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[Objective] To investigate the effects and mechanisms of Nrf2-ARE (nuclear factor erythroid-2 related factor-anti-oxidant response element) pathway on uremic serum-mediated endothelial dysfunction in human aortic endothelial ceils.[Methods] Human aortic endothelial cells were incubated in endothelial cell medium containing 10％ normal serum,10％ non-diabetic nuremic serum or 10％ diabetic uremic serum respectively,and 20 μmol/L tertiary butyl hydroquinone (tBHQ) were pretreated with cells to active Nrf2-ARE pathway.The cells apoptosis rate were measured by flow cytometry,and the synthesis of NO was detected by flow cytometry and immune fluorescent confocal,while the expression of P-eNOSer1177/eNOS,and quinone oxidoreductase-1 (NQO1) were measured by western blotting.The levels of malondialdehyde,superoxide dismutase,eatalase,and glutathione in these cells were also measured with kits.[Results] Aortic endothelial cells incubated with uremic serum had a higher level of apoptosis rate and MDA (P ＜ 0.05),and a lower level of NO systhesis,P-eNOSSer1177/eNOS expression,CAT,SOD,GSH (P ＜ 0.05).Pretreated with tBHQ can reduce the apoptosis rate and MDA level (P ＜ 0.05),improve the amount of NO systhesis,the expression of P-eNOSSer1177/eNOS,the levels of CAT,SOD,and GSH in these cells (P ＜ 0.05).[Conclusion] Activation of Nrf2-ARE pathway can improve endothelial dysfunction in aortic endothelial cells induced by uremic serum,and its mechanism might be related with enhancement of the antioxidant stress.
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[ ABSTRACT] AIM: To investigate whether L-carnitine ( LC) inhibits the eryptosis effect of uremic serum on erythrocytes.METHODS:Erythrocyte suspension (2%) was cultured and divided into 3 groups in vitro: control group ( C group) , uremic serum group ( U group, 30%uremic serum) , and uremic serum+LC group ( L group, 30%uremic serum+200 μmol/L LC) .Erythrocytes were collected at 24 h and 48 h.Eryptosis ( phosphatidylserine expression repre-sents eryptosis) was estimated by flow cytometry with Annexin V staining.The content of reactive oxygen species ( ROS) was also detected.Glutathione ( GSH) was measured by ELISA.RESULTS:Eryptosis in C group was increased as the in-cubating time extended.Eryptosis in U group was higher than that in C group, while that in L group was lower than that in U group.Meanwhile, ROS content was higher and GSH was lower in U group than those in C group.ROS content was low-er and GSH was higher in L group than those in C group.CONCLUSION:LC inhibits uremic serum-induced eryptosis by decreasing ROS and increasing GSH, thus attenuating oxidative stress.
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Aim To investigate the effects of Panax Notoginsenosides(PNS) on the gene expression and protein excretion of TGF-?_1 and CTGF of human renal tubular epithelial cell induced by uremic serum in vitro.Methods Forty sera from CRF patients and twenty sera from healthy volunteers were collected,gently mixed,inactived and separated respectively in steriled condition.HK-2 Cells were cultured in RPMI-1640 medium with 10% newborn calves serum and subcultured routinely.They were differentiated by phase contrast microscope and scanning electron microscope detection and cytokeratin18(CK-18) immunohistochemistry method.The protein levels of TGF-?_1 were examined by enzyme-linked immunoadsordent assay(ELISA).The protein levels of CTGF were examined by Western Bloting assay.The gene expression of TGF-?_1 and CTGF was detected by Semi-quantitative reverse trainscriptase polymerase chain reaction(RT-PCR).Results TGF-?_1 and CTGF gene expression and protein level were increased in 10% uremic serum groups compared with that of normal control group(P
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AIM: To explore the effects of uremic serum on proliferation and trans-differentiation of human renal tubular epithelial cells. METHODS: Human renal tubular epithelial cell line (HK-2) was cultured in RPMI-1640 medium. The proliferation effects of uremic serum at different concentrations were evaluated by methylene blue assay (MTT method) and flow cytometry. The positive cells percentage of ?-smooth muscle actin(?-SMA)in different concentration uremic serum medium was also measured by flow cytometry in vitro. RESULTS: Absorbance 490 (A 490) was increased in 5%-20% uremic serum groups compared with that in normal controls with the use of MTT. Cells in G 1 phase were decreased, but proliferation index (PI) was increased in 10%-20% uremic serum groups compared with that in normal controls with the use of flow cytometry. No significant difference of cell proliferation index was found among uremic serum groups. The positive percentage of ?-SMA cells was increased significantly in uremic serum groups compared with that in normal controls, and increased in parallel with the increasing of uremic serum concentration. CONCLUSION: Uremic toxin may accelerate renal tubulointerstitial fibrosis through promoting renal tubular epithelial cell proliferation and trans-differentiation in patients with chronic renal failure.