The Mechanism Of Metformin Regulating Trophoblastic Glucose Metabolism Through ATXN7L3 In Preeclampsia

Part Ⅰ Metformin Regulates Trophoblastic Glucose Metabolism via TLR4/NF-κB/PFKFB3 Signal PathwayObjectiveTo compare the expression and localization of TLR4 and PFKFB3 in placenta of normal pregnancy and preeclampsia women.To confirm that TLR4 regulates PFKFB3 expression through NF-κB signaling pathway.To clarify that pharmacologic concentration of Metformin(MET)regulates glucose metabolism homeostasis in trophoblast cells through TLR4/NF-κB/PFKFB3 signaling pathway and its molecular mechanism in vitro.Methods1.qRT-PCR,Western Blotting and immunofluorescence were used to detect the expressions and location of TLR4 and PFKFB3 in the placenta of preeclampsia and normal pregnancy,and the correlation between the mRNA expressions of TLR4 and PFKFB3 in the preeclamptic placentas was investigated by Spearman grade analysis.2.The classical NF-κB signaling pathway inhibitor BAY11-7085(10 μM)was pretreated for 1h to inhibit NF-κB signaling pathway,and then LPS(100ng/mL,24h)was used to stimulate HTR-8/SVneo cells in vitro.The PFKFB3 expression was detected via qRT-PCR and Western Blotting.3.Normal HTR-8/SVneo cells were induced by pharmacological gradient(10-40 μM)MET,and apoptosis and proliferation of cells in each group were detected by flow cytometry and CCK8.4.HTR-8/SVneo cells were stimulated by LPS(100ng/mL,24h)in vitro,MET(10μM,24h)was selected for treatment in the treatment group,and TLR4 was overexpressed by overexpressing TLR4 plasmid on the MET treatment.5.The protein expression levels of TLR4/NF-κB signaling pathway and PFKFB3 in each group were detected by Western Blotting.6.Seahorse analyzer and Lactic acid secretion were used to detect cellular glycolysis.7.Transmission electron microscopy,Red Mito-tracker,JC-1 fluorescent probe and Seahorse analyzer were used to detect cellular oxidative phosphorylation8.Adenosine triphosphate(ATP)kit was used to detect celluar ATP content.9.LPS(100ng/mL,24h)was used to stimulate HTR-8/SVneo cells in vitro,and MET(10μM,24h)was selected as the treatment group.In addition,the overexpressed NF-κB1 plasmid was used to overexpress NF-κB1,and the overexpressed NF-κB1 plasmid was treated with MET(10μM,24h).The degree of nuclear translocation of transcription factor NF-κB1 in each group was detected by immunofluorescence.The binding degree of the transcription factor NF-κB1 to the promoter of PFKFB3 was detected by Ch-IP and dual luciferase assay.The transcription level of PFKFB3 in each group was detected by qRTPCR.Results1.Compared with the normal placenta,the mRNA and protein expression of TLR4 and PFKFB3 in preeclamptic placenta were significantly increased.Besides,the mRNA expression of TLR4 and PFKFB3 were positively correlated;2.With the increase of concentration gradient and time gradient of LPS stimulation,the protein expression levels of TLR4 and PFKFB3 increased gradually,in a concentrationdependent and time-dependent manner.3.TLR4 may regulate the expression of PFKFB3 in trophoblast cells through NF-κB signaling pathway;4.Pharmacological concentrations of MET(10-40 μM)had no significant adverse effects on the cytoactivity of trophoblast cells in vitro;5.LPS significantly activated TLR4/NF-κB signaling pathway and increased the expression of PFKFB3,while MET(10μM)inhibited TLR4/NF-κB signaling pathway and decreased the expression of PFKFB3.TLR4 was overexpressed on the basis of MET treatment,and it was found that TLR4/NF-κB signaling pathway was significantly activated again,and PFKFB3 expression significantly increased again.6.LPS and overexpressed transcription factor NF-κB 1 significantly increased the expression of NF-κB1,promoted the transfer of NF-κB 1 from cytoplasm to nucleus,increased the binding of NF-κB 1 to PFKFB3 promoter,and promoted the transcription of PFKFB3.Pharmacological concentration of MET(10μM)can inhibit the above effects.7.LPS significantly activated basal glycolysis capacity,maximal glycolysis capacity and lactate secretion of trophoblast cells,while pharmacological concentration of MET(10μM)significantly inhibited these effects.After further overexpression of TLR4 on the basis of MET treatment,the basal glycolysis capacity,maximum glycolysis capacity and lactate secretion of