| Objective:1. To explore whether HMGB1 and RAGE involved in the pathogenesis of unexplained recurrent spontaneous abortion(URSA), we detect the protein expression level of HMGB1, RAGE, Th17 and Treg cells related cytokines in patients peripheral blood with URSA, and analysis the expression meaning of HMGB1 and RAGE, and correlation between HMGB1 and RAGE, Th17, Treg cells related cytokines.2. To investigate the adjustment function of HMGB1 to Th17 and Treg cells and preliminaryly discuss the possible mechanism of HMGB1 in URSA.3. To provide new theoretical and new thought for URSA pathogenesis, HMGB1 is likely to be a new therapeutic targets for the treatment of URSA.Methods:Selected 42 cases early pregnant women who had a history of unexplained recurrent spontaneous abortion(URSA group) and 32 normal early pregnant women with no history of abortion(NP group). Flow cytometry was used to detect the frequency of Th17 and Treg cells in the peripheral blood of both groups. ELISA were applied to detect the concentrations of HMGB1, RAGE, IL-17, IL-6, TGF-β and IL-10 in the peripheral blood of both groups. Fluorescence quantitative PCR was used to detect the relative m RNA expression of HMGB1, RAGE, Foxp3, IL-17 in the two groups. Sorting CD4+T cells by positive immune magnetic bead separation method and cultured in vitro. Recombinant human HMGB1(100ng/ml) stimulation CD4+T cells and culture CD4+T cells withdifferent time and detected the proportion of Th17 and Treg cells with FCM and test the relative expression of RAGEm RNA. Using Graph Pad Prism5.0 statistical software to analyze data and drawings.Results:1. Treg cells proportion in URSA group was lower than NP group(P < 0.01), while the proportion of Th17 cells and Th17/Treg ratio of URSA group were higher than NP group(P < 0.01).2. The concentration of HMGB1 and RAGE in the peripheral blood of URSA group was higher than NP group(P < 0.05); Compared with NP group, the expression level of IL-17 and IL-6 in the peripheral blood of URSA group were increased(P < 0.05), while the expression levels of TGF-β and IL-10 was decreased(P < 0.05).3. Compared with NP group, the relative expression of HMGB1, RAGE, IL-17 m RNA in the peripheral blood of URSA group was increased(P < 0.01); While Foxp3 m RNA expression was decreased(P < 0.01); IL-17/Foxp3 m RNA ratio increases significantly(P < 0.01).4. ELISA results showed that the expression level between HMGB1 and RAGE were positively correlated in the peripheral blood of URSA group(r = 0.70, P < 0.01);The expression level of HMGB1 was positively correlated with IL-17 and IL-6(r = 0.79,r = 0.84, P < 0.01), while HMGB1 expression level was negatively correlated with TGF-β and IL-10(r =-0.90, r =-0.83, P < 0.01).5. Recombinant human HMGB1 stimulates CD4+T cells with 0h, 6h, 12 h, 24 h.Compared with 0h and 6h group, RAGEm RNA expression was no statistical difference(P > 0.05), 12 h and 24 h group were higher than 0h and 6h group(P < 0.01);Th17 and Treg cell proportion and Th17/Treg ratio of 0h and 6h group was no statistical significance(P > 0.05), compared with 0h and 6h group, Th17 cells proportion and Th17/Treg ratio of 12 h and 24 h group were higher(P < 0.01); wlile Treg cells proportion was reduced significantly(P < 0.01).Conclusion:1. The happening of URSA may be associated with Th17, Treg cells and Th17/Treg imbalance.2. ELISA results show that HMGB1 and RAGE are closely related to the occurrence and development of URSA. The abnormal expression of Th17 and Treg cell related cytokines play an important role in the pathogenesis of URSA.3. PCR results show that HMGB1 has a close relationship with URSA and RAGE;the key cytokines Th17 and Treg cells and abnormal expression of IL-17/Foxp3 imbalance plays an important role in the occurrence of URSA.4. Enhanced HMGB1 may stimulate increased RAGE expression; HMGB1 by promoting Th17 cells important proinflammatory cytokine release play a proinflammatory role, through down regulating Treg cells immune inhibitory factor weaken the immune suppression function, which impact Th17/Treg balance of URSA.5. HMGB1 time-dependently increased RAGE expression, and promoted Th17 cell proliferation and inhibited Treg cell differentiation. HMGB1 may act on Th17 and Treg cells, aggravating the immune inflammatory reaction and weakening the immune suppression function, resulting in Th17/Treg imbalance, breaking the maternal fetal immune tolerance, eventually led to the maternal immune system on fetal immune rejection. |
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