Plasma Olink Proteomics Identifies CCL20 as a Novel Predictive and Diagnostic Inflammatory Marker for Preeclampsia.

Inflammation is generally thought to be involved in the occurrence and development of preeclampsia (PE), but its specific effect on PE remains unclear. In the present study, the expression levels of 92 inflammation-related proteins were measured in the late pregnancy maternal plasma from patients with PE (n = 15) and normal pregnant controls (n = 15) using the Olink inflammation panel based on the highly sensitive and specific proximity extension assay technology. A total of 28 inflammation-related markers differed between the PE and control groups. Among them, fibroblast growth factor 21 (FGF-21) and cysteine-cysteine motif chemokine ligand 20 (CCL20) had the largest fold changes. We further validated the levels of CCL20 in the late (43 with PE and 44 controls) and early (37 with PE and 37 controls) pregnancy maternal plasma using enzyme-linked immunosorbent assay (ELISA). To the best of our knowledge, for the first time, CCL20 was found to be upregulated in the late and early pregnancy plasma of patients with PE and had an area under the curve (AUC) of 0.753 and 0.668, respectively. In conclusion, patients with PE had increased levels of most inflammatory markers, and CCL20 might be a novel potential predictive and diagnostic biomarker for PE.

Activation of PAR-1/NADPH oxidase/ROS signaling pathways is crucial for the thrombin-induced sFlt-1 production in extravillous trophoblasts: possible involvement in the pathogenesis of preeclampsia.

BACKGROUNDS/AIMS: Preeclampsia was characterized by excessive thrombin generation in placentas and previous researches showed that thrombin could enhance soluble Fms-like tyrosine kinase 1 (sFlt-1) expression in first trimester trophoblasts. However, the detailed mechanism for the sFlt-1 over-production induced by thrombin was largely unknown. The purpose of this study was to explore the possible signaling pathway of thrombin-induced sFlt-1 production in extravillous trophoblasts (EVT). METHODS: An EVT cell line (HRT-8/SVneo) was treated with various concentrations of thrombin. The mRNA expression and protein secretion of sFlt-1 in EVT were detected with real-time polymerase chain reaction and ELISA, respectively. The levels of intracellular reactive oxygen species (ROS) production were determined by DCFH-DA. RESULTS: Exposure of EVT to thrombin induced increased intracellular ROS generation and overexpression of sFlt-1 at both mRNA and protein levels in a dose dependent manner. Short interfering RNA (siRNA) directed against PAR-1 or apocynin (an inhibitor of NADPH oxidase) could decrease the intracellular ROS generation and subsequently suppressed the production of sFlt-1 at mRNA and protein levels. CONCLUSIONS: Our results suggested that thrombin increased sFlt-1 production in EVT via the PAR-1 /NADPH oxidase /ROS signaling pathway. This also highlights the PAR-1 / NADPH oxidase / ROS pathway might be a potential therapeutic target for the prevention of preeclampsia in the future.

[Protective effect of estrogen on acute lung injury after hemorrhagic shock in pregnant rabbit].

