| PurposeThe subject, by establishing anticardiolipin antibody (ACA) positive abortion mice model, observes the influences of Anziheji on the JAK/STAT3 signaling pathways of the placenta and decidua of ACA positive abortion mice, and discusses the pathological mechanism for the therapy of ACA positive abortion with Anziheji, in order to provide a new theoretical and experimental basis for using Anziheji to treat ACA positive abortion.Method1. Divide BALB/C mice into 5 groups at random: Blank control group, model group, low-dose Anziheji group, high-dose Anziheji group and aspirin group. Dissolve human β2-GP I in sterile PBS, and adjust the concentration to 100ug/0.25ml. On day 1, inject 50ul mixed solution of human (32GP-1 and CFA (1:1) in the abdominal cavity for the model group, low-dose Anziheji group, high-dose Anziheji group and aspirin group, and inject 50ul normal saline for the blank control group; on day 8, strengthen the immune with IFA instead of CFA; on day 18, cage the female and male mice of each group together as per the proportion 1:1, and take the day when vaginal plug is seen as day 0 of pregnancy. On day 15 of pregnancy, kill pregnant mice, pick the eyeballs and take the blood, calculate the embryo loss rate, and detect the ACA and IL-6 titer of peripheral blood by means of ELISA.2. Take fresh placenta and decidua tissues, rinse them with normal saline for several times, add a corresponding volume of lysate for homogenization, and after lysis, remove supernatant through centrifugal, and detect the ACA and IL-6 concentration of villi and decidua on the maternal-fetal interface by means of ELISA.3. Take placenta and decidua tissues, fix them with paraformaldehyde, and embed the sections with paraffin. Detect the JAK, GP130, SOCS, P-STAT3 and PCNA content of placenta and decidua on the maternal-fetal interface with immunohistochemical method.4. Take frozen placenta and decidua tissues, and detect the P-STAT3 and STAT3 content of placenta and decidua on the maternal-fetal interface with Western-blot method.Results1. The research has been conducted with β2-GP I immune mice, and this method is more stable than previous modeling method. Both the embryo loss rate and blood ACA titer of the model group were obviously higher than those of blank control group (P<0.05), proving that the modeling was successful.2. The low- and high-dose Anziheji and aspirin could distinctively lower embryo loss rate (P <0.05); and the embryo loss rate of low- and high-dose Anziheji group was equivalent to that of the blank control group (p>0.05).3. The AC A titer of the serum, placenta and decidua of the model group rose obviously in comparison with that of the blank control group (P<0.01); low- and high-dose Anziheji and aspirin could distinctively lower the AC A titer of the serum, placenta and decidua of mice (P< 0.01). The curative effect of low-and high-dose Anziheji groups was equivalent to that of Aspirin group.4. The serum IL-6 titer of the model group rose obviously in comparison with that of the blank control group (P<0.01); high-dose Anziheji could lower IL-6 titer (P<0.05), while low-dose Anziheji group, aspirin group and model group did not have obvious difference in IL-6 tilter (P >0.05). Each group had no significant difference in terms of IL-6 concentration of placenta and decidua (P> 0.05).5. The JAK of placenta and decidua of the model group dropped obviously in comparison with that of the blank control group (P<0.05). Aspirin could obviously raise the JAK level of placenta and decidua of model mice; low-and high-dose Anziheji could raise the JAK level of placenta (P<0.05), and had no influence on the JAK of decidua (P> 0.05).6. The GP130 of placenta and decidua of the model group dropped obviously in comparison with that of the blank control group (P<0.05). Aspirin could obviously raise the GP130 of placenta and decidua (P<0.01), and high-dose Anziheji could raise the GP130 of decidua (P< 0.01), but low-dose Anziheji had no influence on GP130 (P>0.05).7. The SOCS of placenta and decidua of each group did not change (P>0.05).8. The p-STAT3 of the model group dropped obviously in comparison with that of the blank control group (P<0.01). Low-and high-dose Anziheji and aspirin could obviously raise the p-STAT3 level of placenta and decidua (P<0.01). The curative effect of low-and high-dose Anziheji groups was equivalent to that of Aspirin group.9. The PCNA of the model group dropped obviously in comparison with that of the blank control group (P<0.05). Low-and high-dose Anziheji and aspirin could obviously raise the PCNA level of placenta and decidua (P<0.05).ConclusionsOne of the pathomechanisms for AC A to induce pregnancy loss is that ACA could directly act on the JAK/STAT3 signaling pathways of placenta and decidua on the maternal-fetal interface, make related protein expression decreased, lower the proliferation activity of nutrient cells and decidual cells, thus cause the damage of tissues on the maternal-fetal interface, and result in pregnancy loss. Aspirin could raise the expression of related proteins JAK, GP130 and STAT3 of the JAK/STAT3 signaling pathways on the maternal-fetal interface, but it has no influence on IL-6 of serum as well as the IL-6 and SOCS on paternal-metal interface, proving that the mechanism for aspirin to treat ACA positive abortion is realized by activating JAK/STAT3 signaling pathways and promoting the proliferation of nutrient cells and decidual cells.Anziheji could raise the JAK of placenta, boost the expression of STAT3 of placenta and decidua, and promote the proliferation of nutrient cells and decidual cells, and its influences on the IL-6 of serum and the IL-6, GP130 and SOCS on the maternal-fetal interface are not obvious. We consider that, the mechanism for Anziheji and aspirin to treat ACA positive abortion is similarly realized by activating JAK/STAT3 signaling pathways and promoting the proliferation of nutrient cells and decidual cells, and the JAK/STAT3 signaling pathways are not activated through IL-6 signaling pathway. Whereas the network relationship of signaling pathways and the influences of many cell factors on them, as well as the multi-target characteristic of Chinese herbal compound, we consider that maybe some other factors and even signaling pathways have participated in the change of JAK/STAT3 signaling pathways, and their function mechanism is still to be further researched. |
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