Effect Of HMGB1 In Pathogensis Of Severe Preeclampsia And Regulation Of Glaucocalyxin A On The Expression Of HMGB1 In Trophoblast

Background Preeclampsia(PE)is a common,pregnancy-specific disease that belongs to the family of hypertensive disorders in pregnancy and is characterized by hypertension,proteinuria and other systemic disturbances at or after 20 weeks of gestation.PE is a kind of common disease in obstetrics.The incidence is 3%~5%,which has a inceased trend in recent years.Severe preeclampsia(s PE)is a major contributor to maternal and fetal morbidity and mortality.The precise mechanisms of PE pathogenesis remain unknown.It is widely acknowledged that the placenta is the central organ in the pathogenesis,and PE is caused by maternal responses to placentation and associated with an increased inflammatory state.It has been further proposed that preeclampsia is an excessive maternal inflammatory response to pregnancy,involving proinflammatory cytokines such as tumour necrosis factor,interleukin and HMGB1.HMGB1 is a kind of non-histone protein binding chromosomal and presenting in eukaryotic cells.In recent years,it has been demonstrated that intranuclear HMGB1 plays pivotal roles in the inflammatory responses,and extracellular HMGB1 secretes inflammatory stimulation function as a kind of pro-inflammatory cytokine,and triggers pro-inflammatory responses.HMGB1 binds to several transmembrane receptors,including the receptor for advanced glycation end-products(RAGE)that isthe specific receptor identified by extracellular HMGB1.Other receptors,such as Toll like receptors,have also been confirmed to contribute to HMGB1 mediated signaling pathway.The binding of HMGB1 to its receptors is found to be associated with induction of nuclear factor-κB(NF-κB)activation and release of pro-inflammatory cytokines and chemokines such as TNF,IL-1,IL-6 and IL-8.NF-κB family is an upstream regulator of multiple labour-associated processes.How to regulate the expression of HMGB1 and its signaling pathway has an important value in predicting and clinical treatment for PE.There are many kinds of medicine in regulating inflammatory reaction.But most of them have toxic side-effect.Finding low toxicity and high efficacy of the active ingredient from natural animals and plants from natural animals and plants has become research focus of the domestic and foreign scientists.A number of diterpenoids were found in Rabdosia amethystoides,such as Glaucocalyxin A(GLA).The compounds have better biological activities,such as antibacterial and antiviral effects,anti-tumor activity,regulating immune function and against oxidative stress.The mechanisms of its antibacterial,anti-inflammatory and anti-tumor activity remain unclear and need to be explored.Its anti-inflammatory activity has aroused great attention of domestic and foreign scientists.However,no statement is available on the expression of HMGB1-RAGE signaling pathway in the placenta and maternal blood in severe PE.To better explore the various mechanisms of severe PE,mRNA and protein expression of HMGB1,RAGE,as well as NF-κB activity in the placenta were investigated,and the concentration of HMGB1 in the maternal venous serum was evaluated.Subsequently,HMGB1-RAGE signaling pathway is detected in trophoblasts in vitro,to further clarify the mechanisms of HMGB1 on regulating inflammatory reaction and biological behavior of trophoblasts.Moreover,study of GLA on regulating the expression of HMGB1 in trophoblasts was taken on,which is in order to better clarfy the role of nucleus-cytoplasm transition of HMGB1 in the pathologic process of PE,and explore the mechanisms in anti-imflammation of GLA.The resource of Rabdosia amethystoides is extremely rich,which has good development prospects.PartⅠ HMGB1 signaling pathway in the placenta of severe preeclampsiaObjectives Elucidate the mechanism of HMGB1 signaling pathway,including RAGE and NF-κBp65,in the pathogenesis of PE.Provide experimental basis to further explore the mechanism of preeclampsia.Methods 1.64 women with severe PE and 61 women with normal pregnancy from the Third Affiliated Hospital of Zhengzhou University,from October 2010 to April 2014,were recruited in the study.64 severe PE women were classified as the severe PE group,while 61 normal pregnant women constituted the control group.The placental biopsies and maternal venous serum were collected.2.villous trophoblast(VT)and extravillous trophoblast(EVT)tissue microarray was constructed by Tumor Biology Laboratory in Harvard University.3.The mRNA levels of reative gene of HMGB1 pathway,HMGB1,RAGE and NF-κB p65,were analyzed by real time PCR.4.Levels of HMGB1 and RAGE protein were detected in frozen placental specimens by western blotting.5.The locations of HMGB1 and RAGE were evaluated in the well-characterized tissue microarray by immunohistochemistry.6.ELISA was further used to detect HMGB1 level in maternal serum.7.All the analyses were treated by SPSS 21.0 statistic software.The data were expressed as mean±SD.Statistical significance was assessed using independent two-tailed Student t-tests or Mann-Whitney’s test for independent data.Immunohistochemical results were analyzed using the Pearson chi square test.