Treatment Of Hydroxychloroquine In Obstetric Antiphospholipid Syndrome And Related Experimental Study

Antiphospholipid syndrome(APS)is a non-specific autoimmune disease characterized by thrombosis,pathologic pregnancy and thrombocytopenia.The main serological characteristic was that antiphospholopid antibodies(aPL)were consistently positive.Obstetric APS(Obstetric APS)is obstetric antiphospholipid syndrome(OAPS),where complications include abortion,preterm birth,preeclampsia,fetal intrauterine growth restriction,fetal death in utero,placental insufficiency.Currently,Low molecular weight heparin(LMWH)combined with Low dose aspirin(LDA)is the main treatment for OAPS.Although it can improve the live birth rate of patients,30%of patients still suffer serious complications.Early pregnancy is easy to induce recurrent spontaneous abortion(RSA),which has become an urgent problem in the field of obstetrics.Therefore,it is extremely urgent to seek effective treatment for patients with refractory GAPS and explore the molecular mechanism of OAPS complicated with RSA.This study elaborated the molecular mechanism of GAPS associated RSA,explored the protective mechanism of hydroxychloroquine(HCQ)in treating refractory OAPS associated RSA,revealed the clinical efficacy and drug safety of HCQ in treating refractory OAPS,and provided certain theoretical basis for the clinical application of HCQ.The experimental method1.Complete clinical data of 21 patients with refractory OAPS were collected,and these patients were treated with HCQ on the basis of anticoagulant therapy.The clinical efficacy and drug safety of HCQ were evaluated by T test or χ2 test,and factors related to the occurrence of abortion were analyzed by single factor.2.Serum IgG was extracted by NabTM Protein A Plus Spin kit,and IgG concentration was determined by BCA kit.3.Extraction of human umbilical vein endothelial cells(HUVECs)and decidual vascular endothelial cells(DVECs).The effects of aPL on proliferation,migration and angiogenesis of vascular endothelial cells were detected by CCK-8 assay,scratch assay and angiogenesis assay.4.RT-PCR and western blot were used to detect the effects of aPL on the expression of VEGF and MMP-2 in vascular endothelial cells.5.Western blot was used to detect the effect of aPL on the expression of VEGF and MMP-2 in vascular endothelial cells through ERK pathway.6.Western blot,scratch test and angiogenesis test were used to study the effect of aPL on vascular endothelial cell migration and angiogenesis through ERK pathway.7.CCK-8 assay was used to detect the effect of HCQ on vascular endothelial cell viability.The effect of HCQ on aPL-inhibited vascular endothelial cell migration and angiogenesis was investigated by scratch test and angiogenesis test.8.RT-PCR and western blot assay were used to detect the effects of HCQ on aPL-inhibited ERK phosphorylation,VEGF and MMP-2 expression in vascular endothelial cells.Experimental results:1.Clinical efficacy and safety of HCQ in the treatment of refractory OAPS patients complicated with RSAThe results showed that HCQ combined with anticoagulant could effectively reduce the incidence of abortion in patients with refractory OAPS compared with anticoagulant therapy.It can significantly reduce the positive rate of aCL and LAC in serum.It can significantly reduce the average titer level of aCL and aβ2GPI in serum.The positive rate of aβ2GPI,aCL,LAC and aPL at medium and high titer level were decreased significantly.Univariate analysis showed that the positive rate of aβ2GPI,aCL,LAC and aPL were related to RSA induced by aPL.Anticoagulation combined with HCQ does not increase the incidence of adverse reactions in OAPS patients.2.aPL inhibits the proliferation of vascular endothelial cellsCCK-8 assay showed that the inhibition of aPL on proliferation of HUVECs and DVECs was time-dependent and concentration-dependent.3.aPL inhibits migration and tubulogenesis of vascular endothelial cellsThe results of cell laceration test and canalization test showed that the migration area of HUVECs and DVECs was significantly reduced after aPL treatment,and the number of tubule nodes,nets and branches were also significantly reduced,indicating that aPL could significantly inhibit the migration and canalization ability of HUVECs and DVECs.4.aPL inhibited VEGF and MMP-2 expression levels in vascular endothelial cellsRT-PCR and western blot results showed that the expression levels of VEGF and MMP-2 in HUVECs and DVECs were significantly inhibited by aPL.5.aPL may reduce the expression of VEGF and MMP-2 in vascular endothelial cells and inhibit the migration and angiogenesis of vascular endothelial cells by inhibiting ERK pathwayThe results showed that when aPL down-regulated ERK phosphorylation in HUVECs and DVECs,VEGF and MMP-2 expression levels were also decreased,and the migration area and tubule formation number of HUVECs and DVECs were also significantly reduced.When ERK activator upregulated ERK phosphorylation level inhibited by aPL,the expression levels of VEGF and MMP-2 increased,and the migration area and tubule formation number of HUVECs and DVECs also increased significantly.These results suggest that aPL may inhibit the expression of VEGF and MMP-2 in HUVECs and DVECs by inhibiting ERK pathway,and inhibit cell migration and angiogenesis.6.Effect of HCQ on vascular endothelial cell viabilityCCK-8 experiment showed that HCQ had no significant effect on HUVECs and DVECs viability at the concentration of 0-10 μg/mL,while HUVECs and DVECs viability were significantly inhibited at the concentration of 100 μg/mL.These results indicate that HCQ can affect vascular endothelial cells in a certain concentration range,and high concentration will lead to decreased cell viability.In this experiment,1 μg/mL was selected as the functional concentration of HCQ in subsequent experiments,which was consistent with the concentration applied in previous studies.7.HCQ improves the inhibitory effect of antiphospholipid antibody on vascular endothelial cell migration and angiogenesisThe results showed that aPL reduced the migration area of HUVECs and DVECs,the number of tubule nodes,the number of mesh and the number of branches,while HCQ significantly improved the migration area,the number of tubule nodes,the number of mesh and the number of branches.These results indicated that HCQ could significantly improve the inhibitory effect of aPL on HUVECs and DVECs migration and angiogenesis.8.HCQ improves the inhibitory effect of aPL on ERK phosphorylation,VEGF and MMP-2 expression levels in vascular endothelial cellsRT-PCR and western blot showed that HCQ could restore the inhibition of aPL on ERK phosphorylation,VEGF and MMP-2 expression in HUVECs and DVECs,and there was no significant difference between HCQ and NC group.In the absence of aPL,HCQ did not affect ERK phosphorylation,VEGF and MMP-2 expression in HUVECs and DVECs.Conclusions:1.HCQ can significantly reduce the incidence of abortion and the level of aPL titer in patients with refractory OAPS.Moderate and high titer aβ2GPI,moderate and high titer aCL,LAC and aPL triple positive were the related factors of aPL causing RSA.It is expected to be a basic drug for the treatment of refractory OAPS.2.aPL may inhibit the expression of VEGF and MMP-2 in HUVECs and DVECs by regulating the ERK pathway,thus inhibiting the proliferation,migration and angiogenesis of vascular endothelial cells in the decidua of uterus.3.HCQ may play a protective role in the migration and angiogenesis of vascular endothelial cells in the decidua tissue by improving the inhibitory effect of aPL on ERK phosphorylation,VEGF and MMP-2 expression of vascular endothelial cells,providing a objectivity foundation for the clinical application of HCQ in aPL induced RSA.