Ursodeoxycholic acid in serum and liver tissue in patients with end-stage cholestatic liver cirrhosis
Yoshihiro KitaHiroo SakakuraM. HirataYasushi HariharaHiroko TanakaMitsuhiro ItoHiroyuki YoshinoToshio TakayamaKiyoshi KubotaKohei HashizumeMasatoshi Makuuchi
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Ursodeoxycholic acid
Objective
Cellular senescence of endothelial cells has been proposed for its involvement in endothelial dysfunction and atherogenesis. This study investigates the effects of ginsenoside Rb1, a major constituent of ginseng, on H2O2-induced endothelial senescence and molecular changes in primary human umbilical vein endothelial cells.Methods
Prematurely senescent human umbilical vein endothelial cells (HUVECs) were induced by treatment with H2O2 as judged by senescence-associated β-galactosidase assay (SA-β-gal), cell morphological appearance, and plasminogen activator inhibitor-1 (PAI-1. expression. Total nitric oxide (NO) production was measured using Griess reaction. Endothelial NOS (eNOS), PAI RNA expressions were analysed by real time PCR. Total eNOs, pS1177 eNOS and pT495 eNOS protein expressions were analysed by westernblotting.Results
Treatment with 40∼100μM H2O2 caused 26.8∼63.8% of the cells to be SA-β-gal positive. Pretreated with Rb1 markedly inhibited SA-β-gal activity dose-dependently. Also, Rb1 can reduce the expression of PAI which was increased in the H2O2 treated group. In H2O2 treated groups, eNOS mRNA expression decreased, while Rb1 can effectively restore its mRNA expression. eNOS activity of HUVECs was inhibited by decreasing eNOS phosphorylation at Ser-1177 and increasing eNOS phosphorylation at Thr-495 in H2O2 treated groups. While in Rb1 pretreated groups, the both exhibited opposite changes. Consistent with these findings, Rb1 does in fact increase NO levels. All the inhibitory effects of Rb1 on senescence were completely obliterated by L-NAME, the NOS inhibitor.Conclusion
Rb1 can effectively protect HUVEC from senescence through modulating the expression of eNOS.Senescence
Endothelial Dysfunction
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Objective To investigate the effects of advanced glycation end products (AGEs) on activity and expression of endothelial nitric oxide synthase in cultured human umbilical vein endothelial cells (HUVECs). Methods AGE-modified bovein serum albumin (AGE-BSA) was prepared by incubated BSA with glucose at 37?℃ in vitro. HUVECs were co-incubated with different concentrations of AGE-BSA for 3, 6, 12, 24 and 48 hours respectively. The activity of eNOS is determined by the convertion of radiolabelled 3H-L-arginine to 3H-L-citrulline and eNOS expression levels were measured by Western blotting method. Results The activity of eNOS in HUVECs was partially activated under basic condition, and it was markedly increased by histamine (P0.001). AGE-BSA significantly inhibited the activity of eNOS in a concentration and time-dependent manner. The expression of eNOS had a significant reduction when HUVECs were incubated with AGE-BSA (200?mg·L -1) for 24 hours (P0.001)and a further decrease (P0.05, 48?h vs 24?h) for 48 hours. Conclusions AGE-BSA significntly inhibit the activity of eNOS,and this inhibited effect may be partially due to reduced eNOS expression.
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Objective To investigate the effect of uric acid(UA) on the expression of endothelial nitric oxide synthase(eNOS) and the production of nitric oxide in cultured human umbilical vein endothelial cells(HUVECs).Methods HUVECs were incubated with different concentrations of UA(0,0.5,1,1.5,2 mg/L) and 50 mg/L ox-LDL(as positive control) for 24,48 and 72 h.Then,eNOS mRNA was detected by RT-PCR.The expression of eNOS protein was observed by Western blot.NO in supernatant medium was analyzed by enzymic method.ResultsUA 5 mg/dl group increased the expression of eNOS mRNA of HUVECs as compared with that from placebo control group(P0.05).While the concentration of UA increasing and time runing,the expression of eNOS and the production of NO and ox-LDL decreased significantly(72 h NO2-/NO3-is 0.52±0.18 versus 1.00±0.1 for 2 mg/L versus control,P0.05).Conclusion UA inhibits the expression of eNOS and the production of NO of HUVECs in a dose-dependent and time-dependent manner.It suggests that UA may damage the function of endothelial cells and may influence the genesis and development of cardiovascular diseases.
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Objective To evaluate the effects of PGE_1 on Production of No and the activity of eNOS in human umbilical vein endothelial cells(HUVECs).Methods Nitric oxide(NO) production and the activity of nitric oxide synthase(NOS) in HUVECs were measured by No and NOS assay kits,after HUVECs incubated with PGE_1 alone at different concentrations(0.04-0.4 μg/ml) and different times(2-4h) or HUVECs incubated with PGE_1 as pretreatment before TNF-α 10ng/ml.Results(1) Activity of eNOS and the expression of NO were up-regulated by PGE_1 in concentration-dependent manner.(2) Short time intervention with PGE_1 had little effects on both activity of eNOS and the expression of NO.However,the expression of NO was increased after 12h incubation and the activity of eNOS was enhanced after 24h treatment.(3) PGE_1 could prevent eNOS activity inhibition induced by TNF-α.Conclusions PGE_1 has a protection effect on endothelial cells by enhancing eNOS activity and increasing NO production,besides,it could prevent eNOS activity inhibition induced by TNF-α.