trophoblastic cells were significantly increased again.8.LPS significantly damaged mitochondrial structure,decreased mitochondrial membrane potential,and decreased basal and maximum respiration rate of cells.Pharmacological concentration of MET(10μM)reverses these effects significantly.When TLR4 was overexpressed on the basis of MET treatment,the mitochondrial structure of trophoblastic cells was destroyed again,the mitochondrial membrane potential was reduced again,and the basal and maximum respiration rates of cells were significantly inhibited again.9.LPS significantly reduced trophoblast ATP production,and the pharmacologic concentration of MET(10μM)could significantly reverse this effect.Further,the overexpression of TLR4 on the basis of MET treatment significantly reduced trophoblast ATP production again.ConclusionsTLR4/NF-κB/PFKFB3 signaling pathway is involved in the regulation of trophoblast glucose metabolism,which may be the mechanism of the imbalance of placental glycometabolism homeostasis in preeclampsia.Pharmacological concentrations of MET correct trophoblastic glycometabolism disorders through the TLR4/NF-κB/PFKFB3 pathway,which may be used to ameliorate placental glucose metabolism homeostasis and placental dysplasia in preeclampsia.Part Ⅱ:Metformin Regulates TLR4/NF-κB/PFKFB3 Signal Pathway Through ATXN7L3-mediated H2Bub1 Modification to Affect Trophoblastic Glucose MetabolismObjectiveThe HTR-8/SVneo trophoblastic cells in the control group,LPS group and LPS+MET group were tested proteomically,and the biological functions and pathways involved in the differential proteins were preliminarily bioinformatics analyzed.To further explore the role of MET in regulating TLR4/NF-κB/PFKFB3 pathway on glucose metabolism in trophoblast cells and the specific molecular mechanism.Methods1.Proteomic detection of HTR-8/SVneo trophoblast cells in control group,LPS group and LPS+MET group was performed by TMT-labeled two-dimensional liquid chromatography-mass spectrometry,and GO annotation analysis and KEGG pathway enrichment analysis were performed to analyze the biological functions that different proteins might be involved in.2.The candidate protein ATXN7L3 screened by proteomics was verified by Western Blot.3.LPS(100ng/mL,24h)was used to stimulate HTR-8/SVneo cells in vitro.MET(10μM,24h)was selected for the treatment group,followed by si-ATXN7L3,and ATXN7L3 was knocked down on the basis of MET treatment.4.Protein expression levels of ATXN7L3,TLR4/NF-κB signaling pathway and PFKFB3 in each group were detected by Western Blotting.The degree of nuclear translocation of transcription factor NF-κB 1 in each group was detected by immunofluorescence.5.The binding degree of ATXN7L3 and H2B protein in each group was detected by coIP;The modification level of total H2Bub1 in cells of each group was detected by Western Blotting and immunofluorescence.The recruitment degree of ATXN7L3 and modification level of H2Bub1 in TLR4 promoter region were detected by Ch-IP.TLR4 transcription levels were detected by qRT-PCR.6.Seahorse analyzer and Lactic acid secretion were used to detect cellular glycolysis.7.Transmission electron microscopy,Red Mito-tracker,JC-1 fluorescent probe and Seahorse analyzer were used to detect cellular oxidative phosphorylation8.ATP kit was used to detect celluar ATP content.Results1.Proteomic results showed that compared with the control group,a total of 17 differentially expressed proteins were detected in LPS group,including 7 up-regulated proteins and 10 down-regulated proteins.A total of 41 differentially expressed proteins were detected in LPS+MET group compared with LPS group,including 32 up-regulated proteins and 9 down-regulated proteins.Compared with the control group,ATXN7L3 protein was significantly decreased in LPS group.Compared with LPS group,ATXN7L3 protein was significantly increased in LPS+MET group,suggesting that ATXN7L3 protein may not only be a new perspective of preeclampsia,but also a new target of pharmacological action of MET,which is of great research value.2.LPS significantly decreased the expression level of ATXN7L3 protein,and significantly activated the TLR4/NF-κB/PFKFB3 signaling pathway;This effect was significantly inhibited after MET treatment.After further lowering ATXN7L3 on the basis of MET treatment,it