| BackgroundObstetric antiphospholipid syndrome(OAPS)is a systemic autoimmune disease mediated by antiphospholipid antibodies(aPLs),with recurrent abortion,severe preeclampsia,fetal growth restriction and other placental pathological pregnancies as the main clinical characteristics,with or without thrombosis,which seriously threatens the health of pregnant women.According to the diagnostic criteria of 2006 Sydney international consensus statement on antiphospholipid syndrome,the pathological pregnancy outcomes of OAPS were described as recurrent miscarriage less than 10 weeks of gestation,miscarriage and stillbirth more than 10 weeks of gestation,and preterm birth due to placental dysfunction such as fetal growth restriction and preeclampsia.The pathogenesis of OAPS has not been clarified,and it is usually considered to be related to coagulation dysfunction and immune-mediated injury.The occurrence of complications of OAPS is related to the impairment of placental function.APLs is a group of autoimmune antibodies against negatively charged phospholipids.The main target antigens are β2 Glycoprotein 1 antigen(β2-GPI)and prothrombin.APLs mainly include IgG and IgM anticardiolipin antibody(ACL),lupus anticoagulation(LA)and anti β2 glycoprotein 1 antibody(anti-β2GP1 Ab),among which anti-β2GP1 is the main antibody causing OAPS.The clinical heterogeneity of OAPS is extremely high.Some atypical OAPS patients only meet the clinical or laboratory standards of the classification criteria,which makes the disease have many bottlenecks in diagnosis and treatment.First-line therapy with aspirin and lowmolecular-weight heparin only alleviated some adverse pregnancy outcomes,increasing the rate of normal pregnancy outcomes from 30%to 70-80%.However,about 20%-30%of patients have no effect on this treatment scheme.Complications of this refractory OAPS are not uncommon,such as preeclampsia(2%-17%)and thrombosis(2%-3%).Therefore,it is urgent to explore the molecular mechanism of OAPS and find new diagnostic and therapeutic targets for OAPS.Hydroxychloroquinone(HCQ)is a small molecule compound with the molecular formula of C18H26C1N3O and molecular weight of 335.87.It was originally used as an antimalarial drug.Because of its anti-inflammatory,immunomodulatory and antiplatelet properties,it has been widely used in the clinical treatment of systemic lupus erythematosus(SLE),rheumatoid arthritis(RA)and other autoimmune diseases.As a non first-line drug for the treatment of OAPS,HCQ has low toxicity and the ability to cross the placenta.Currently,it is only used for OAPS patients who fail to receive routine treatment,OAPS patients with SLE or other systemic autoimmune diseases,and patients with high-risk aPLs spectrum.In the treatment of obstetric patients with refractory OAPS in Shandong Provincial Hospital,HCQ showed significant efficacy and less side effects.Based on the lack of consistent research on the therapeutic effect of HCQ on OAPS,the mechanism of action of HCQ has not been clarified,and its safety,efficacy,and therapeutic mechanism are still controversial.Therefore,it is of certain clinical significance to study the therapeutic effect and mechanism of HCQ on OAPS,which can provide theoretical reference for the treatment of OAPS.This study is intended to explore the therapeutic mechanism of HCQ on OAPSinduced placental pregnancy from four aspects:clinical research,OAPS animal model,cell model and bioinformatics analysis.To explore the therapeutic target of HCQ,optimize the therapeutic scheme of HCQ on OAPS,and find new diagnostic and therapeutic targets.Part Ⅰ Clinical characteristics and related factors of HCQ in patients with OAPS ObjectiveThe clinical data were analyzed to determine the scope of use of hydroxychloroquine(HCQ)in obstetric antiphospholipid syndrome(OAPS).The pregnancy outcome of HCQ/no HCQ treatment group was compared,the therapeutic effect of HCQ on OAPS was evaluated,and the drug safety of HCQ was discussed.Method1 The medical records of 117 inpatients with antiphospholipid syndrome(APS)admitted to the Obstetrics Department of Provincial Hospital from 2019 to 2020 were retrospectively analyzed.The basic information of the enrolled patients was statistically analyzed.2 Case analysis was used to explore the safety of HCQ medication during pregnancy.3 Chi-square test was used to analyze the statistical differences in pregnancy outcomes between HCQ group and non-HCQ group.Logist regression analysis was used to explore confounding factors.Result1 Retrospective analysis of 117 individual patients