Research On Material Basis Of Yinchenhao Decoction For Treatment Of Maternal-fetal ABO Incompatibility

Objective:To explore the effects and molecular mechanisms of Yinchenhao Decoction on Maternal-fetal ABO incompatibility.Methods:1.Measurement of maternal serum IgG anti-A levels and spontaneous abortion was used to evaluated the therapeutic effects of Yinchenhao Decoction on Maternal-fetal ABO incompatibility;The Chinese medicine group was treated with oral Yinchenhao Decoction(granules)while the control group was treated with vitamin E+vitamin C+ phenobarbital+oxygen on 149 pregnant women.2.The chromatograms of Yinchenhao granules at three wavelengths of 254 nm,280 nm and 324 nm were investigated by HPLC.The control fingerprints were established by the Chinese Traditional Medicine Chromatographic Fingerprint Similarity Evaluation System Software(2012A)to evaluate the stability of the components of Yinchenhao Granules.The HPLC analysis was used to identify and characterize the bioactive compounds from Yinchenhao Decoction3.The common peaks were identified and the target pharmacodynamic components were screened through the comparison of Yinchenhao granules fingerprint,the retention time of reference substance and ultraviolet spectrogram.4.Since human erythroleukemia K562 cells in the state of pluripotent myeloid progenitor cells were selected as research objects,the K562 cells were employed to evaluated the therapeutic effects of copounds on the eaythroid differentiation.The staning of biphenyl benzylamine were used to evaluated the effects of compounds on the erythroid differentiation of K562 cells.5.Western Blot(WB)and immunofluorescence(IF)were used to detect expression of the fetal erythroid differentiation marker proteins in compounds treated K562 cells.6.CCK-8 and flow cytometry were used to detect the K562 cells proliferation and cell cycle distribution.7.The expression of FOXO3 and cell cycle-associated protein in compounds treated K62 cells were detected by WB and IF.Results:1.Compared with the western medicine group,the Chinese medicine group can significantly reduce the IgG antibody titer of pregnant women(P<0.01).The two groups of pregnant women did not show signs of spontaneous abortion which suggests that Yinchenhao Decoction(granules)is effective and its effect is better than western medicine.2.The similarity of different batches of Yinchenhao Decoction(granules)was high and its quality was stable,which was confirmed by the comparison of fingerprints of HPLC method.3.9 main chemical components of Yinchenhao Decoction(granules)are identified through the comparison of the retention time of the reference substance and the ultraviolet spectrum,which finally screen the 6,7-dimethoxycoumarin(6,7-DOC)as the target medicinal ingredient.4.The result of biphenyl benzylamine staining showed that compound 6,7-DOC makedly iduced the erythroid differentiation of K562 cells in a dose and time dependent manner.5.The results of WB and IF showed that compound 6,7-DOC induced the expressions of erythroid markers including α-globin,β-globin and γ-globin in K562 cells with dose dependent manner.6.The results of CCK-8 showed that there was no significant difference between the low-dose group and the control group(P>0.05).The cell proliferation in the middle and high-dose groups was significantly inhibited compared with the control group(P<0.05),while the time and dose were observed to be correlation.Flow cytometry results showed that compared with the control group,the low-dose group did not change significantly.The medium-and high-dose groups increased the proportion of cells in G0/G1 phase,while the proportion of cells in S phase decreased.7.WB test results showed that the expression of FOXO3 and p27 had not significantly changed in the low-dose group compared with the control group while the expression levels of FOXO3 and p27 were significantly increased in the middle and high-dose groups,and there was no change in the expression of total CDK4/CDK6,on this promise,pCDK4/pCDK6 was significantly inhibited.The results of IF test showed that compared with the control group,the expression of FOXO3 in the low dose group had not significantly changed while the expression of FOXO3 in the high and middle dose groups significantly had increased in a dose-dependent manner.Conclusion:1.Maternal-fetal ABO incompatibility can be effectively treated by Yinchenhao Decoction.2.6,7-DOC is the active ingredient of Yinchenhao Decoction for the treatment of Maternal-fetal ABO incompatibility.3.6,7-DOC can promote the differentiation of erythroid cells,which may be related to the increase of erythroid differentiation marker expression induced by the transcription factor FOXO3,also may related to the inhibiting proliferation which indirectly promote differentiation in G0/G1 phase.