was found that the expression level of TLR4 and transcription factor NF-κB1 increased again,nuclear translocation was enhanced,p-p65/p65 ratio increased again,and the expression level of key glycolysis enzyme PFKFB3 increased again significantly.3.The binding degree of ATXN7L3 protein and histone H2B decreased in LPS group,while the binding degree of them increased significantly in LPS+MET group.Further knockdown of ATXN7L3 resulted in the binding degree of ATXN7L3 and histone H2B decreased again.4.The total H2Bub1 level of trophoblast cells in LPS group was significantly increased,while that in LPS+MET group was significantly decreased.Further knocking down ATXN7L3 showed a partial rise of total H2Bub1 level.5.The recruitment degree of ATXN7L3 in the TLR4 promoter region of LPS group was significantly reduced,while the enrichment degree of H2Bub1 modification was significantly increased.This effect was significantly reversed in the MET group.After further knocked down ATXN7L3 on the basis of MET treatment,the enrichment level of ATXN7L3 in TLR4 promoter region decreased again,and the modification level of H2Bub1 increased obviously.6.TLR4 transcription expression level in LPS group was significantly increased,and TLR4 transcription expression level in LPS+MET group was significantly decreased.Further knockdown of ATXN7L3 led to a significant increase in TLR4 transcription level again7.LPS significantly activated basal glycolysis capacity,maximal glycolysis capacity and lactate secretion of trophoblast cells,while pharmacological concentration of MET(10μM)significantly inhibited these effects.Further,on the basis of MET treatment,ATXN7L3 was knocked down,and the basic glycolysis capacity,maximum glycolysis capacity and lactate secretion of trophoblastic cells were significantly increased again.8.LPS significantly damaged mitochondrial structure,decreased mitochondrial membrane potential,and decreased basal and maximum respiration rate of cells.Pharmacological concentration of MET(10μM)reversed these effects significantly.When ATXN7L3 was further knocked down on the basis of MET treatment,mitochondrial structure of trophoblastic cells was destroyed again,mitochondrial membrane potential was reduced again,and basal and maximum respiration rates of cells were significantly inhibited again.9.LPS significantly reduced trophoblast ATP production,and the pharmacological concentration of MET(10μM)could significantly reverse this effect.Further,on the basis of MET treatment,ATXN7L3 was knocked down,and trophoblast ATP production was significantly reduced again.ConclusionMET may regulate TLR4 transcriptional expression through AtxN7L3-mediated H2Bub1 modification,and then regulate the activity of NF-κB signaling pathway,affecting the expression of glycolysis PFKFB3,and thus affecting trophoblast glucose metabolism.This study elucidates the possible new pharmacological mechanism of MET from the perspective of epigenetic modification,and provides a possible new idea for the study of the mechanism of MET improving placental glucose metabolism in preeclampsia.Part Ⅲ:Metformin Regulates Placental Glucose Metabolism in Preeclamptic Rats through ATXN7L3/TLR4/NF-κB/PFKFB3 Signal PathwayObjectiveA classic LPS-induced preeclampsia rat model was established.In vivo experiments were to confirm that MET improves placental sugar metabolism in preeclampsia pregnant rats through ATXN7L3/TLR4/NF-κB/PFKFB3 pathwayMethods1.Normal control group:normal pregnant rats were intraperitoneally injected 0.9%normal saline;Preeclampsia group:Normal pregnant rats were intrabacially injected 20μg/kg LPS every day from GD13 to GD18 to establish a preeclampsia pregnant rat model.MET treatment group:GD13 to GD18,intraperitoneally injected 20 μg/kg LPS daily,MET dissolved in water from the same day,MET 500 mg/kg·d by gavage at 18 PM every night;2.Body weight of pregnant rats was recorded on GD4,GD7,GD10,GD13,GD15 and GD18,respectively3.Blood pressure of rats was measured on GD13 and GD19,and urine was collected to measure the contents of urinary protein and creatinine.4.Pregnancy outcomes of rats in each group were compared:placenta and fetal rats were statistically analyzed;5.HE sections were prepared to observe and compare the pathological changes of kidney and placenta in each