The Effect Of Low Dose Aspirin On Suppressing Trophoblasts Pyroptosis In Preeclampsia

ObjectivePreeclampsia(PE)refers to a placental-deriveddisease andspecific to pregnancy,which seriously endangers the health of mothers and infants.Studies have shown that placental factors play an important role in the pathogenesis of PE,inadequate invasion of trophoblasts causes maternal uterine spiral arterial remodeling disorder in early pregnancy,placental hypoperfusion,ischemia and hypoxia,and thus triggers the oxidative stress response,releasing a variety of placental factors,which enter into the maternal blood circulation to cause organ damage and related clinical symptoms,eventually promotes the occurrence and development of preeclampsia.It has been demonstrated that oral administration of lowdose aspirin from early pregnancy to 36 weeks of pregnancy can reduce the incidence of preeclampsia and significantly improve adverse pregnancy outcomes for high-risk groups of preeclampsia,but the specific molecular mechanism is unknown.ROS,an oxidative stress product,can induce stimulation of NLRP3 inflammasome and participate in the activation of cell pyroptosis pathways.The previous experiments of research group have proved that activation of pyroptosis pathway exists in patients with preeclampsia.Therefore,this study analyzed the correlation between trophoblastspyroptosis in placental tissues and the pathogenesis of preeclampsia;A model of trophoblasts pyroptosis in the pathogenesis of PE induced by oxidative stress was constructed in vitro;Starting with cells pyroptosis,it laid a foundation for studying the mechanism of low-dose aspirin in preventing the occurrence and development of PE.The completion of this project provided new ideas for studying the pathogenesis and prevention of preeclampsia,and developing new drugs for treatment.Materials and Methods1.Collecting clinical data and experimental specimens to detect activation of pyroptosis pathway in placentas Placenta specimens of 25 PE patients with single fetus and single diseasewere collected,placental specimens of early onset severe preeclampsia(EOSP,17 cases,gestational week <34weeks),age(32.6 ± 5.1)years,parity(1.5 ± 0.5)times,pre-pregnancy BMI(23.2 ± 3.5)kg/m2;Placental specimens of late onset severe preeclampsia(LOSP,8 cases,gestational week ≥ 34 weeks),age(28.9 ± 3.0)years,parity(1.3 ± 0.4)times,pre-pregnancy BMI(20.9± 2.5)kg/m2;In addition,placental specimens of late pregnancy with normal blood pressure during the same period were selected as the control group(NT,10 cases),age(31.2 ± 4.0)years,parity(1.8 ± 0.4)times,and pre-pregnancy BMI(21.0 ± 2.4)kg/m2.The placenta specimens were preserved at-80℃.Detecting the expression levels of pyroptosis-related molecules in clinical specimens by western blotto explore the correlation between trophoblasts pyroptosis and the pathogenesis of preeclampsia,and analyzed the clinical significance of its expression in different stages of preeclampsia.2.Establishment of trophoblasts-pyroptosis model induced by H2O2In this study,human trophoblast cell line HTR-8/SVneo was selected.Trophoblasts were treated with different concentrations of H2O2(100 μM,150 μM,200μM,250 μM)for different time(2h,4h,6h,12h),the protein expression levels of caspase1,Cleaved caspase1,GSDMD,and IL-1β were detected by western blot;RT-q PCR was used to examine the m RNA levels of NLRP3,caspase1,GSDMD,IL-1β and IL-18.The morphological changes of trophoblasts after H2O2 treatment were observed under thelight microscope and theelectron microscope.3.Preliminary study on the molecular mechanism of lowdose aspirin in inhibiting trophoblasts pyroptosis On the basis of the successful construction of H2O2-induced trophoblasts HTR-8/SVneo pyroptosis model,the trophoblasts were treated with different concentrations of aspirin(1m M,5m M)for different time(8h,20h),and then treated with H2O2(150μM)for 4h,the protein levels of NLRP3,caspase1,Cleaved caspase1,NF-κB p65 and p-NF-κB p65(phospho S536)were detected by western blot;The m RNA levels of NLRP3,caspase1,GSDMD,IL-1βand IL-18 were examined by RT-q PCR.The morphological changes of trophoblasts pyroptosis model group induced by H2O2 after intervention with lowdose aspirin were observed under the light microscope.Results1.The activation of the trophoblasts pyroptosis pathway was detected in placental specimens,there was no correlation between the degree of trophoblasts pyroptosis and the patient’s age,parity and pre-pregnancy BMI,andthe difference was not statistically significant(p>0.05);The protein levels of caspase1,Cleaved caspase1,GSDMD,and IL-1β in the EOSP group were higher than those in the NT group and the LOSP group,and the difference was statistically significant(p<0.05);The degree of trophoblasts pyroptosis: EOSP> LOSP;The protein levels of caspase-1,Cleaved caspase-1,GSDMD and IL-1β in the LOSP group were not statistically significant compared with the NT group(p>0.05).2.The optimal conditions for H2O2-induced pyroptosis injury model of trophoblastic cell line HTR-8/SVneowere 150μM and 4h.Under these conditions,the protein levels of NLRP3,caspase1,Cleaved caspase1,GSDMD,and IL-1β in trophoblasts were significantly higher than those in the control group;Compared with the control group,the m RNA levels of NLRP3,caspase1,GSDMD,IL-1β and IL-18 were all up-regulated with statistical significance(p<0.05);Typical changes of pyroptosis such as cell swelling,cell fragmentation and plasma membrane bubble formation could be clearly seen under the light microscope.Under the electron microscope,the destruction of cytoskeleton stability,chromatin breakage,and the movement of large organelles in cytoplasm from intracellular to extracellular through NT-GSDMD pores on the membrane could be observed.3.After intervention with lowdose aspirin in advance,in the trophoblasts pyroptosis model group,the protein levels of NLRP3,caspase1,Cleaved caspase1,NF-κB p65 and p-NF-κB p65(Ser536)were distinctly suppressed;In addition,the m RNA levels of NLRP3,caspase1,GSDMD,IL-1β,and IL-18 were evidently down-regulated with statistical significance(p<0.0001);Under the light microscope,it was observed that the morphologies of trophoblasts pyroptosis model group tended to be normal,with polygonal shape,tight arrangement,clear boundary and obvious proportion of nucleus and cytoplasm.Conclusions1.Clinical specimen studies have shown that trophoblasts pyroptosis is related to the pathogenesis of early onset severe preeclampsia,and the degree of trophoblasts pyroptosis is positively correlated with the severity of the disease in different stages of preeclampsia;2.The optimal conditions for H2O2 induced trophoblasts HTR-8/SVneo pyroptosis model is150μM and 4h,in vitro experiments successfully constructthe trophoblasts pyroptosis modelinduced by oxidative stress response;3.The best conditions for aspirin to inhibit trophoblasts pyroptosis are5 m M and 12h;4.Aspirin can prevent NF-κB expression and its Ser536 phosphorylation,and repressH2O2-induced trophoblasts pyroptosis via the NF-κB-GSDMD signaling pathway.