OBJECTIVE: The paper is an attentative effort to evaluate the reaction and mechanism of estrogen on pregnant rabbits with acute lung injury caused by hemorrhagic shock. METHODS: Sixty pregnant New Zealand white rabbits were randomly divided into 6 groups, with 10 rabbits in each group, namely normal control group (NG group, with anesthesia only), estrogen group (E(2)G group, with additional estrogen injection at 60 min) and the other four hemorrhagic shock groups underwent hemorrhagic shock (i.e. E(2)SG, FSG, SBSG, E(2)SBSG group; mean blood pressure- 40 mmHg (1 mmHg = 0.133 kPa) by phlebotomy for 15 min. After maintenance of the pressure for 45 min, the rabbits were treated with E(2) (0.37 mg/kg), fructose injection (5%, 2 ml/kg), the p38 mitogen-activated protein kinases (p38MAPK) inhibitor SB-203580 (2 mg/kg) or E(2) plus SB-203580. Tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) were measured at different time points (0 min, 60 min, 80 min and 260 min), lung tissue methane dicarboxylic aldehyde (MDA) level, lung tissue myeloperoxidase (MOP), superoxide dismutase (SOD) activity, lung tissue dry weight/wet weight (DW/WW) value were measured after the experiment was finished, pulmonary pathology of the rabbits was observed. RESULT: (1) Serum TNF-α level of NG group and E(2)SG group were not significantly different compared with the other four groups at the 0 min and 60 min. At 80 min and 260 min of experiment, serum TNF-α level of all the four shock groups were increased, E(2)SG group [(172.4 ± 16.0) and (216.7 ± 18.6) ng/L], FSG group [(171.6 ± 9.1) and (263.9 ± 7.8) ng/L], SBSG group [(172.8 ± 7.2) and (300.6 ± 4.8) ng/L], E(2)SBSG group [(167.9 ± 4.8 ) and (261.8 ± 9.6) ng/L], and significantly higher than NG group and E(2)G group, separately (P < 0.05). (2) Serum IL-6 level of NG group and E(2)SG group were not significantly different compared with the other four groups at the 0 min, 60 min and 80 min. At 260 min, the serum IL-6 level [(98.3 ± 0.9) and (110.4 ± 1.8) ng/L; (120.9 ± 2.3) and (109.8 ± 2.6) ng/L] of the four shock groups (E(2)SG, FSG, SBSG, E(2)SBSG group) were significantly higher than NG group and E(2)G group (P < 0.05). (3) Lung tissue MDA level [(2.20 ± 0.12), (2.57 ± 0.11), (3.17 ± 0.08), (2.75 ± 1.09) nmol/mg] and MPO activity [(4.45 ± 0.25), (6.65 ± 0.56), (9.55 ± 0.30), (6.78 ± 0.11) U/mg] of the four shock groups (E(2)SG, FSG, SBSG, E(2)SBSG group) were higher than NG group and E(2)G group (P < 0.05). (4) Lung tissue SOD activity [(51.8 ± 1.8), (40.2 ± 1.5), (30.0 ± 1.7), (41.2 ± 2.0) U/mg] was significantly higher in the four shock groups (E(2)SG, FSG, SBSG, E(2)SBSG group) compared with NG group and E(2)G group (P < 0.05). (5) Lung tissue DW/WW value (0.143 ± 0.008, 0.127 ± 0.008, 0.109 ± 0.006, 0.125 ± 0.008) was significantly lower in the four shock groups (E(2)SG, FSG, SBSG, E(2)SBSG group) compared with NG group and E(2)G group (P < 0.05). (6) Lung tissue of the rabbits in NG group and E(2)G group is basically normal without obvious pathology changes. Lung tissue pathological damage of rabbits was observed in the four shock groups, and the pathological damage of rabbits in SBSG group was most serious. CONCLUSION: Estrogen can reduce acute lung injury of pregnant rabbits with hemorrhagic shock, the p38MAPK pathway plays a critical role in mediating the salutary effects of E(2) on shock-induced acute lung injury.

[Relationship between pigment epithelium-derived factor expressed in placentas and the pathogenesis of preeclampsia disease].