group.6.TUNEL staining was performed on the placenta sections to compare the changes of cell death levels in the placenta of rats in each group.7.Immunofluorescence staining was performed on the placenta,and CD31 was used to label the placental vascular endothelial cells,and the changes of placental microvascular density in the placenta of rats in each group were compared.8.Western Blot was used to detect ATXN7L3 protein expression level,H2Bub1 modification level,TLR4 protein expression level,transcription factor NF-κB1 protein expression level,the ratio of p-p65/p65,an important component of NF-κB signaling pathway,and the PFKFB3 protein expression in placenta of rats in each group.9.Mitochondrial morphology of fetal placental cells in each group was observed by transmission electron microscopy.10.Lactic acid content in placenta of rats in each group was detected by lactic acid kit;11.ATP content in placenta of rats in each group was detected by ATP kit;Results1.During pregnancy,no significant difference was found among the three groups in body weight2.At GD13,the blood pressure,urinary protein and creatinine of pregnant mice in the three groups had no statistical significance;On GD19,blood pressure,urinary protein and creatinine of pregnant mice in the LPS-induced preeclampsia group were significantly higher than control group,while blood pressure,urinary protein and creatinine of pregnant mice in the metformin group were significantly lower than preeclampsia group.3.Serious glomerular pathological injury occurred in the LPS-induced preeclampsia model group;No significant pathological changes were observed in the control group and the metformin treatment group.4.The body weight and body length of fetal mice in the LPS-induced preeclampsia group were lower than control group,while those in the MET group were higher than preeclampsia group.5.The diameter and weight of placenta in the LPS-induced preeclampsia group were lower than control group,and those in the MET treatment group were significantly higher than preeclampsia group.6.The TUNEL staining level of placenta in the LPS-induced preeclampsia group was significantly increased,while that in the MET treatment group was significantly decreased;7.Placental microvascular density in the LPS-induced preeclampsia group was significantly lower than normal control group,and the placental microvascular density in the MET treatment group was significantly higher than preeclampsia group.8.The expression level of ATXN7L3 protein in placenta of LPs-induced preeclampsia rats was significantly decreased,the modification level of H2Bub1 was significantly increased,the TLR4 expression of was increased significantly,the NF-κB1 protein expression was increased significantly,and the ratio of p-p65/p65 in placenta was significantly increased.The PFKFB3 protein expression was significantly increased.In the MET treatment group,the above trends were significantly reversed,the placental ATXN7L3 protein expression level increased significantly,the H2Bub1 modification level was significantly decreased,the TLR4 expression level decreased significantly,the NF-κB1 protein expression level was significantly decreased,and the placental p-p65/p65 ratio was significantly decreased.The PFKFB3 protein expression was significantly decreased.9.Mitochondria of normal rat placenta cells were long cords and thin strips.Mitochondrial fission of placental cells in preeclampsia rats induced by LPS showed several fragments,mitochondrial swelling and crista damage,indicating the mitochondrial structure was damaged.In the MET group,the mitochondrial structure of placental cells was partially recovered,mainly showing elongated form.10.Placental lactic acid content of LPS-induced preeclampsia rats was significantly increased,while that of MET treated rats was significantly decreased;11.LPS-induced placental ATP production of preeclampsia rats was significantly decreased,while the content of placental ATP in MET group was significantly increased;ConclusionsTo verify that MET may regulate placental glucose metabolism in PE pregnant mice through ATXN7L3/TLR4/NF-κB/PFKFB3 pathway at the overall level,thus adding a possible new theory for the pathogenesis of PE and providing possible new evidence for MET to be a candidate treatment for preeclampsia.