| Preeclampsia is a serious hypertensive disorder of pregnancy,which is a leading cause of maternal death and major contributor to maternal and perinatal morbidity.However,the triggering factors and underlying mechanisms responsible for the pathogenesis of preeclampsia remain elusive.The leading hypotheses strongly rely on disturbed placental functions.Studies have supported the two-stage model.Stage one is inadequate placental perfusion,and stage two is the maternal syndrome resulting from inadequate placental perfusion.The consequences of inadequate perfusion are intermittent hypoxia and the generation of oxidative stress,which lead to the release of antiangiogenic proteins and inflammation.In addition to apoptosis,autophagy is another programmed cell death pathway.It is a reparative and life-sustaining process by which cytoplasmic components are sequestered in double-membrane vesicles and degraded on fusion with lysosomes under stress to maintain cellular homeostasis.Autophagy-related protein beclin-1 forms an early complex that promotes the synthesis and growth of pre-autophagosomal membranes.LC3 is synthesized as pro LC3 and integrated into the membranes of autophagosomes.Thus,beclin-1 and LC3 are considered as markers for detecting autophagy.Autophagy plays important roles in immunity and inflammation.The beneficial and detrimental effects of immunity and inflammation can be balanced by autophagy that may protect against infections as well as autoimmune and inflammatory diseases.Recently,autophagy has become a highly researched field in obstetrics,Preeclampsia displays many characteristics,including hypoxia and inflammatory responses,which are associated with autophagy.Oxidized low density lipoprotein(ox LDL)is the oxidative modification of native LDL in numerous disease states as a result of oxidative stress and vascular endothelial injury.Ox LDL binding to its receptor,lectin-like oxidized low density lipoprotein receptor 1,causes endothelial dysfunction and could play a significant role in the pathobiology of atherosclerosis,diabetes,hypertension,and preeclampsia.Previous studies have shown that the cytotoxicity induced by ox LDL is different in diverse cell types.Previous studies have shown that the cytotoxicity induced by ox LDL differs indiverse cell types.A study of human umbilical vein endothelial cells(HUVECs)suggested that ox LDL increases the autophagic level in a concentration-dependent manner.Ox LDL inhibits autophagy in macrophages and smooth muscle vascular cells.However,there are only a few studies on the effects of ox LDL in autophagy of human trophoblasts.Objective:We aimed to investigate variations of autophagy in preeclampsia,the influence of ox LDL on autophagy in the JEG-3 cell line,and whether autophagy can protect against ox LDL-mediated inflammation.Methods:1.Placental tissues and serum were obtained from 26 women with preeclampsia(PE,n=26)and 20 women with normal pregnancy(NP,n=20),who delivered in the Department of Obstetrics,Affiliated Hospital of Qingdao University Medical College,From June 2014 to January 2015.Serum were isolated from venous blood,and immediately frozen at-70°C until analysis.All the placenta samples were collected across each placenta and pooling of theses for one sample,placed on ice,transported to the laboratory,and immediately frozen at-70°C until analysis.2.Enzyme-linked immunosorbent assay(ELISA)was used to measure the plasma concentrations of ox LDL and inflammatory cytokenes(TNF-?and IL-6)in these serum samples.We used immunohistochemistry to assess the presence and location of TNF-?and IL-6,western blotting to analyze the expression of TNF-?and IL-6 in placentas.3.Immunohistochemical analyses showed that beclin-1 was mainly localized in cytotrophoblasts,while LC3 was localized in cytotrophoblasts and syncytiotr phoblasts.We found that m RNA and protein expression(Beclin 1 and LC3)is decreased in the preeclampsia placenta compared with normotensive pregnancies,as assessed by western blotting and q PCR.4.Immunofluorescence was used to assess the presence and location of the LC3 protein in the JEG-3 cells treated with various concentrations(25,50,100,150mg/l)and time point(6,12,24,48h)of ox LDL.5.Quantitative real-time PCR were used to analyze the expression of TNF-? and IL-6 in the JEG-3 cells treated with different various concentrations(25,50,100,150mg/l)of ox LDL for 6h.6.Quantitative real-time PCR and western blotting were used to analyze the expression of autophagy proteins(Beclin 1 and LC3),TNF-? and IL-6 in the JEG-3 cells pretreated by rapamycin and following ox LDL treatment.7.Data were analyzed using the Statistical Package for Social Sciences 17.0.Data are presented as the mean±SE.A t-test was used to determine statistical significance.A probability level of less than 0.05 was considered to be statistically significant.Results:1.ELISA analyses showed that the concentrations of ox LDL,TNF-? and IL-6 in PE were significantly higher compared with control group.Immunohistochemical analyses showed that TNF-? and IL-6 were mainly localized in cytotrophoblasts,TNF-? and IL-6 immunoreactivities were dense in placentas from PE group.We found that protein expression(TNF-? and IL-6)is increased in the preeclampsia placenta compared with normotensive pregnancies,as assessed by western blotting.2.Immunohistochemical analyses showed that beclin-1 was mainly localized in cytotrophoblasts,while LC3 was localized in cytotrophoblasts and syncytiotr phoblasts.We found that m RNA and protein expression(LC3,Beclin 1)is decreased in the preeclampsia placenta compared with normotensive pregnancies,as assessed by western blotting and q PCR.3.Confocal microscopy images showed that neither ox LDL treatment at 25,50,100,or 150 mg/l for 6 h or varying the ox LDL treatment time(100 mg/l for 6,12,24,or48 h)could induce autophagy in the JEG-3 cells.When the cells were pretreated with rapamycin for 1 h and then exposed to ox LDL,the puncta from LC3 labeling was apparent and an enlarged nucleus at 24 and 48 h.Therefore,we chose 6 h as the optimal time point for the experiments.4.We detected the levels of tumor necrosis factor-?(TNF-?)and interleukin-6(IL-6)induced by treatment with various concentrations of ox LDL for 6 h.The results showed that ox LDL at 50 and 100 mg/L produced an obvious increase in TNF-? and IL-6 levels.Furthermore,the expression of m TOR was not affected by 100 mg/l of ox LDL.Therefore,we chose 100 mg/l of ox LDL as the appropriate concentration to use.5.ox LDL at 100 mg/L produced an obvious increase in TNF-? and IL-6 levels.when Cells were pretreated with rapamycin(100 n M)for 1 h and then exposed to 100 mg/l of ox LDL for 6 h.We found significant increases in the expression of beclin-1 LC3 and as well as remarkable decreases in the levels of TNF-? and IL-6.Conclusion:1.Increased levels of ox LDL,TNF-?and IL-6 may be involved in the pathoanatomical process of preeclamsia.2.The expression levels of inflammatory cytokines increased following ox LDL treatment,while ox LDL could not induce autophagy in trophoblasts.3.ox LDL induced high levels of inflammatory cytokines in trophoblasts and that autophagy induction protected against this inflammatory response.We conclude that impaired autophagy in preeclampsia might result in decreased protection for trophoblasts from oxidative and inflammatory stress,and this may contribute to the pathogenesis of preeclampsia.Future studies should focus on therapeutic interventions for patients with preeclampsia.How to switch on the protective effects of autophagy is a strategy worth pursuing and a challenge for clinicians. |
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