OBJECTIVE: To investigate the effect of pigment epithelium-derived factor (PEDF) on the pathogenesis of preeclampsia disease, by detecting the expression of PEDF in the placentas, as well as the relationship between PEDF and the production of placental vessels. METHODS: A study was performed in 60 cases of pregnant women with preeclampsia in the obstetrical department of Nanfang Hospital affiliated to southern medical university from October 2011 to January 2013, in which 30 cases were patients with mild preeclampsia(mPE) and other 30 cases were those with severe preeclampsia (sPE).40 normal pregnant women who also been hospitalized and delivered were selected as control group. The expression of PEDF and micro-vessel density (MVD) in placentas were assayed by using western blot and SP immunohistochemical method, then the relationship between PEDF and MVD was analyzed. RESULTS: (1) The pathological changes of placentas:the placental weight were lightened obviously in the mild preeclampsia and severe preeclampsia groups, the reduced blood vessels and luminal stegnosis were found in chorionic villus, basement membrane of trophocytes were thickening. The hyperplasia syneytiotrophoblast were like nodosity, with focus infarction, fibrinoid necrosis, or thrombogenesis.While there was no the above mentioned pathological alteration in normal control group. (2)The levels of PEDF expression in mild and severe preeclampsia group were 0.63 ± 0.09, 0.93 ± 0.07, while 0.47 ± 0.04 in control group, which in mild and sever preeclampsia groups were significantly higher than that in normal group (P < 0.05). Compared to mild preeclampsia group, the expression of PEDF was significantly increased in severe preeclampsia group, there was statistical significance between the difference (P < 0.05).(3) The amount of microvessel density (MVD) in mild and severe preeclampsia group were 106 ± 9, 93 ± 8, while 136 ± 9 in control group, which were significantly reduced in mild and severe preeclampsia group, compared to that in normal control group (P < 0.05). And it was significantly lower in severe preeclampsia group than that in mild preeclampsia group (P < 0.05). (4) The expression of PEDF was negatively correlated with the amount of MVD in mild and severe preeclampsia group (r = -0.426, P < 0.05; and r = -0.646, P < 0.05 respectively), which was also negative in control group (r = -0.589, P < 0.05). CONCLUSION: Increased PEDF expression in placentas of women with preeclampsia induce the dysfunction of the placental vascular reconstruction and the pathological alteration like ischemic and hypoxia in placentas, which may be involved in pathogenesis and pathogenic progress of preeclampsia.

Expression of SASH1 in Preeclampsia and Its Effects on Human Trophoblast.

AIM: To explore the involvement of SASH1 in preeclampsia. METHODS: Expression of SASH1 was determined by qPCR, WB, and immunohistochemistry in the placenta of both normal and preeclamptic pregnancies. The SASH1 gene of human HTR-8/SVneo cells was overexpressed by transfection of pEZ-Lv206-SASH1. After that, the CCK-8 assay, EdU assay, transwell assay, and flow cytometry were used to examine the cell proliferation, migration, invasion, and apoptosis. RESULTS: Higher expression of SASH1 was detected in placental tissues collected from patients with preeclampsia, compared with those from gestational age-matched control samples. The expression of SASH1 was significantly enhanced by transfection with pEZ-Lv206-SASH1 in HTR-8/SVneo cells. In addition, the HTR-8/SVneo cells transfected with pEZ-Lv206-SASH1 exhibited significantly reduced proliferation, migration, and invasion ability compared to the cells in the empty vector group and normal group. Flow cytometry analysis demonstrated that the apoptosis rate of cells transfected with pEZ-Lv206-SASH1 was significantly higher than that of cells transfected with empty vector and untreated cells. CONCLUSIONS: SASH1 is significantly upregulated in the placenta of preeclampsia, and overexpression of SASH1 can inhibit the proliferation, migration, and invasion, but induce apoptosis of trophoblast cells in vitro.

[Diagnostic value of CA125, HE4 and Copenhagen Index in differentiating benign from malignant epithelial ovarian tumors].