‘Alpha equals 0.05’ was considered to be statistically significant.Results 1.VT and EVT tissue microarray: 98.98%(97/98)VT and 86.36%(76/88)EVTwere well constructed.2.Real time PCR:Compared with matched control placentas,the mRNA levels of HMGB1,RAGE and NF-κB p65 were increased in severe preeclamptic placentas(P<0.05).3.Western blotting: Compared with matched control placentas,HMGB1 and RAGE protein expression were increased in severe preeclamptic placentas(P<0.05),especially the RAGE protein was elevated more obviously(P<0.01).4.Immunohistochemistry: In severe preeclamptic placentas,HMGB1 and RAGE immunoreactivity were strengthened in the cytoplasm of trophoblast cells when compared with the controls(P<0.05).5.In addition,compare with the matched controls,there was an increased level of HMGB1 in the maternal serum of severe PE patients(P<0.05).Brief Summary HMGB1-RAGE signaling pathway is involved in the pathogenesis of severe preeclampsia.HMGB1 is probably a new molecular marker of severe preeclampsia.PartⅡ Research of HMGB1 effect on inflammatory reaction and biological behaviour of trophoblastObjectives In part one,the results showed that mRNA and protein level of HMGB1,RAGE and NF-κB in placenta of preeclampsia were different from those of controls.In this part,further exploring the effect of HMGB1 on trophoblast biological behaviour and the role of HMGB1 in mechanism of preeclampsia,in order to provide the new experimental and theoretical basis.Methods 1.The human choriocarcinoma cell line JAR cells were incubated in vitro.2.HMGB1 eukaryotic expression vector(p HMGB1-EGFP)and HMGB1-siRNA were constructed to transfect JAR cells.3.Real time PCR and western blotting were used to confirm the best effect time of p HMGB1-EGFP and HMGB1-siRNA,also the best sequence of HMGB1-siRNA.4.Real time PCR and western blotting were used to detect RAGE mRNA and protein expression after transfection of HMGB1 eukaryotic expression vector.JAR cells proliferation was tested by WST-1 assay.Flow cytometry assay were used to detect apoptosis of JAR cells.In the same time,non-transfected cells group and empty plasmid transfected cells were established as the contrast groups.5.Real time PCR and western blotting were used to detect RAGE mRNA and protein expression after transfection of HMGB1-siRNA.JAR cells proliferation was tested by WST-1 assay.Flow cytometry assay were used to detect apoptosis of JAR cells.In the same time,non-transfected cells group was established as the contrast groups.6.All the analyses were treated by SPSS 21.0 statistic software.The data were expressed as mean ± SD.Statistical significance was assessed using independent two-tailed Student t-tests or Mann-Whitney’s test for independent data.Analysis of variance was used in multi group comparison.‘Alpha equals 0.05’ was considered to be statistically significant.Results 1.Transfection of HMGB1 plasmid and HMGB1-siRNA in JAR cells Transfection efficiency of p IRES2-EGFP、p HMGB1-EGFP on JAR cells was evaluated by real time PCR,it showed that the best transfection efficiency was at 48 h.After JAR cells transfected with the HMGB1-siRNA1,interfering RNA was evaluated by real time PCR,it showed that HMGB1 mRNA and protein were obviously decreased at 72 h.2.Influence of transfection of HMGB1 gene on RAGE and NF-κB expression 48 h after JAR cells was transfected by p HMGB1-EGFP,total RNA and proteinwere extracted,then RAGE mRNA and protein were evaluated by real time PCR and western blotting.Compared with untransfected contol group and empty plasmid control group,the expression levels of RAGE mRNA and protein in HMGB1 plasmid transfected group were statisticly increased(P<0.05,0.01).But there was no statistical difference between untransfected control group and empty plasmid control group(P>0.05).72 h after JAR cells was transfected by HMGB1-siRNA1,total RNA and protein were extracted,then RAGE mRNA and protein were evaluated by real time PCR and western blotting.Compared with untransfected control group,the expression levels of RAGE mRNA and protein in HMGB1-siRNA1 transfected group were significantly decreased(P<0.01).48 h after JAR cells was transfected by p HMGB1-EGFP,NF-κB activation was detected.Compared with untransfected control group and empty plasmid control group,NF-κB relative expression was obviously increased in HMGB1 plasmid transfected group(P<0.05).But there was no statistical difference between untransfected control group and empty plasmid control group(P>0.05).3.Influence of the expression of HMGB1 gene on the proliferation and apoptosis of JAR cells After JAR cells’ being transfected with p HMGB1-EGFP and HMGB1-siRNA respectively,cell proliferation rates were measured by WST-1.Proliferation ratio had no difference in untransfected control group,empty plasmid control group,HMGB1 plasmid transfected group and HMGB1-siRNA1 transfected group(P>0.05).After