Functional Regulation And Mechanism Of DNK Cells-derived PEDF At Maternal-Fetal Interface And Pregnancy Maintenance During Early Pregnancy

Part I Expression and biological function of PEDF/PEDF-R at normal maternal-fetal interfaceObjective:To study the expression of PEDF in pNK cells,dNK cells and DSCs of normal pregnant women.To investigate the distribution of PEDF-R on decidual tissue and DSCs of normal early pregnancy and the biological function of PEDF at maternal-fetal interface.Methods:In this study,flow cytometry was used to detect the expression of PEDF in pNK cells,dNK cells,and DSCs in normal pregnant women.The changes of PEDF in pNK cells after co-culture with DSCs or HTR-8/SVneo cells(immortalized human extravillous trophoblast cell line)or three kinds of cells co-cultured group was detected.To analyze the effect of rhPEDF,normal dNK cells-derived PEDF on DSCs apoptosis and inflammatory response induced by LPS.The protein levels of PEDF-R on normal decidual tissue and DSCs were detected by immunohistochemistry and Western blot.Results:(1)About 80%of dNK cells express PEDF,while only about 50%of pNK cells express PEDF,the difference is statistically significant(P<0.01).(2)pNK cells were co-cultured with DSCs or HTR-8/SVneo cells(immortalized human extravillous trophoblastic cell line)or three kinds of cells co-cultured,the results showed that compared with pNK cells alone,the expression levels of PEDF in pNK cells were significantly increased in the three cell co-culture group and the two cell types co-culture group,and the difference was statistically significant(P<0.01).(3)The expression of PEDF-R was found on the normal decidua by immunohistochemistry,and then the expression of PEDF-R on DSCs was confirmed by Western blot.Flow cytometry showed that DSCs hardly secreted any PEDF,compared with normal dNK cells,which was statistically significant(P<0.001).(4)We added rhPEDF to DSCs for 12h,and then treated these cells with LPS.The flow cytometry results showed that the proportion of total apoptotic cells of DSCs increased after LPS was added.and the proportion of total apoptotic cells was decreased in the presence of rhPEDF.The difference was statistically significant(P<0.001).PEDF-R was silenced on DSCs with three different plasmids and the plasmid sh-PEDF-R3 was selected with the best silencing effect for subsequent experiments.The results of Flow cytometry displayed that the proportion of total apoptotic cells of DSCs increased after the stimulation of LPS.The culture supernatant of dNK cells from normal early pregnant women attenuated the proportion of total apoptotic cells of DSCs,while the proportion of apoptotic cells increased after silencing PEDF-R,significantly higher than the negative plasmid control group.The difference was statistically significant(P<0.01).(5)After pre-adding rhPEDF to the plate for 12h,the DSCs were treated with LPS.The proportion of TNF-?+ DSCs cells increased after LPS treatment,while the proportion of TNF-a+DSCs cells decreased after the administration of rhPEDF.The difference was statistically significant(P<0.01).But for IL-1?,IL-8,IL-17A,there are no significant effect(P>0.05).The culture supernatant of dNK cells from normal pregnant women was pre-added into PEDF-R knockout DSCs and control DSCs culture system,the results showed that the proportion of TNF-?+DSCs cells increased after LPS treatment and the effcet was abrogated by the pre-addition of rhPEDF.Compared with the negative plasmid control group,the proportion of sh-PEDF-R cells on DSCs increased obviously and the difference was statistically significant(P<0.01).The same method was used to detect IL-1?,IL-8,IL-17A secreted by DSCs,and its secretion was not significantly changed(P>0.05).Conclusion:(1)Compared with pNK cells,the expression of PEDF in dNK cells in normal early pregnant women was significantly higher than that in pNK cells,which may be related to the local microenvironment of decidua at maternal-fetal interface.(2)PEDF-R was expressed on normal decidua and normal DSCs,but PEDF was not expressed in DSCs.(3)PEDF produced by dNK cells can inhibit LPS-induced apoptosis and inflammatory of DSCs through the interaction with PEDF-R.Part II The mechanism of PEDF exerting biological function at maternal-fetal interfaceObjective:To analyze the signaling pathway of PEDF protecting DSCs from LPS-induced damage at the maternal-fetal interface.Methods:Western blot was used to detect the changes of the protein levels of MAPK family members(ERK1/2 and P38),STAT family members(STAT6),I?B,NF-?B,and flow cytometry was used to further analyze the signaling pathway of PEDF in biological function.Results:(1)MAPK family members(ERK1/2 and P38)and STAT family members(STAT6)were detected by Western blot.The results showed that the phosphorylation of ERK1/2 was significantly decreased by the application of LPS,and this effect was weakened by the addition of rhPEDF in advance.LPS stimulation led to the up-regulation of the protein levels of phosphorylated I?B and phosphorylated NF-?B,and rhPEDF pre-treatment significantly inhibited the up-regulation of LPS-induced protein levels.No changes of P38 and STAT6 protein levels were detected by Western blot.