PLGF Alleviates Pyroptosis Induced By High Glucose Through Regulating Mitophagy Via Bnip3-pink1-parkin Pathway In HTR8-S/Vneo

Preeclampsia(preeclampsia,PE)is a kind of hypertensive disorder of pregnancy,which manifested by increased systolic blood pressure(≥140mm Hg)and/or diastolic blood pressure(≥90mm Hg)after 20 weeks of pregnancy,with or without proteinuria.PE combined with dysfunction of each system can only be treated by termination of pregnancy at present,and its cause and mechanism are not yet clear.Present researches suggest that its occurrence may involve abnormal trophoblast cell death,secretion of inflammatory factors and oxidative stress.Both gestational diabetes mellitus(GDM)and diabetes complicated with pregnancy are accompanied by an increase in blood glucose of pregnant women.Hyperglycemia is closely related to the occurrence of PE and is considered to be a high risk factor for PE,but its mechanism is not yet clear.Placenta growth factor(PLGF)is a secreted protein that promotes cell proliferation and angiogenesis.Its normal level is of great significance to placenta and infant development.However,PLGF decline is significant in PE patients.Therefore,it is very important to explore the relationship between hyperglycemia and PE and the role of PLGF in it.Our previous studies have shown that after high glucose(HG)treatment,exogenous supplementation of PLGF can restore the proliferation and migration of trophoblast cells;in vivo experiments have shown that injection of PLGF can relieve the symptoms of PE in model rat.Those results mean that PLGF has a potential role in treating preeclampsia.Hence,exploring the effect of PLGF under the influence of high glucose is of great significance to the treatment of PE.The release of inflammatory mediators and the elimination of damaged cells induced by hyperglycemia is a common cause for eclampsia.Clearing damaged cells and inhibiting the release of inflammatory factors caused by hyperglycemia are important strategies for the treatment of PE.Pyroptosis is a kind of pro-inflammatory cell death and an important pathological feature of PE.The increase of pro-inflammatory mediators caused by autophagy inhibition is one of the factors that cause pyroptosis.The pyroptotic cell and decrease of autophagy levels can be detected in preeclampsia accompanied by hyperglycemia.Therefore,the purpose of this study is to explore whether PLGF can affect trophoblast cell pyroptosis caused by HG through regulating autophagy,and to explore its underlying mechanism.Part Ⅰ: the effect of PLGF on PE pathological process[Objective] To observe the effect of PLGF on PE pathological process.[Methods] First,to clarify the effect of hyperglycemia on the placenta,20 pregnant women are divided into 2 groups basing on the "Guidelines for Diagnosis and Treatment of Diabetes in Pregnancy" of the American Diabetes Association:(1)Control group: diagnosed as healthy pregnant women;(2)GDM group: gestational diabetes mellitus is diagnosed according to " Guidelines for Diagnosis and Treatment of Diabetes in Pregnancy ".Record information about the medical history of pregnant women.After partum,take root of the umbilical cord in the placenta and wash quickly and put it on dry ice for storage.H&E staining to observe the pathological changes of the placenta.Western blot and immunofluorescence are used to detect the expression of pyroptosisrelated molecules.Then,the placental trophoblast cells HTR8-S/Vneo are cultured,for observing the effect of high glucose on the proliferation and migration of trophoblast cells.Hypertonic group is set with mannitol(25m M).After treat HTR8-S/Vneo cells with different concentrations of D-glucose(5,15,25 m M)(24h),CCK8,cell scratch,transwell are used to detect cell proliferation,migration and invasion capacity.Subsequently,the effect of high glucose on the senescence and oxidative damage of trophoblast cells are tested.HTR8-S/Vneo cells are treated with different concentrations of D-glucose(5,15,25 m M)(24h),stained with senescence associated b-galactosidase(senescence associated b-galactosidase,SA-bGal),and SOD and MDA to detect the senescence and oxidative damage levels of trophoblast cells.To determine whether high glucose causes pyroptosis of trophoblast cells,HTR8-S/Vneo cells are cultured in vitro,hypertonic control is set with mannitol(25m M),and treated with 25 m M D-glucose for 24 h.Western blot,qRT-PCR and ELISA are used to detect pyroptosis-related molecules.The detection of LDH and PI/Hoechst33342 double staining positive cells to measure the level of cell death.For confirming whether high glucose decrease the expression of PLGF in trophoblast cells are needed.Trophoblast cells are cultured,and treating with mannitol(25m M)is set up as hypertonic control.After treating HTR8-S/Vneo cells with different concentrations of D-glucose(5,15,25,30 m M)for 24 h,western blot and qRT-PCR are used to detect the expression of PLGF at protein and mRNA levels.Furthermore,to track the localization of PLGF,the cells are treated with high glucose for 24 hours.Then,the distribution of PLGF is detected by immunofluorescence,and the expression levels of cytoplasm and cytoplasm are detected by western blot.