showed that HCQ tended to be used in patients with a history of adverse pregnancy and other autoimmune diseases.Patients with a history of abortion less than 10 weeks were more likely to receive HCQ treatment(75.3%VS57.7%,P<0.05).Patients diagnosed with nonstandard OAPS were less likely to receive HCQ therapy(1.5%VS11.5%,P<0.05).In laboratory diagnosis,high antibody titer and high positive rate were more likely to use HCQ treatment.Among the lupus anticoagulant(LA),anti-cardiolipin antibody(ACL),and anti-β2Gpi antibody positive categories,the proportion of HCQ and non-HCQ groups was 30.8%vs 23.1%,50.8%vs 40.4%,46.2%vs 32.7%,respectively.At the same time,patients with double positive and triple positive antibodies used more HCQ(29.3%vs 19.2%,9.2%vs 3.8%).2 In 117 patients,no retinal or cardiac toxicity of HCQ was observed in pregnant women or fetuses.We Only observed one patient with abnormal liver function.The patient had elevated liver enzymes on admission.However,HCQ was not clear as the direct cause of liver injury due to the complicated medication.3.A total of 104 patients were statistically analyzed for pregnancy outcomes after removing confounding factors such as SLE.HCQ increased the live birth rate of OAPS patients from 87.5%to 98.2%(P<0.05).Although there was no statistical significance,treatment with HCQ was observed to reduce the incidence of pathological pregnancy outcomes such as preeclampsia,fetal distress,and oligohydramnios(8.9%vs 14.6%;7.1%VS10.4%;8.9%VS10.4%).After HCQ treatment,the incidence of amniotic fluid muddy decreased significantly(P<0.05).Part Ⅱ Evaluation of the therapeutic effect about HCQ on the mouse model of OAPSObjectiveThe animal model of OAPS was established to explore the pathogenesis of OAPS and its pathological effects on placental development.HCQ was used to treat OAPS mice,to evaluate the therapeutic effect of HCQ on OAPS,and to determine the pre vention and treatment of HCQ on pathological pregnancy and the protection of HCQ on placenta.Method1 C57BL6j mice were injected with monoclonal antiphospholipid antibody(aPL)through tail vein to establish a mouse model of OAPS and its adverse pregnancy outcomes were analyzed.2 The animal model of OAPS was treated by tail vein injection of HCQ,and the pathological pregnancy outcome was analyzed.3 Placental tissue was removed and the tissue sections were stained with HE.Pathological analysis was performed to study the therapeutic effect of HCQ from histology.Result1 Construct the animal model of OAPS by using aPL.Under the influence of aPL,OAPS model mice can successfully fit the clinical symptoms of OAPS patients,among which abortion and stillbirth are the main manifestations.The fetal resorption frequency has increased to 31%.At the same time,there were risks of pathological pregnancy such as placental hematoma and fetal growth restriction.2 Treatment with HCQ effectively reduced the fetal resorption frequency of OAPS mouse from 31%to 6.7%,which was the same as 4.5%in the control group(P<0.01).At the same time,the treatment of HCQ improved the fetal growth restriction and placental dysplasia of OAPS mice caused by aPL.HCQ increased the average weight of intrauterine fetus from 345.8mg to 445.3mg in OAPS model,and the placental weight was increased from 92.3mg to 104.3mg(P<0.01).There was no significant difference between HCQ treatment group and control group.However,after HCQ treatment,the proportion of no pregnancy after embolization decreased from 32.2%to 18.2%,which was partially improved,but failed to completely recover to the level of the control group(11.8%).3 According to pathological analysis,placental injury caused by aPL often occurs at the placental maternal-fetal interface near the decidua,and there are multiple large necrotic areas combined with vascular collapse lesions.The necrotic lesions were distributed in strips or sheets,often accompanied by fibrinoid degeneration,calcium deposition,eosinophil aggregation,etc.The placental vessel degenerates,the vascular wall thickened to form a necrotic focus,and some endothelial cells fall into the vascular cavity.Although HCQ could not completely prevent the occurrence of placental lesions,after treatment,the necrotic area was reduced,the area became smaller,and the vascular lesions were less severe.The lesion changed from the original large area to punctate or mass.At the same time,there are fewer diseased vessels,and the lesion area was more mild,showing sporadic punctate necrosis of endothelial cells.This suggests that HCQ has a good effect in treating OAPS