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Objective:To investigate the effects of C-peptide on expression of endothelium nitric oxide synthase(eNOS)by cultured human umbilical vein endothelial cells(HUVEC)in hyperglycemia.Methods:HUVEC were incubated at different concentrations of glucose and C-peptide for72hours.Using semi-quantitative RT-PCR techniques,the expression of eNOS mRNA in HUVEC was measured.Results:In physiologic concentration of glucose(5.5mmol/L),low concentration of C-peptide(0.3nmol/L)reduced the expression of eNOS mRNA,and physiologic(3nmol/L)and high(30nmol/L)concentration of C-peptide could increase it(P0.01).In high concentration of glucose(33.3mmol/L),the expression of eNOS mRNA was decreased(P0.01),and the expression of eNOS mRNA increased after added physiological and high concentration of C-peptide(P0.01).Conclusion:C-peptide can affect the expression of eNOS mRNA in HUVEC.Physiological and high concentration of C-peptide can reduce the inhibition of high glucose on exˉpression of eNOS in HUVEC.This suggests that C-peptide can be important on the treatment of diabetic macrovascular diseases.
C-peptide
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Objective To investigate the effect of TNF-α on production of nitric oxide (NO) and activity of nitric oxide synthase(eNOS) in human umbilical vein endothelial cells(HUVECs). Methods HUVECs were incubated with TNF-α at different concentrations (2-20ng/ml) and different times(2-48h) or pretreatment with L-NMMA or DXM. Nitric oxide production and activity of nitric oxide synthase in HUVECs were measured by NO and NOS assay kits. Results (1) Activity of eNOS was down-regulated by TNF-α in a concentration-dependent manner, while production of NO was upregulated. (2) The activity of eNOS attenuated after long-time treatment with TNF-α, but marked increased production of NO was detected after 24h. (3) The induction of NO by TNF-α can be inhibited by L-NMMA and DXM. Conclusion TNF-α can reduce activity of eNOS and induce iNOS, so that production of NO is increased.
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AIM: To observe the direct effect of LPS on expressions of ET-1, eNOS, and iNOS mRNA in human umbilical vein endothelial cells, and further research the molecular mechanism of effect of LPS on production of ET-1 and NO. METHODS: The third passage of cultured human umbilical vein endothelial cells was incubated with low concentration (100 μg/L) of LPS for 6 hours. Total RNA was extracted. The expressions of ET-1, eNOS, and iNOS mRNA were analyzed by semi-quantitative RT-PCR method. RESULTS: ET-1 mRNA experession increased significantly, while expression of eNOS mRNA decreased significantly, and there was no significant change in expression of iNOS mRNA. CONCLUSIONS: In human umbilical vein endothelial cells, low concentration of LPS enhanced the expression of ET-1 mRNA, inhibited the expression of eNOS mRNA, and had no significant effect on the expression of iNOS mRNA.
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Objective: To discuss the effects of hyperglycemia on human umbilical vein endothelial cell (HUVEC) apoptosis and analyze the relationship between apoptosis and expressions of bcl-x, bax and eNOS. Methods: HUVECs were incubated at different concentration of glucose for 72 hours, and total RNA was extracted. The expressions of bcl-x, bax and eNOS were measured by SQ-RT-PCR technique. The nature of endo thelial cells apoptosis was determined by AO/EB straining. Results: Hyperglycemia increased HUVEC apoptosis (P 0.01) and decreased the expressions of eNOS and bcl-x. There was a negative correlation between HUVEC apoptosis and the eNOS expression. Conclusion: Hyperglycemia can induce ECs apoptosis in DM, and it may be related to the decrease of eNOS and bcl-x expressions.
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The activity of eNOS was singificantly depressed by a high concentration of glucose in a concentration-and time-dependent manners after incubation of HUVECs with different concentrations of glucose and with high glucose plus insulin for different times.The physiological concentration of insulin can partially reverse the inhibitions of the activity and expression of eNOS induced by high concentration of glucose.
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Objective To investigate the effects of 2,3,5 4'-tetrahydroxystilbene-2-O-β-D-glucoside(THSG) on content of nitric oxide(NO) and activity of endothelial nitric oxide synthase(eNOS) and the mechanisms.Methods The human umbilical vein endothelial cells(HUVECs) were cultured and divided into four groups:the control,THSG(1,10,100 μmol·L-1)groups.Spectrophotometry endothelium assay was used to measure NO and activity of eNOS.RT-PCR was used to analyze the eNOS mRNA expression and Western Blot was used to analyze eNOS protein expression level in HUVECs.Results THSG significantly increased the production of NO,activated NOS and up-regulated expression of eNOS mRNA and protein in a concentration-dependent manner compared with the control group.Conclusion THSG raises expression of eNOS mRNA and protein,which may result in activating NO production and relaxing blood vessels.
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