[Results] The number of vascular lumens in the GDM group decreased comparing with the control group.And,the expression levels of pyroptosis-related proteins NLRP3,caspase1 and IL-1β increased in GDM group,indicating that hyperglycemia can inhibit the growth and development of the placenta and promote the expression of inflammatory mediators.In vitro experiments,when treating HTR8-S/Vneo cells with Dglucose(25m M),cell activity is inhibited(P<0.01),and the cellular damage did not recover in 48h(24h: P<0.05;48h: P<0.05).The cell scratch experiment showed that under the condition of D-glucose(25m M),the cell migration ability decreased,and the injury did not recover within 48h(24h:P<0.001;48h: P<0.001).The transwell experiment showed that compared with the hypertonic group,the cell invasion ability decreased after treatment with 25 m M D-glucose for 24h(P<0.001).The results of SA-bGal staining showed that the positive area increased in 25 m M group compared to the hypertonic control group,indicating that the level of cell senescence increased.While,the level of SOD decreased and the content of MDA increased.Therefore,the D-glucose concentration of 25 m M will be comfirmed as a high glucose in subsequent studies.After high glucose treatment,the expression levels of pyroptosis-related proteins NLRP3,caspase1,GSDMD and IL-1β increased.The results of qRT-PCR and ELISA showed that high glucose also promoted the intracellular expression and release of IL-1β in trophoblast cells.The results of LDH detection and PI/Hoechst33342 double staining showed that the level of cell death increased after high glucose treatment.we also observed that PLGF was inhibited in cytoblast and cytosol.[Summary] pyroptosis was activated in placenta of GDM patients;High glucose promotes pyroptosis in trophoblast cells and inhibits the expression of PLGF.Part Ⅱ: The Potential Mechanism of FGF21 Against AS[Objective] To investigate the effect of PLGF in alleviating the damage of high glucose to trophoblast cells and its potential molecular mechanism.[Methods] First,Subsequently,to explore the effect of PLGF could alleviate cell damage caused by high glucose,cells are divided into 4groups:(1)control group;(2)PLGF group(100ng/ml);(3)high glucose group;(4)high glucose + PLGF group.Western blot,qRT-PCR and ELISA are used to detect pyroptosis-related molecules.LDH and PI/Hoechst33342 double-stained cells to measure cell death.In order to determine whether PLGF alleviates cell damage through regulating mitochondrial function,cultured trophoblast cells are divided into 4 groups:(1)control group;(2)PLGF group(100ng/ml);(3)high glucose group;(4)high glucose + PLGF group.mito SOX detects reactive oxygen species(ROS)levels;kit used to detect m PTP;Fluo-4 AM for detecting intracellular calcium levels;and flow cytometry for detecting MMP;kit for detecting ATP level,qRT-PCR to detect mitochondrial gene replication level,western blot to detect mitochondrial biogenesis related protein level.To verify whether the protective role of PLGF is related to autophagy.GFP-m RFP-LC3 system,western blot and transmission electron microscope are used to analyze the effect of PLGF on autophagy(flux)under the influence of high glucose.Subsequently,analyzing whether the molecular mechanism of PLGF regulating autophagy is related to BNIP3.We transfected BNIP3 siRNA in trophoblast cells to analyze the effect of BNIP3 knockdown on autophagy(flow)and pyroptosis,and detected ROS level,GFP-m RFP-LC3 system,western blot and transmission electron microscopy are used to analyze changes in autophagy(flow),western blot is also used to detect the level of pyroptosis-related proteins.In order to explore whether the PLGF-regulated autophagy is also affected by the PINK1/Parkin pathway,cells are transfected with PINK1 siRNA to analyze the effect of PINK1 knockdown on autophagy(flow)and pyroptosis,and the ROS Level,GFP-m RFP-LC3 system,western blot and transmission electron microscopy to analyze changes in autophagy(flow),western blot is also used to detect the level of pyroptosis-related proteins.Finally,in order to explore the connection between BNIP3 and PINK1/Parkin pathways,trophoblast cells transfected with BNIP3 siRNA are also detected with Parkin expression changes.Mito-Track Green is used to labeled mitochondria,and then the localization and expression changes of PINK1 are detected by immunofluorescence and western blot.[Results] Compared with the high glucose group,the pyroptosisrelated proteins NLRP3,caspase1,GSDMD and IL-1β decreased after PLGF treatment;the expression and release of IL-1β decreased;LDH content and PI/Hoechst33342 double-stained cell area decreased,indicating that PLGF can alleviate the level of cell death after high glucose treatment.High glucose treatment leads mitochondrial dysfunction.Compared with the control group,the ROS level,abnormal open levels of m PTP and intracellular Ca2+ levels increase;mitochondrial membrane potential is disturbed;ATP production and mt DNA decrease,and NRF1,NRF2 and PGC-1α decreased.While,the abnormal can be reversed by PLGF treatment.The GFP-m RFP-LC3 system showed that the yellow spots are reduced after high glucose treatment but for PLGF treatment.Western blot was observed with decreased of beclin1,the conversion rate of LC3-I to LC3-II,while the expression of p62 increased in HG group but opposite for PLGF treatment.Transmission electron microscopy results showed that the autophagic vesicles increased for PLGF treatment comparing with HG group.Transfection of BNIP3 siRNA increase ROS level and decreased the yellow spots marked by the GFP-m RFP-LC3 system.The expression of beclin1,the conversion rate of LC3-I to LC3-II decreased in the HG+PLGF+BNIP3KD group,and the expression of p62 increased comparing with the HG+PLGF group;and the number of autophagic vesicles also decreased.While,decreased NLRP3,caspase1,GSDMD and IL-1β expression by PLGF treatment were eliminated by BNIP3 KD treatment,under HG condition.However,the results of transfection with PINK1 siRNA show the same change comparing to the results of transfection with BNIP3 siRNA.The expression of PINK1 in mitochondria decreased after transfection of BNIP3 siRNA.[Summary] High glucose promotes mitochondrial dysfunction in trophoblast cells through inhibiting the expression of BNIP3,PINK1,Parkin and mitophagy,and leads increasing of ROS which causing trophoblast cell pyroptosis.PLGF leads mitophagy in trophoblast cells through the BNIP3/PINK1/Parkin pathway and relieve the scorch caused by high glucose.