in vivo.Part Ⅲ Study on the Mechanism of HCQ in Treating OAPSObjectiveExplore the effect of HCQ on aPL stimulated cell function in vitro.The effect of HCQ treatment on the proliferation,tube formation,invasion and migration of trophoblasts under the stimulating of aPL is to investigate.Bioinformatics analysis was used to study the potential targets of HCQ in treating OAPS and explore the molecular mechanism of HCQ in treating OAPS.Method1 Human extravillous trophoblast HTR8 cell line was cultured,and the cytotoxicity and proliferation of HCQ/aPL at different concentration gradients were determined by CCK8 assay.2 The effects of aPL/aPL+HCQ on the invasion,migration and tube formation of HTR8 cell line were detected by transwell migration assay,Scratch wound-healing assay and tubule formation test,respectively.3 GEO data set GSE36083 was downloaded,and bioinformatics analysis was performed to analyze the differential gene expression and the pathway enrichment of OAPS.4 The target protein was analyzed by Discovery Studio software,and its binding site and binding energy with HCQ were detected in the form of molecular docking.Thus,the molecular targets of HCQ for the treatment of OAPS can be inferred.5 PCR and WB were used to verify changes in mRNA and protein expression of multiple target molecules in aPL/aPL+HCQ cell samples.Result1 high concentrations of aPL(≥50 μg/ml)and HCQ(≥100 μg/ml)can significantly inhibit the viability of trophoblast cells,resulting in cytotoxicity.When the concentration of antibody was ≤20ug/ml,the proliferation ability was about 90%in the control group.When HCQ was added,the cell proliferation ability was not recovered.It was observed that aPL could reduce trophoblast proliferation in a dose-dependent manner rather than a time-dependent manner.2 aPL can affect the vascular remodeling ability of trophoblast cells.After 20 μg/ml antibody stimulation,the number of trophoblast tubule nodes,the number of mesh formation and the mesh area decreased to 23.3%,12.7%and 16%respectively in the control group,with significant statistical significance.At the same time,with the increase of antibody concentration,the degree of reduction was proportional to the dose.With the addition of HCQ treatment,the inhibitory effect of OAPS to trophoblast tube formation was mostly reversed.According to the tubule nodes,the number of mesh formation and the mesh area,HCQ increased the antibody-induced inhibitory effect to 78.5%,75.2%and 99.1%of the control group,respectively.3 APL can affect the invasion and migration of trophoblast cells.Under the aPL treatment,the 24h migration area of cells decreased(67.8%VS 54.3%,P<0.01).At the same time,the invasive ability was reduced,and about 59.5%of the cells failed to penetrate the inferior ventricular surface within 24 hours compared with the control group.After HCQ treatment,the number of trophoblast HTR8 cells passing through the Matrigel from the upper chamber increased to 90.2%.Compared with the antibody group,the rate of cell passing through the membrane recovered.After HCQ treatment,the migration area of trophoblast HTR8 was 63.4%within 24 hours,which recovered to 94%of the control group.4 After analysis of GSE36083 dataset in GEO database,11 differential genes(P<0.05)were selected.The molecular interaction between the differential genes and HCQ showed that 6 of them had HCQ binding sites,suggesting that they were potential targets of HCQ in the treatment of OAPS.Through PCR and WB experiments,it was preliminarily found that HCQ could reverse the down-regulation effect of NOG,TNFSF10 and CXCR4 under aPL stimulation.These results suggest that NOG,TNFSF10 and CXCR4 may be potential targets of HCQ in the treatment of OAPS.Conclusion1 The use of HCQ in the treatment of OAPS can improve the pregnancy outcome,increase the live birth rate,and reduce the incidence of pregnancy complications such as amniotic fluid muddy.2 APL can affect normal pregnancy in mouse model,increase the risk of stillbirth and abortion,and pathological changes of placenta occur.HCQ can reduce the incidence of adverse pregnancy outcomes and improve placental function.3 HCQ can improve the damage of trophoblast function caused by aPL,and improve the invasion ability,migration ability and vascular remodeling ability of trophoblast cells,but it cannot restore the mildly inhibited proliferation ability by aPL of trophoblast cells.4 Through bioinformatics analysis and in vitro experimental verification,NOG,TNFSF10 and CXCR4 were found to be potential targets of HCQ in the treatment of OAPS. |
