ABSTRACT
Abstract Background: Phospholipase C-like 1 (PLCL1), a protein that lacks catalytic activity, has similar structures to the PLC family. The aim of this research was to find the function and underlying mechanisms of PLCL1 in fibroblast-like synoviocyte (FLS) of rheumatoid arthritis (RA). Methods: In this study, we first analyzed the expression of PLCL1 in the synovial tissue of RA patients and K/BxN mice by immunohistochemical staining. Then silencing or overexpressing PLCL1 in FLS before stimulating by TNF-α. The levels of IL-6, IL-1β and CXCL8 in FLS and supernatants were detected by Western Blot (WB), Real-Time Quantitative PCR and Enzyme Linked Immunosorbent Assay. We used INF39 to specifically inhibit the activation of NLRP3 inflammasomes, and detected the expression of NLRP3, Cleaved Caspase-1, IL-6 and IL-1β in FLS by WB. Result: When PLCL1 was silenced, the level of IL-6, IL-1β and CXCL8 were down-regulated. When PLCL1 was overexpressed, the level of IL-6, IL-1β and CXCL8 were unregulated. The previous results demonstrated that the mechanism of PLCL1 regulating inflammation in FLS was related to NLRP3 inflammasomes. INF39 could counteract the release of inflammatory cytokines caused by overexpression of PLCL1. Conclusion: Result showed that the function of PLCL1 in RA FLS might be related to the NLRP3 inflammasomes. We finally confirmed our hypothesis with the NLRP3 inhibitor INF39. Our results suggested that PLCL1 might promote the inflammatory response of RA FLS by regulating the NLRP3 inflammasomes.
ABSTRACT
Placentation,which is critical for maternal-fetal exchange of nutrients and gases,is a complicated process comprising stepwise vasculogenesis and angiogenesis.Hypoxia caused by impairedtrophoblast invasion may cause various angiogenic abnormalities in human placenta.The Notchl signaling pathway plays an important role in the regulation of angiogenesis.The angiogenesis of human umbilical vein endothelial cells (HUVECs) under normal/hypoxic conditions and the mRNA/protein level of Notchl/Dell4/Jaggedl were investigated in this study.The effects of DAPT/JAG-1 on the migration of HUVECs were also assessed by cell wound healing assay,so as to discover the possible role of notchl signaling pathway in the angiogenesis of human placenta.The results showed that angiogenic ability of HUVECs was seriously reduced under hypoxic conditions.The mRNA and protein levels of Notchl/Dell4/Jaggedl were decreased in the hypoxic group compared to the control one.In addition,the migration capability of HUVECs was significantly obstructed when treated with DAPT and under hopoxic condition,but promoted when treated with JAG-1.The above results demonstrate that hypoxia downregulates the angiogenesis in human placenta via Notch 1 signaling pathway.
ABSTRACT
The mechanism of injury on the human glomerular endothelial cells (ciGENC) induced by preeclampsia serum was investigated.Concentration of maternal serum sFlt-1 protein was detected by ELISA.Fluorescently-labeled bovine serum albumin infiltrating through lower chamber of Transwell was measured by multifunction microplate reader.Morphologic change of ciGENC was observed under inverted phase contrast microscope.The concentration of sflt-1 in preeclampsia groups was significantly increased as compared with control group (P＜0.01).Permeability in preeclampsia groups was significantly increased as compared with control group (P＜0.01).By contrast with severe preeclampsia group,the permeability of ciGENC monolayer in mild preeclampsia group was decreased significantly (P＜0.05).Intervention of exogenous VEGF significantly decreased permeability of ciGENC in preeclampsia groups.It was concluded that sFlt-1 increased ciGENC permeability by damaging integrity of endothelial barrier function.
ABSTRACT
This study examined the effect of over-expression of sFlt-1 by trophoblasts on the barrier function of glomerular endothelial cells and the role of VEGF in this process in order to explore the pathogenesis of glomerular disease in preeclampsia.SFlt-1 expression in the human trophoblasts (TEV-1 cells) was enhanced by transfecting sFlt-1 plasmid DNA into TEV-1 cells.The monolayer barrier function of glomerular endothelial cells (ciGEnCs) was determined by measuring the fluorescence intensity of bovine serum albumin (BSA) that crossed the monolayer of glomerular endothelial cells.The results showed that the over-expression of sFlt-1 by TEV-1 cells led to the barrier dysfunction of ciGEnCs,and the exogenous VEGF could alleviate the ciGEnCs dysfunction resulting from the over-expression of sFlt-1 to a certain extent.It was concluded that the dysregulation of sFlt-1 and VEGF in preeclamptic pregnancy may contribute to the barrier dysfunction of glomerular endothelial cells,and VEGF may play an important role in maintaining the barrier function of glomerular endothelial cells,but it may not be the sole factor.
ABSTRACT
This study examined the association of expression of vascular endothelial growth factor(VEGF),a promoter of angiogenesis,with endothelium dysfunction in preeclampsia.The level of VEGF protein and mRNA in the placenta and peripheral blood samples of 30 preeclampsia patients and 30 normotensive pregnant women was measured by immunohistochemistry,real-time reverse transcriptase-polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA),respectively.VEGF expression in the human umbilical vein endothelial cells(HUVECs)was blocked by small interfering RNAs(siRNAs).The monolayer barrier function of HUVECs was determined by measuring the fluorescence intensity of BSA that crossed the HUVEC monolayers.The cell proliferation and cell-secreted nitric oxide(NO)level were detected by MTT method and nitrate reductase assay,respectively.The results showed that VEGF was expressed in the syncytiotrophoblasts and endothelial cells of vessels and capillaries in the placenta tissue.The serum level of VEGF in the preeclampsia patients was significantly decreased as compared with that in normal pregnant subjects,although VEGF mRNA expression in the placenta tissue of preeclampsia patients remained still high.Moreover,VEGF deficit could lead to endothelium cell dysfunction,and the administration of VEGF could protect endothelium cells from injury.It was concluded that lack of VEGF contributes to endothelium dysfunction,which may lead to the occurrence and development of preeclampsia.
ABSTRACT
The effects of leptin on cytotrophoblast proliferation and invasion activity were investigated.Immunohistochemistry was used to determine the placental expression of leptin in first-trimester preg-nancy. By using RT-PCR and quantitative real-time PCR, the expression of leptin in cytotrophoblast and the effect of leptin on cytotrophoblast secretion were detected. The potential of cell proliferation, inva-siveness and migration was assessed by MTT, Transwell invasion assay and migration assay respec-tively when the cytotrophoblast was cultured with different concentrations of leptin. The results showed that: (1) Leptin was distributed diffusely around cell membrane, in cytoplasma, and on nuclear mem-brane of cytotrophoblast; (2) Leptin mRNA was expressed in cytotrophoblast. Ten ng/mL leptin could promote the secretion of cytotrophoblast significantly (P<0.01); (3) After culture with different concen-trations of leptin for 24 h or longer, the proliferation of cytotrophoblast was inhibited, while in 24 h leptin could promote cytotrophoblast invasion and migration. Leptin at a concentration of 500 ng/mL could promote cytotrophoblast invasiveness and migration significantly as compared with controls (P<0.05). It was suggested that leptin could inhibit cytotrophoblast proliferation, and promote cytotro-phoblast invasion and migration activity.