(2)Specific signal transduction inhibitors were used to analyze DSCs apoptosis,the results revealed that the proportion of apoptosis of DSCs increased significantly after LPS stimulation,but the effect was abrogated by the pre-administration of rhPEDF,and SCH772984(ERK1/2 inhibitor)could notably reversed PEDF-mediated inhibition of apoptosis.The difference was statistically significant(P<0.05),but there was no significant difference in the proportion of apoptosis of DSCs between CAPE(NF-?B inhibitor)group and LPS group(P>0.05).(3)Specific signal transduction inhibitors were also used to detect cytokine secretion of DSCs,the results showed that the level of TNF-? secreted by DSCs increased significantly after LPS treatment,but the proportion of TNF-? DSCs in the pre-added rhPEDF group was declined.The difference was statistically significant(P<0.01),but the level of TNF-? secretion after blocking ERK1/2 signaling pathway with SCH772984(ERK1/2 inhibitor)had no significant change compared with rhPEDF group(P>0.05).However,the expression of TNF-? in DSCs in CAPE(NF?B inhibitor)group was markedly lower than that in LPS group(P<0.001).Conclusion:PEDF may protect DSCs from apoptosis by activating ERK1/2 signaling pathway,and inhibit the pro-inflammatory process of DSCs induced by LPS by inhibiting NF-?B signaling pathway,Part ? Abnormal expression of PEDF/PEDF-R at maternal-fetal interface in pregnant women with abortion and its effect on DSCsObjective:Comparison of PEDF expression between dNK cells of normal pregnant women and dNK cells of pregnant women with abortion.To investigate the protein expression levels on normal decidual tissue and DSCs,on abnormal decidual tissue and DSCs,and the difference of biological function between normal dNK cells and abnormal dNK cells.Methods:Flow cytometry and Western blot were used to detect the expression of PEDF in dNK cells of normal pregnant women and dNK cells of abortive women.Immunohistochemical technique and Western blot were used to confirm the expression of PEDF-R on normal decidual tissue and DSCs,on abortive decidual tissue and DSCs.The biological function of PEDF secreted by normal dNK cells and abortive dNK cells were analyzed by flow cytometry.Results:(1)It was found by flow cytometry that approximately 80%of dNK cells from normal pregnant women expressed PEDF,whereas only 45%of dNK cells from abortion were PEDF positive.Moreover,western blot showed that the expression level of PEDF in dNK cells from women with miscarriage was obviously lower in comparison with normal pregnancy.The difference was statistically significant(P<0.001).(2)Western blot results indicated that PEDF-R bands were almost not found at 55kDa on DSCs from women with abortion,but PEDF-R bands were significantly observed on DSCs in normal early pregnant women group.Quantitative analysis also showed that the expression of PEDF-R on DSCs of abortive women was significantly lower than that of normal early pregnant women(P<0.001).At the same time,the results detected by immunohistochemistry were consistent with those detected by western blot.(3)The results of flow cytometry showed that the total apoptosis rate of DSCs increased after adding LPS,and pre-supplementation with the dNK-cell supernatant attenuated LPS-induced apoptosis of DSCs.But the effect was more evident with pre-addition of the normal dNK-cell supernatant(NP-sNK)in comparison with the abnormal dNK-cell supernatant(AP-sNK).The difference was statistically significant(P<0.05).(4)The flow results revealed that the TNF-a secreted by DSCs increased significantly after adding LPS,and the rate of TNF-a+DSCs was decreased when the dNK-cell culture supernatant was added in advance,but the rate of TNF-a secreted by DSCs was higher than that in NP-sNK group when AP-sNK was pre-added.The difference was statistically significant(P<0.01).Conclusion:(1)The expression of PEDF in abortive dNK cells was significantly lower than that in normal dNK cells.Accordingly,the expression of PEDF-R in abortive decidual tissue and DSCs decreased.This suggests that the decrease of PEDF/PEDF-R expression may be related to spontaneous abortion.(2)Compared with the normal supernatant of dNK cells,anti-inflammatory and anti-apoptosis abilities of the abortion supernatant of dNK cells were weakened.This suggests that in abortion,dNK cells have decreased anti-inflammatory and anti-apoptotic abilities because of the down-regulated PEDF and PEDF-R.Therefore,dNK cells and PEDF secreted by these cells may be essential to maintain normal pregnancy.