OBJECTIVETo examine the effect of angiotensin II (Ang II) on nuclear factor-kappa B (NF-kappaB) activation in human endothelial cell line ECV304 and the molecular mechanism by which Ang II activates NF-kappaB.

METHODSECV304 cells were transiently cotransfected with an NF-kappaB/luciferase reporter gene and inactive NF-kappaB-inducing kinase (NIK), IkappaB kinase alpha (IKK(alpha)), IkappaB kinase beta (IKK(beta)) mutants or vectors, respectively. The effect on NF-kappaB was detected by using an electrophoretic mobility shift assay (EMSA) and overexpression of the mutants enabled blocking of reporter gene activation induced by Ang II. With immunofluorescence and immuno-electronic microscope techniques, including confocal microscopy and gold particle labeled electronic microscopy, definite cytoplasmic-to-nuclear translocations of NF-kappaB activation were detected using subunits p50 and p65 induced by Ang II.

RESULTSThe translocation of p50 in nuclei was highly remarkable 2 hours after Ang II stimulation, and the activity was somewhat reduced 6 hours after stimulation to the 18th hour. Northern blot also showed PDGF-B mRNA increased by stimulation of Ang II for 18 hours.

CONCLUSIONAng II is effective in stimulating NF-kappaB activation through a pathway dependent on NIK, IKK(alpha) and IKK(beta), and induces PDGF-B transcription in the endothelial cell line, ECV304.v

Angiotensin II ; pharmacology ; Cell Line ; Electrophoretic Mobility Shift Assay ; Endothelium, Vascular ; drug effects ; metabolism ; Humans ; NF-kappa B ; pharmacology ; Proto-Oncogene Proteins c-sis ; genetics ; Transcription, Genetic ; drug effects

【Objective】 To analyze the effects of IgG subtypes(IgG 1 and IgG3) of antibodies contained in infant serum and erythrocyte eluates on hemolytic disease of the newborn(HDN), so as to provide reference for its early clinical diagnosis and treatment. 【Methods】 49 newborns with HDN in our hospital from June 2019 to March 2020 were detected for three hemolytic tests(direct antiglobulin test, elution test and indirect antiglobulin test), as well as the components of IgG1 and IgG3 in eluates. The correlation analysis was conducted by combining birth hours (physiological jaundice) and hemolytic degrees (total bilirubin, indirect bilirubin, and hemoglobin). 【Results】 In the 44 cases of IgG1 and IgG3 subtype detection of infant RBC eluates, regression equations could be established between total bilirubin, indirect bilirubin and birth hours, and between hemoglobin and elution test, and linear regression relationships were found (P<0.05). In the 28 cases of IgG1 and IgG3 subtype detection of infant serum, regression equations could be established between total bilirubin, indirect bilirubin, birth time and IgG3 subtype, and between hemoglobin and IgG1 subtype (P<0.05), and linear regression relationships were found (all P<0.05). Three infants, presenting IgG1 and IgG2 subtypes(+ ) and three hemolysis tests(-), were all second pregnancy, constituted by Rh-HDN of 2 case and other-system-HDN 1. 【Conclusion】 The degree of HDN is directly related to IgG1 and IgG3 antibodies in infant blood plasma. In addition to the total bilirubin and indirect bilirubin, the changes of IgG3 antibodies in infant plasma and IgG1 antibody in anemic infants should be monitored. If IgG1 and IgG3 antibodies are yielded even with all negative ABO-HDN hemolysis tests, non-ABO-HDN should be considered in time to achieve accurate diagnosis and treatment.