Mechanism Of PINK1-mediated Mitophagy Regulating Trophoblast Pyroptosis In The Pathogenesis Of Preeclampsia

BackgroundPreeclampsia(PE)is characterized by gestational hypertension with or without multi-organ involvement,which puts a great threat to both mothers and fetuses.One of the most profound mechanisms underlying the pathogenesis of PE is systemic inflammatory response induced by spiral artery remodeling disorder.Previous studies have found that PINK1-mediated mitophagy is of vital importance to the maintenance of functional mitochondria via eliminating impaired mitochondria and accumulating ROS.Cell pyroptosis has recently been considered an important mechanism in inflammation,with Caspase-1 at its core.AIM2,NLRC4 and NLRP3,one of whose activators is ROS,might play central roles in activating Caspase-1.Therefore,it is speculated that PINK1-mediated mitophagy exerts protective effect in PR via reducing ROS induced inflammasome and pyroptosis.Part 1 Role of PINK1-mediated mitophagy in pathogenesis of PE ObjectiveLevels of AIM2,NLRC4,NLRP3,as well as cleaved-caspase-1,GSDMD-N,IL-1β,IL-18 were detected and compared between placentae of PE group and healthy controls.The effects of trophoblast hypoxia/reoxygenation on PINK1-mediated mitophagy were then further explored to assess the correlation between PINK1-mediated mitophagy and the immunopathogenesis of PE.Methods1.To what extent the mitochondrial autophagosome were observed by transmission electron microscope.2.Placentae of 20 PE and 20 healthy controls were dissected.PINK1 positioning in placenta was examined by Immunohistochemical staining.Protein level of PINK1 and m RNA level of PARKIN was detected using western blotting and real time-PCR,respectively.3.HTR-8 cell line was used to generate a hypoxia/reoxygenation(H/R)model in order to simulate the ischemia and hypoxia environment in PE placental.By then,western blotting was used to assess the protein level of PINK1 and PARKIN in both groups;cell immunofluorescence was used to detect mitophagy;DCFH-DA for ROS level.Results1.There was several mitochondrial autophagosomes and no swelling in mitochondrial morphology in trophoblast cells of normal placenta under transmission electron microscope.However,there were no obvious mitochondrial autophagosomess and wollen,light-colored mitochondria with blurred crest were observed in PE group.2.PINK1 was expressed in both extra-villous trophoblast cells and villous trophoblast cells of two groups.The expression level of PINK1 and PARKIN m RNA was significantly less in PE group than healthy controls.3.Compared with controls,mitophagy and protein expression level of PINK1 and PAIKIN were significantly decreased in H/R group;ROS increased.ConclusionPINK1-mediated mitophagy deficiency in PE placenta,PINK1-mediated mitophagy deficiency that result in increased ROS levels might play a central role in the pathogenesis of PE.Part 2 Inflammasome-induced pyroptosis in PEObjectiveThe expression level of inflammasome including AIM2,NLRC4 and NLRP3,as well as molecular featured in pyroptosis such as Cleaved-caspase-1、GSDMD-N、IL-1β、IL-18 was detected in both groups.H/R model was then applied to explore its influence on inflammasome and pyroptosis-associated molecules.Methods1.Immunofluorescence was used to detect the co-localization of inflammasomes mentioned above and pyroptosis executive protein,GSDMD.To what extent GSDMD is regulated by inflammasomes is further analyzed.Both expression levels of inflammasomes and GSDMD were detected in PE groups and healthy controls.2.Expression of AIM2,NLRC4,NLRP3,Cleaved-caspase-1 and GSDMD-N at m RNA and protein levels was detected by western blotting and real time-PCR,as well as m RNA expression of IL-1β、IL-18 by real time-PCR.They were then compared between PE group and healthy controls.3.Morphology of cell pyroptosis in HTR8 cells were observed through scanning electron microscope.4.Protein level of AIM2,NLRC4,NLRP3,Cleaved-caspase-1 and GSDMD-N was assessed by western blotting;IL-1β、IL-18 m RNA by real-time PCR.Differences between healthy controls and H/R-treated group were compared.Results1.AIM2,NLRC4,NLRP3 and GSDMD were all found expressed in PE group and healthy controls,but levels are significantly higher in PE group than that of healthy controls.Their locations are basically consistent.2.The expression of AIM2、NLRC4、NLRP3、Cleaved-caspase-1 和 GSDMD-N at both m RNA and protein levels,as well as expression of IL-1β、IL-18 m RNA,were significantly higher in PE group than that of healthy controls.3.Pyroptosis was observed in H/R-treated group through scanning electron microscope.4.Protein level of AIM2、NLRC4、NLRP3、Cleaved-caspase-1 and GSDMD-N and m RNA level of IL-1β、IL-18 were significantly higher in H/R treated group than those of normal HTR-8 group.ConclusionPyroptosis mediated by inflammasomes including AIM2、NLRC4、NLRP3 was significantly observed in trophoblast cells,which potentially underlies regulation of inflammation in PE.Part 3 Mechanisms underlying pyroptosis via PINK1-mediated mitophagy in PEObjectiveThe correlation between PINK1-mediated mitophagy and pyroptosis of trophoblast cells was to be assessed through in vitro experiment.Methods1.Generation of PINK1-overexpression and knockdown models was constructed by plasmid and si RNA transfected HTR-8/Svneo extra-villous trophoblast cells.After that,both groups were treated with hypoxia and reoxygenation.To be more specific,groups were then divided into 5 subdivisions:(1)H/R group: original HTR-8/Svneo extra-villous trophoblast cells were H/R treated(2)Vector + H/R group: 48 hours after vector transfection,HTR-8/Svneo was H/R treated(3)PINK1 + H/R group:48 hours after PINK1 transfection,HTR-8/Svneo was H/R treated(4)si-PINK1 + H/R group : 48 hours after si-PINK1 transfection,HTR-8/Svneo was H/R treated(5)negative control group: 48 hours after si-NC transfection,HTR-8/Svneo was H/R treated.2.ROS level in five subdivided groups were detected using DCFH-DA.3.The protein level of PINK1,AIM2,NLRC4,NLRP3,Cleaved-caspase-1 and GSDMD-N was detected by western blotting,IL-1β 、 IL-18 m RNA by real time-PCR.Results1.ROS level in group 3 was significantly lower than that in group 1 versus group 2,ROS level in group 4 was significantly higher than that in group 1 versus group 5.2.Protein expression level of PINK1 were found to be significantly up-regulated in group 3 than that of group 1 versus group 2,but the protein expression level of AIM2、NLRC4、NLRP3、Cleaved-caspase-1 and GSDMD-N and m RNA level of IL-1β、IL-18 were found to be significantly down-regulated in group 3 than that of group 1 versus group 2;Protein expression level of PINK1 were found to be significantly down-regulated in group 4 than that of group 1 versus group 5,but the protein expression level of AIM2、NLRC4、NLRP3、Cleaved-caspase-1 and GSDMD-N and m RNA level of IL-1β 、 IL-18 were found to be significantly up-regulated in group 4 than that of group 1 versus group 5.ConclusionTrophoblast cell pyroptosis is regulated by PINK1-mediated mitophagy,which can be further explained by inflammasomes activated by altered ROS levels.To recapitulate,deficiency in PINK1-mediated mitophagy that result in trophoblast pyroptosis can be deemed as an important mechanistic pathway underlying oxidative stress and inflammatory activation in the pathogenesis of PE.