Study On The Effect And Mechanism Of Bushenguchong Treatment To Regulate Function And Differentiation Of Subsets Of NK Cells In Treating The URSA Of Kidney Deficiency Type

Objective:To investigate the mechanism of NK cells in the pathogenesis of URSA,explained mechanism of the Bushenguchong Treatment "Jiaweishoutai Wan" by regulating the function and NK cells subsets differentiation to induce immune tolerance in the treatment of URSA,and provided targeted therapeutic targets and theoretical basis for traditional Chinese medicine treatment of URSA.Methods:This study was divided into three parts.Initially,was the theory research,systematically expounding the TCM etiology and pathogenesis of URSA,treatment of URSA in TCM and Western medicine,the relationship between NK cells and fetal immune tolerance.Furthermore,was the experimental study,to research the activity of NK cells,the differentiation of subpopulations,the secretion of cytokines and the level of mRNA transcription of URSA pregnancy loss group by flow cytometry,ELISA method,RT-PCR technology and other modern molecular biology techniques,compared with healthy pregnant women.To observe the changes of NK cell activity and function in patients with URSA before and after the treatment of Bushen Guchong Treatment.The serum levels of E2,P and P-HCG in the URSA group were detected by chemiluminescence method and compared with those of HEP group.And studied the correlation of E2,P and P-HCG levels with NKG2D and NKG2A with URSA.And finally,was the discussion,to investigate the role of NK cells in the pathogenesis of URSA,and to explore the mechanism of the treatment of URSA by regulating the function and the differentiation of the sub groups of NK cells.Results:Compared with the HEP group,the expression of NKG2D of NK cells in URSA pregnancy loss group was higher and NKG2A was lower(P<0.01).Before treatment,the ratio of CD3-CD56+TNF-?+,CD3-CD56+IFN-?+cell subsets in the URSA pregnancy loss group was significantly higher than that of the HEP group while the proportion of CD3"CD56+IL-4+ and the CD3-CD56+IL-10+NK cells subsets was significantly lower(P<0.01).The TNF-a,IFN-y and mRNA transcription levels of pNK cells in URSA pregnancy loss group were significantly higher than those in the HEP group while the IL-4,IL-10 and mRNA transcription levels of pNK cells were significantly lower(P<0.01).The serum E2,P,(3-HCG levels had no significant difference of URSA pregnancy group after treatment with Bushenguchong Method compared with the same period in the HEP group(P>0.05).The expression of NKG2D in pNK cells was significantly lower than that before treatment in URSA pregnancy group while the NKG2A was significantly higher(P<0.01).The proportion of CD3"CD56+TNF-?+,CD3-CD56+IFN-?+NK cells subsets was significantly lower than that before treatment while the propo-rtion of CD3"CD56+IL-4+ and CD3-CD56+IL-10+NK cells subsets was significantly higher(P<0.01).The levels of TNF-?,IFN-? and mRNA secreted by pNK cells were significant-ly decreased compared with those before treatment while the levels of IL-4,IL-10 and mRNA were significantly higher(P<0.01).The TNF-a mRNA level of dNK cells in HEP group was higher than that of pNK cells(P<0.05),there was no difference in the level of IFN-ymRNA compared with pNK cells(P>0.05),and IL-4 and IL-10mRNA was signify-cantly higher than that of pNK cells(P<0.01).The TNF-a,IL-4 and IL-10mRNA levels in dNK cells of URSA pregnancy loss group were significantly higher than those in pNK cells(P<0.01),and there was no difference of IFN-ymRNA between pNK and dNK cells(P>0.05).The E2,P and ?-HCG levels of URSA pregnancy group had a negative linear correlation with the expression of NKG2D,while had a positive linear correlation with the expression of NKG2A.Conclusions:The activity,the differentiation of sub group,the secretion of cytokines and the level of mRNA transcription NK cells played an important role in the pathogenesis of NK.Bushenguchong method was the basic treatment of kidney deficiency of URSA,and Jiaweishoutai Wan was an effective prescription for the treatment of URSA with kidney deficiency.Jiaweishoutai Wan inhibited the maternal fetal immune rejection by downregulating the expression of NKG2D and upregulating the expression of NKG2A,inducing immune tolerance by adjusting the ratio of cell subsets,the secretion level of downregulating of Thl type cytokines TNF-?,IFN-y and the corresponding transcription level of mRNA,increasing the level of secretion of Th2 cytokines IL-4,IL-10 and mRNA transcription.The effectiveness of Bushenguchong method to treat the URSA was proved from the protein level and gene level.The trend of mRNA transcription level of TNF-a?IFN-??IL-4?IL-10 secreting by dNK cells were consistent with them from pNK cells.The level and the transcription level of mRNA secretion of TNF-a and IFN-y was higher,and lower of IL-4,IL-10,which mediated maternal immune rejection,involved in the pathogenesis of URSA.NK cells mediated pregnancy immune tolerance status during may be realized mainly through the immune function of maternal fetal interface.The peripheral blood NK cells to a certain extent also reflected the immunological characteristics of the maternal fetal interface,through the detection of peripheral blood NK cells could indirectly know the immune status of the decidua.The E2,P,p-HCG levels and NKG2D,NKG2A expression had a linear correlation,respectively the negative correlation and positive correlation.The results indicates that the method of Bushenguchong could regulate the secretion of E2,P,?-HCG by regulating the activation of NK cells in URSA patients.