OBJECTIVE: To analyze diagnostic value of Copenhagen Index based on pretreatment serum CA125, HE4 and age in differentiating benign and malignant epithelial ovarian tumors. METHODS: The clinical data were analyzed for 208 consecutive patients with epithelial ovarian tumors (including 100 with malignant and 108 with benign tumors) treated in our department between September, 2014 and September, 2016. The receiver-operating characteristic curve was drawn based on the golden standard of pathological diagnosis for calculation of the diagnostic sensitivity and specificity of CA125, HE4 and the Copenhagen Index. RESULTS: In the overall cases, early stage cases and advanced stage cases, the prediction probabilities of CA125, HE4 and Copenhagen Index were all significantly higher for malignant than in benign tumors (P<0.001). The sensitivities of CA125, HE4, Copenhagen Index for differentiating benign and malignant tumors were 81.0%, 86.0% and 91.0% in the overall cases, 64.0%, 68.0% and 72.0% in early stage cases, and 86.7%, 92.0% and 97.3% in advanced stage cases, and their diagnostic specificities were 88.0%, 93.5% and 96.3%, respectively. Copenhagen Index had the highest diagnostic sensitivity (but not in early stage cases) and specificity followed by HE4 and then by CA125 (P<0.001) (P>0.05). CONCLUSION: Copenhagen Index combined with CA125, HE4 and age hase better diagnostic value than HE4 or CA125 alone for differentiation between benign and malignant epithelial ovarian tumors, and can be used clinically to improve the early diagnostic rate of epithelial ovarian cancer.

[Effects of docosahexaenoic acid on cell apoptosis, invasion and migration of cervical cancer cells in vitro].

OBJECTIVE: To investigate the effect of docosahexaenoic acid (DHA) on apoptosis, migration and invasion of cervical cancer cell lines. METHODS: cervical cancer cell lines Hela and Siha in logarithmic phase were treated different concentrations of DHA. The morphological changes of the cells were observed microscopically and cell apoptosis was observed using Hoechst 33258 fluorescent staining. MTT assay was used to evaluate the effect of DHA in suppressing cell growth, and flow cytometry was employed to analyze the changes of cell apoptotic rate following DHA stimulations. Wound healing assay and Transwell migration assay were used to evaluate the migration of the cell lines. The expression levels of Bax, Bcl-2 cleaved caspase3, MMP-9 and VEGF proteins were detected by Western blotting. RESULTS: DHA exposure of the cells caused obvious morphological changes and dose-dependently increased the number of apoptotic bodies in the cells. MTT assay showed that DHA inhibited the growth of the cancer cells in a time- and concentration-dependent manner. DHA also effectively suppressed migration and invasion of the cancer cells. The cells exposed to DHA showed significantly down-regulation of Bcl-2, MMP-9 and VEGF proteins and up-regulation of cleaved-caspase 3 and Bax. CONCLUSION: DHA can promote cervical carcinoma cell apoptosis by down-regulating the anti-apoptotic proteins Bax, Bcl-2 and cleaved-caspase3 and suppress cell invasion by decreasing MMP-9 and VEGF expressions.

[Effects of estrogen on renal function of pregnant rabbits with hemorrhagic shock].

OBJECTIVE: To evaluate the effects of estrogen on renal function of pregnant rabbits with hemorrhagic shock. METHODS: Forty pregnant New Zealand white rabbits were randomized into 4 groups, namely normal control group (NG group, with anesthesia only), estrogen group (E2 group, with additional estrogen injection at 60 min), estrogen-hemorrhagic shock (E2SG) group and fructose-hemorrhagic shock (FSG) group. In the latter two groups, the rabbits were subjected to phlebotomy for 15 min to induce hemorrhagic shock with a blood pressure of 40 mmHg; after maintenance of the pressure for 45 min, intravenous injections of estrogen or fructose were given before resuscitation 20 min later. Blood urea nitrogen (BUN) and creatinine (Cr) concentration were measured at different time points and renal pathology of the rabbits was observed. RESULTS: No significant differences were founding serum BUN and Cr levels between NG and E2G groups during the experiment. In FSG and E2SG groups, serum BUN level began to increase at 80 min after hemorrhagic shock and was significantly higher in FSG group (P<0.05); serum Cr level increased progressively from the start of the experiment and began to decrease at 60 min, with a faster rate of reduction in E2SG group (P<0.05). CONCLUSION: Estrogen can effectively lower serum BUN and Cr levels and ameliorate renal pathologies to offer protective effect in pregnant rabbits against hemorrhagic shock.