The Mechanism Of Chemerin In Regulatin Of HUVECs-induced-NO And The Relationship Between Chemerin And Preeclampsia

Preeclampsia is a commonly idiopathic disease in pregnancy. Severe preeclampsia is one of the main cause of morbidity and mortality of mothers and newborns. It was identified that the central link of the pathogenetic of preeclampsia is the activation and/or damage of endothelial cell of the whole body of pregnant. Since the adipokine impairs glucose metabolism and preeclampsia is associated with metabolic disease, we regarded that chemerin may play a role in the pathogenesis of preeclampsia. Now the data mostly derived from the research in internal medicine diseases. Data are infrequent available concerning chemein in preeclampsia, furthermore rare data in endothelial cell function. We will discuss the expression of chemerin in preeclampsia and the role of chemerin in humanumbilical vein endothelial cells.PART ONEThe changes of expression and significance about chemerin in preeclampsiaObjective:To investigate the expression of chemerin which was identified as a new adipokine in the peripheral blood and placenta of preeclampsia (PE), in order to investigate its roles on the pathogenesis and the development of PE.Methods:Peripheral blood and placenta tissues were obtained from60patients with PE(30with moderate PE(MPE group),30with severe PE(SPE group) and28normal pregnant women (NP group) in the operation room for Caesarean birth. We tested the concentrations of chemerin by ELISA in the peripheral blood, meanwhileFluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blot(WB) were used to evaluate the expression of chemerin mRNA and protein in placenta tissues. Furthermore, we detected the markers of renal function, glucose and lipid metabolism, and so on. As the last step, statistical analysis was carried on.Results:1. The serum chemerin in SPE group was (493.83±105.23) ng/ml, while the MPE group was (330.23±56.22) ng/ml, and the NP group was (220.00±50.78) ng/ml, pairwise comparisons were statistically signigicant (P<0.01).2.The relative expression of chemerin mRNA in placenta tissues of the SPE group was (4.00×10-4±1.11×10-4), while the MPE group was (3.04×10-4±0.87×10-4), and the NP group was (1.13×10-4±0.54×10-4), pairwise comparisons were statistically significant (P<0.01).3. The relative expression of chemerin protein in placenta tissues of the SPE group was (1.84±0.40), while the MPE group was (1.20±0.17), and the NP group was (0.57±0.15), pairwise comparisons were statistically significant(P<0.01).4. There was a positive correlation between chemerin serum levels and systolic blood pressure, diastolic blood pressure, BMI and serum insulin, r=0.690(P<0.001),0.655(P<0.001),0.724(P<0.001),0.627(P<0.001) and negative correlation between chemrin and eNOS, r=-0.0.727(P<0.001)Conclusions:Chemerin was expressed in peripheral serum and placenta of NP woman and PE. All of above showed that chemerin might participate in the pathogenesis and the development of PE. Part twoEffects of chemerin on the production of NO of HUVECsObjective:To explore how to harvest and identify HUVECs in vitro and investigate the effects of chemerin on the production of NO of HUVECs.Methods:We obtained fresh umbilical cord to cultivate original human umbilical vein endothelial cells through enzyme digestion. Then we used immune-cytochemistry and immunofluorescence to identify human umbilical vein endothelial cells. HUVECs were cultured and treated with different concentrations of chemerin (0,10,100,500,1000ng/ml) for12,24,48hours. The measurement of culture media NO of HUVECs was performed using the Griess reagent. Culture media was collected for the measurement of mitrite (NO2-) content, which was the stable breakdown product of NO.Results:The cells we cultured was in good condition, monolayer, and flagstone shaped. The results of immunofluorescence cytochemistry showed factor Ⅷ positively stained. The cultured cells were identified as human umbilical vein endothelial cells. Chemerin promoted culture media NO content of HUVECs between0～500ng/ml chemerin(P<0.05), chemerin could dose-dependently induce NO generation.There were no statistically difference of culture media NO content between500ng/ml and1000ng/ml.Conclusins:(1)We have successfully cultivated original human umbilical vein endothelial cells.(2) Chemerin increased NO generation of HUVECs, And with concentrations between10ng/ml and500ng/ml, chemerin could dose dependently induce NO generation. Part threeStudy on the methanisms of chemerin in regulating HUVECs-induced-NO Objective:The aim of this study was to investigate the possible signaling pathways of chemerin on HUVECs-induced-NO.Methods:(1) The phosphorylation status of eNOS serine-1177(Ser-1177) and Akt serine-473(Ser-473) of HUVECs after treatment with different concentration of chemerin (0,10,100,500,1000ng/ml) for24hours were detected by specific antibodies respectively using Western Blot.(2) In order to confirm the signaling pathway, HUVECs were treated with chemerin and a PI3K inhibitor LY294002or a NOS inhibitor L-NAME, and expression of Akt Ser-473and eNOS phosphorylation were determined by Western Blotting.Result:(1) Chemerin increased phosphorylated eNOS Ser-1177and the phosphorylation of Akt Ser-473. And with concentrations between10ng/ml and500ng/ml, chemerin could dose dependently induce increased phosphorylation of eNOS Ser-1177and the phosphorylation of Akt Ser-473, and there were no statistically differences of between500ng/ml and1000ng/ml.(2) The chemerin-induced NO increase was partially restored by a PI3K inhibitor LY294002or a NOS inhibitor L-NAME.Conclusion:Chemerin increased NO generation of HUVECs through PI3K-Akt-eNOS signaling pathway. Part fourChemerin inhibited TNF-a-induced VCAM-1in human umbilical vein endothelial cellsObjective:The aim of this study was to investigate the role and mechanisms of inhibitory effort of chemerin on TNF-a-induced VCAM-1in human umbilical vein endothelial cells.Methods:(1) TNF-a induced the impaired HUVECs model mediated by NF-κB. The protein levels of VCAM-1and NF-κB of HUVECs were detected in the presence and absence of TNF-a (5ng/ml,6hours) and NF-κB inhibitor pyrrolidine dithiocarbamate, PDTC(10μM,30min).(2) Chemerin played a role on TNF-a induced impairment in HUVECs. The protein levels of VCAM-1and NF-κB of HUVECs were detected in the presence and absence of TNF-a(5ng/ml,6hours), chemerin(500ng/ml,24hours), PI3K inhibitor LY294007(5μmol/ml,24hours) and a NOS inhibitor L-NAME(0.5mmol/ml,24hours).Result:(1) TNF-a significantly upregulated the levels of expression of NF-kB and VCAM-1(P<0.05). The effect of VCAM-1could be partially diminished by cotreatment with an NF-κB inhibitor, PDTC (P<0.05).(2) Chemerin significantly inhibited TNF-α induced the expression of VCAM-1and NF-κB in HUVECsCs<0.05). L-NAME and LY294007could partially diminish the inhibitory effect of chemerin on TNF-a-induced VCAM-1expression in HUVECs. Conclusion:(1) TNF-a upregulated the expression of VCAM-1mediated by NF-κB pathway. Chemerin plays an inhibitory role in VCAM-1expression by preventing TNF-a-induced phosphorylation of NF-κB. The anti-inflammatory effect of chemerin is mediated via activation of PI3K-AKT-eNOS-NO pathway.(2) It is suggested that chemerin is a potential drug target against preeclapsia vascular diseases.