JAR cells’ being transfected with p HMGB1-EGFP and HMGB1-siRNA,cell apoptosis rate was measured by flow cytometry assay.Compared with untransfected control group and empty plasmid control group,cell apoptosis ratio was obviously decreased in HMGB1 plasmid transfected group(P<0.05).Compared with untransfected control group,cell apoptosis ratio was obviously increased in HMGB1-siRNA1 transfected group(P<0.05).Brief Summary 1.HMGB1 could efficiently upregulate the mRNA and protein expression of RAGE,activate NF-κB signaling pathway,and strengthen the inflammatory reaction of JAR cells.Our study provides a new experimental basis to clarify the inflammatory mechanisms in preeclampsia.2.Up-regulation of HMGB1 gene in JAR cells had no straight inhition action to cell proliferation,but inhibited cell apoptosis.This phenomenon suggests that HMGB1 maybe have two-sided effect in pathological process of preeclampsia,which provides a new direction in the exploration of preeclampsia.Part Ⅲ Regulation of Glaucocalyxin A on the expression of HMGB1 in trophoblastObjectives Futher explore HMGB1 expression and distribution in trophoblast cells as the cells are stimulated,in order to provide the new experimental and theoretical basis.Explore the effect of Glaucocalyxin A(GLA),an active ingredients fromRabdosia amethystoides,on HMGB1 expression in trophoblast cells to provide experimental and theoretical basis for clinical treatment.Methods The human choriocarcinoma cell line JAR cells were incubated in vitro.JAR cells were stimulated by 0.01μg/ml,0.1μg/ml,1μg/ml and 10μg/ml GLA for 48 hours,then real time PCR and western blotting were used to detect HMGB1 mRNA and protein,respectively.Transfection of luciferase report gene on JAR cells was used to detect NF-κB activation,laser scanning confocal was used to detect the distribution of HMGB1 in trophoblast cells,and ELISA was used to measure the expression of HMGB1 in culture medium.In the same time,non-stimulated by GLA cells groupwere established as the contrast group.All the analyses were treated by SPSS 21.0 statistic software.The data were expressed as mean ± SD.Statistical significance was assessed using independent two-tailed Student t-tests or Mann-Whitney’s test for independent data.Analysis of variance was used in multi group comparison.‘Alpha equals 0.05’ was considered to be statistically significant.Results 1.Effect of GLA on mRNA expression of HMGB1 in JAR cells Compared with the control group,0.01μg/ml and 0.1μg/ml GLA,especially 0.1μg/ml GLA,have the conspicuous effect on increasing HMGB1 mRNA expression.But the HMGB1 mRNA level was dicreased along with the increasing GLA concentration.1μg/ml and 10μg/ml GLA have an obvious effect on diseasing HMGB1 mRNA expression,especially 10μg/ml GLA(P<0.05,0.01).2.Effect of GLA on protein expression of HMGB1 in JAR cells Compared with the control group,0.01μg/ml and 0.1μg/ml GLA have the conspicuous effect on increasing HMGB1 protein expression.But the HMGB1 protein level was dicreased along with the increasing GLA concentration.When compared with the control group,1μg/ml and 10μg/ml GLA have an obvious effect on diseasing HMGB1 protein expression,especially 10μg/ml GLA(P<0.05,0.01).3.Effect of GLA on NF-κB activation of JAR cells Compared with the control group,0.01μg/ml and 0.1μg/ml GLA have the effect on increasing NF-κB activation of JAR cells,especially 0.1μg/ml GLA has the most conspicuous effect(P<0.01).1μg/ml and 10μg/ml GLA have an obvious effect on decreasing NF-κB activation of JAR cells,especially 10μg/ml GLA(P<0.01).4.Effect of GLA on distribution of HMGB1 in JAR cells 0.1μg/ml GLA had effect on HMGB1 nuclear-cytoplasmic translocation and secretion,while HMGB1 mainly existed in the nuclear in the control group.Brief Summary 1.Nuclear-cytoplasmic translocation of HMGB1 may participate in the process of preeclampsia,which provide new experimental basis for clarifying the mechanisms of preeclampsia.2.GLA can regulate cell inflammation through HMGB1 signaling pathway.It has a dose-effect on regulating the expression of HMGB1 and NF-κB,which provide new experimental basis for exploring the anti-inflammation mechanisms of GLA.Conclusions 1.HMGB1-RAGE signaling pathway may be involved in the pathogenesis of severe preeclampsia.HMGB1 is probably a new serum biomarker of severe preeclampsia.2.HMGB1 gene expression in JAR cells could efficiently upregulate the expression of RAGE,and intrigue NF-κB activation,which provide more experimental basis to clarify HMGB1-RAGE inflammatory signaling pathway in physiopathologic mechanisms of preeclampsia.Up-regulation of HMGB1 inhibits JAR cell apoptosis.This phenomenon suggests that HMGB1 probably has a two-sided effect in pathological process of preeclampsia.3.GLA can rose nuclear-cytoplasmic translocation of HMGB1,and regulate trophoblasts’ imflammatory reaction through HMGB1 signaling pathway,which has a dose-effect.This study provide new experimental basis for exploring the anti-inflammation mechanisms of GLA and clinical prevention of PE.