| Objective:To establish endothelial activation and dysfunction model and to explore the protective effect of AGM on lipopolysaccharide(LPS)-induced Human umbilical vein endothelial cells(HUVECs).Methods:1.HUVECs were cultured in vitro.The effect of LPS on survival rate of HUVECs was measured by MTT method.The median lethal dose and the optimal time of HUVECs were investigated according to LD50.HUVECs were intervened with AGM(0,0.125,0.25,0.5,1,2 and 4 mmol/L)for 24 h,and the cell viability was measured by MTT assay.The cell survival rate was calculated according to the optical density at the wavelength of 490 nm.LPS(10 μg/mL)-induced cell viability,treated with AGM(0,0.25,0.5,1 mmol/L)for 24 h,was detected by MTT assay.2.The cells were divided into several groups:control group,LPS group,and AGM treated group:LPS+AGM(0.125,0.5,and 1 mmol/L).To insure the optimal concentration of AGM(1mmol/L),the levels of soluble vascular cell adhesion molecule 1(sVCAM-1),soluble E-selectin(sE-selectin)soluble intercellular adhesion molecule 1(sICAM-1),and monocyte hemoattractant protein 1(MCP-1)at 24 h in the supernatant were detected by ELISA assay.And the levels of interleukin(IL)-6 and 1β at 24 h in the supernatant were detected by ELISA assay.3.The cells were randomly divided into four groups:control,LPS(10μg/mL),AGM(1 mmol/L)and LPS+AGM,cultured for 1,4,6,12,24 h.The HUVECs were pretreated with 10 μgmol/L NF-κB inhibitor(PDTC),10μmol/L p38 inhibitor(SB203580),10 μmol/L ERK inhibitor(PD98059)for 1h prior to LPS stimulation.The levels of reactive oxygen species(ROS)in cells were measured using 2,7-dichlorofluoresce in diacetate(DCFH-DA)method,and the fluorescence levels of ROS were observed with fluorescence inverse microscope.The levels of sVCAM-1,sICAM-1,sE-selectin,MCP-1 IL-6 and IL-1β at 24 h in the supernatant were detected by ELISA assay.The mRNA expressions of IL-6,IL-1β,VCAM-1,ICAM-1,E-selectin,MCP-1 and heme oxygenase-1(HO-1),NAD(P)H:quinone oxidoreductase 1(NQO-1)were investigated by quantitative real-time PCR.The expression levels of ICAM-1,VCAM-1,phospho-p65(p-p65),p65,p-IκBα,IκBα,p-ERK,ERK,p-p38,p38,p-JNK,JNK,Nrf2,p-Akt,Akt were assessed using western blotting analyses.Results:1.According to the principle of LD50,to contrast HUVECs activation and dysfunction model,the optimal dose and the time of LPS were screened for 10 μg/mL.2.The MTT assay results showed that the cell viability was inhibited by 2 and 4mmol/L AGM,and the cell survival rates of them were lower than of normal group(P<0.05)to 78.9±3.2%and 62.8±4.3%,respectively.Treated with 10 μg/mL LPS,the cell viability was decreased to 53±4.6%,having a significance compared with the normal group(P<0.05).Trend of cell survival rate of AGM intervention group approximately agreed with the AGM concentration and there was a positive relationship between them.Compared with LPS group,0.125,0.5,and 1 mmol/L AGM significantly facilitated the survival rate to 73.8±3.2%,78.7±3.1%,and 88.3±2.1%,and there was a statistically significant difference(P<0.05).But the protective effect of 0.125 mmol/L AGM was not obvious.3.ELISA method results showed that 1 mmol/L AGM remarkably decreased sVCAM-1,sICAM-1,sE-selectin,MCP-1,IL-6,and IL-1β at 24 h in the supernatant(P<0.05).And the trend of above molecules concentrations approximately agreed with the AGM concentrations and there was a positive relationship between them.Hence the optimal concentration of AGM was 1 mmol/L.4.The results of flow cytometry showed that the average fluorescence intensity of ROS in LPS group at 24 h was significantly lower than of AGM intervention group(P<0.05).The fluorescence intensity of LPS group was increased observed using fluorescence microscope and the fluorescence of AGM intervention group was obviously darkened.5.qRT-PCR assay results showed that the mRNA expression of IL-6 and IL-1β of LPS group was significantly decreased at 4 h compared with normal group(P<0.05),and the mRNA expression of ICAM-1,VCAM-1 and E-selectin of LPS group were significantly decreased at 6 h compared with normal group(P<0.05),and the mRNA expression of MCP-1 of LPS group were significantly decreased at 12 h compared with normal group(P<0.05).After AGM intervention mRNA expression of above molecules was significantly decreased(P<0.05).6.After LPS stimulation for 6 h,the HO-1 and NQO-1 mRNA expression were no significantly changed.The mRNA expression of HO-1 significantly increased after AGM intervention compared with LPS group(P<0.05).However,NQO-1 mRNA expression was unchanged.7.The results of ELISA test showed that after 10 μmol/L PDTC,10μmol/L PD98059,10 μmol/L SB203580 individually or jointly function for 1 h,sVCAM-1,sICAM-1,sE-selectin,MCP-1,IL-6 and IL-1β at 24 h in the supernatant of LPS group significantly increased than of control group(P<0.05).The levels of above molecules were decreased by different degrees,especially in PDTC group(P<0.05)and PD98059 combination with SB203580 group(P<0.05).8.NF-κB signaling pathway was activated at 24 h after the stimulation of LPS,manifested as phosphorylation of p65 in the nucleus and IκBα in the cytoplasm and inhibition of IκBα expression in the cytoplasm(P<0.05).After AGM intervention,the level of p65 phosphorylation in the nucleus decreased and the expression of IκBα in the cytoplasm increased(P<0.05).Phosphorylation of p38,ERK1/2 and JNK,the members of MAPK family,increased 1 h after the stimulation of LPS,while AGM mainly inhibited phosphorylation of p38 and ERK1/2 and had no obvious effect on JNK.9.The results of gray value showed that after LPS stimulation for 24 h,the level of Nrf2 in cytoplasm was remarkably increased(P<0.05),and there was no change in AGM group and AGM intervention group.In AGM intervention group,the level of Nrf2 in nucleus was remarkably increased compared with LPS group.The phosphoralytion expression of Akt in AGM intervention group was significantly higher than that in LPS group,simultaneouslyConclusion:AGM can effectively inhibit the excessive activation and dysfunction of LPS-induced endothelial cells via improving the cell viability,suppressing the production of IL-1β,IL-6,ICAM-1,VCAM-1,E-selectin and MCP-1,and reducing intracellular ROS accumulation to alleviate the oxidative damage.Its mechanisms may be by inhibiting activation of p38 phosphorylation and its downstream NF-κB signaling pathway,and activating Akt phosphorylation to promote Nrf2 transportation from cytoplasm to nucleus and then to increase its downstream antioxidant enzyme HO-1 expression to produce anti-inflammatory,anti-adhesion and antioxidant effects. |
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