,the difference was statistically obvious(P<0.05),the level of IFN-?decreased,but the difference was not statistically significant(P>0.05);Compared with the low-dose group of traditional Chinese medicine,the levels of IL-2,IFN-?,and TNF-?in the peripheral blood of mice in the high-dose group of Chinese medicine were obviously reduced,The difference was statistically obvious(P<0.05).3.2.3 Expression of IL-4,IL-5,IL-10 in peripheral blood of mice in each groupCompared with the normal pregnancy group,the levels of IL-4,IL-5,and IL-10 in the peripheral blood of the mice in the model group were significantly reduced,and the difference was statistically significant(P<0.01);Compared with the model group,the levels of IL-4,IL-5,and IL-10 in the peripheral blood of mice in the high-dose group of Chinese medicine increased significantly,and the difference was statistically significant(P<0.01);Compared with the model group,the level of IL-4 in the peripheral blood of mice in the low-dose group was obviously increased,and the difference was statistically obvious(P<0.05).The levels of IL-5 and IL-10 were increased,but the difference was not statistically significant(P>0.05);Compared with the low-dose Chinese medicine group,the levels of IL-4,IL-5,and IL-10 in the peripheral blood of mice in the high-dose Chinese medicine group were significantly increased,and the difference was statistically significant(P<0.01).3.2.4 Expression of IL-6,IL-17,IL-23 in peripheral blood of mice in each groupCompared with the normal pregnancy group,the levels of IL-6,IL-17,and IL-23 in peripheral blood of the model group mice were significantly increased,and the difference was statistically significant(P<0.01);Compared with the model group,the levels of IL-6,IL-17,and IL-23 in the peripheral blood of mice in the high-dose group of Chinese medicine were significantly reduced,and the difference was statistically significant(P<0.01);Compared with the model group,the levels of IL-6 and IL-17 in the peripheral blood of mice in the low-dose group of Chinese medicine were significantly reduced,the difference was statistically significant(P<0.01),the level of IL-23 was obviously reduced,and the difference was statistically obvious(P<0.05);Compared with the low-dose group of traditional Chinese medicine,the levels of IL-6,IL-17,and IL-23 in peripheral blood of mice in the high-dose group of Chinese medicine were obviously reduced,and the difference was statistically obvious(P<0.05).3.2.5 Expression of CD4~+CD25~+Foxp3~+/CD4~+,CD4~+Foxp3~+cell ratio in peripheral blood of mice in each groupCompared with the normal pregnancy group,the proportion of CD4~+CD25~+Foxp3~+/CD4~+,CD4~+Foxp3~+cells ratio in peripheral blood of the model group mice was significantly reduced,and the difference was statistically significant(P<0.01);Compared with the model control group,the proportion of CD4~+CD25~+Foxp3~+/CD4~+,CD4~+Foxp3~+cells ratio in peripheral blood of mice in the high-dose group of Chinese medicine increased significantly,and the difference was statistically significant(P<0.01);Compared with the model group,the proportion of CD4~+CD25~+Foxp3~+/CD4~+,CD4~+Foxp3~+cells ratio in peripheral blood of RSA mice in the low-dose group of Chinese medicine increased,but the difference was not significant(P>0.05);Compared with the low-dose group of Chinese medicine,the proportion of CD4~+CD25~+Foxp3~+/CD4~+,CD4~+Foxp3~+cells ratio in peripheral blood of mice in the high-dose group of Chinese medicine increased obviously,the difference was statistically obvious(P<0.05).4 Conclusion4.1 The levels of IFN-?,TNF-?,IL-2,and IL-12 cytokines secreted by Th1 cells were significantly increased in peripheral blood of RSA patients and mice;IL-4,IL-5,and IL-10 secreted by Th2 cells Cytokine levels were significantly reduced in peripheral blood of RSA patients and mice;Th1/Th2 imbalance,Th1 type cytokine secretion increased,and shift to Th1 type,suggesting that Th1/Th2 imbalance is closely related to Recurrent spontaneous abortion.4.2 Th17 cell-related cytokines IL-6,IL-17,and IL-23 levels were significantly increased in the peripheral blood of RSA patients and mice,while the proportion of CD4~+CD25~+Foxp3~+/CD4~+,CD4~+Foxp3~+cells ratio was significantly reduced,indicating that RSA in During the onset of disease,the level of Thl7 cytokines with pro-inflammatory effects increases,while Treg cells with immunosuppressive effects decrease,and the Th17/Treg balance shifts to Thl7 cells,down-regulates the maternal immune suppression of the fetus,causing imbalance in immune balance,leading to miscarriage.4.3 Bushen Antai Granules can significantly reduce the embryo loss rate and abortion rate in a mouse model of Recurrent spontaneous abortion.4.4 Bushen Antai Granules can inhibit the secretion of Th1-type cytokines IL-2,IL-12,IFN-?,TNF-?and Th17-related cytokines IL-6,IL-17,IL-23 in peripheral blood of RSA mice and up-regulate The levels of Th2 cytokines IL-4,IL-5,IL-10 and the ratio of CD4~+CD25~+Foxp3~+/CD4~+,CD4~+Foxp3~+cells ratio in peripheral blood of RSA mice are better,higher doses are more effective.4.5 Possible Mechanism of Bushen Antai Granules on Regulation of Maternal-Fetal Immunity Tolerance4.5.1 Bushen Antai Granules can suppress Th1 cytokine secretion,promote Th2cytokine secretion,correct Th1/Th2 balance shift,reduce maternal-fetal immune rejection,maintain pregnancy,and effectively reduce abortion in Recurrent spontaneous abortion mice Rate and embryo loss rate.4.5.2 Bushen Antai Granules can promote the differentiation of Treg cells and down-regulate the levels of Th17-related cytokines,thereby reversing the Th17/Treg imbalance,inducing maternal-fetal immune tolerance,effectively reducing the abortion rate and embryo loss rate of Recurrent spontaneous abortion mice,and treating RSA.