| Objective:To investigate the role of Intracellular chloride ion channel protein 1(CLIC1)in oxidative damage and inflammation of endothelial cells during atherosclerosis,and to elucidate the molecular mechanism of Tanshinone IIA against atherosclerosis through regulating CLIC1.Methods:1.The apolipoprotein E-deficient(ApoE-/-)mice were randomly divided into ApoE-/-Cont group and ApoE-/-AS group.After 8 weeks of normal diet and high-fat diet respectively,the levels of blood lipids were measured by enzymatic analysis,and the area of atherosclerotic plaque analyzed by H&E staining.After the establishment of atherosclerosis model,the expression of CLIC1 in plaques was observed by immunohistochemistry,while CLIC1 in aorta detected by real-time quantitative PCR and immunoblotting.Subsequently,CLIC1-/-cell lines were cloned by CRISPR/CAS9 double vector lentivirus method to knockout CLIC1 gene in human umbilical vein endothelial cells(HUVECs).For experiment,ECs were divided into control group,H2O2 group,IAA94 group(pretreatment with 40 μM IAA94 for 1 h)and CLIC1-/-group.After treatment with 0.9 mM H2O2 for 12 h,the levels of TNF-α,IL-6 and IL-1β in the supernatant were detected by enzyme-linked immunosorbent assay(ELISA).The content of ROS,MDA and SOD activity were detected in HUVECs and CLIC1-/-cells.ICAM-1,VCAM-1 and CLIC1 expressions were detected by blotting.CLIC1 membrane transfer was measured by Immunofluorescence assay,and the intracellular chloride concentration detected by MQAE method.The role of CLIC1 in oxidative damage and inflammation of endothelial cells was analyzed.The above data were used to analyze the role of CLIC1 in endothelial cell oxidative damage and inflammation.2.ApoE-/-mice were randomly divided into control group,model group,tanshinone IIA sodium sulfonate injection(STS)low dose group and high dose group,6 mice in each group.After 4 weeks of high-fat feeding,STS groups were given the corresponding concentration of drugs(10 or 20 mg/kg/d STS,i.p.).Then after 4 weeks of STS administration,the changes of serum lipids,plaque area and CLIC1 in plaque were observed,meanwhile,changes of oxidative stress and serum inflammatory cytokines as well as expression of ICAM-1,VCAM-1 and CLIC1 were detected,to clarify STS anti-atherosclerotic effect.Subsequently,HUVECs were divided into control group,H2O2 group,LSTS group,MSTS group and HSTS group.After induction with 0.9 mM H2O2,HUVECs were treated with different concentrations of STS(0.3,0.4,0.5 mM)for 24 h,then intracellular oxidative levels,inflammatory cytokine release,and ICAM-1,VCAM-1,CLIC1 expression were detected as well as changes of CLIC1 membrane transfer and intracellular chloride concentration.These above data were used to analyze the role of STS in oxidative damage and inflammation of endothelial cells,and investigate the effect of STS on CLIC1 expression and membrane translocation.3.HUVECs and CLIC1-/-cells were used for experiment,were performed respectively by normal culture,H2O2 injury,MSTS(0.4 mM)administration,then not only intracellular ROS products,MDA content and SOD activity were detected,but also inflammatory factors(TNF-α、IL-6),adhesion molecules(ICAM-1,VCAM-1)and CLIC1 measured.What is more,CLIC1 membrane transfer and intracellular chloride concentration were also measured.These above data were used to analyze the role of STS on oxidative damage and inflammation in CLIC1-/-cells,and explore the role of CLIC1 in STS anti-oxidation and anti-inflammatory,to further study the relationship between CLIC1 membrane translocation and STS anti-atherosclerosis mechanism.Results:1.Compared with the control group,the body weight of AS mice was significantly increased(P<0.05),the blood lipid level obviously increased(P<0.05),the area of aortic plaques and CLIC1 positive staining in plaques significantly increased(P<0.01),as well as CLICl mRNA and protein expression were clearly increased(P<0.01)in aorta.At the same time,the levels of ROS and MDA in HUVECs treated with H2O2 were significantly higher than those in the control group(P<0.01),as well as inflammatory factors(TNF-α,IL-6,IL-1β)and adhesion molecules(ICAM-1,VCAM)(P<0.05).But compared with H2O2 group,both the oxidation products and inflammatory factors and adhesion molecules were significantly decreased(P<0.05),and the activity of SOD increased(P<0.05)after IAA94 pretreatment and CLIC1 gene knockdown.After H2O2 stimulation,intracellular CLIC1 expression was upregulated,immunofluorescence results showed obvious membrane transloction,and Western Blot revealed CLIC1 in the cell membrane was increased.MQAE results showed that the stronger intracellular fluorescence intensity meaned the lower chloride ion concentration,H2O2 treatment increased the chloride ion concentration(P<0.05),while CLIC1 inhibited,CLIC1 membrane transfer was decreased,chloride ion concentration lower.2.Compared with the model group,the body weight and blood lipid level of STS groups were significantly lower(P<0.05),the area of aortic plaque decreased,and the activity of SOD,GSH increased and productions of MDA and GSSG were obviously decreased(P<0.05)as well as serum levels of TNF-α,IL-6 and expression of ICAM-1 and VCAM-1 in aorta(P<0.05),and the expression of CLIC1 both in the plaque and aorta was decreased(P<0.05).Similarly,after STS treatment,the contents of ROS and MDA in HUVECs were significantly lower than those in H2O2 group,while the activity of SOD increased in the HUVECs,and there was a dose-dependent relationship(P<0.05).Release of TNF-α,IL-6(P<0.05)and expression of ICAM-1,VCAM-1 protein were decreased significantly(P<0.05),as well as the expression of intracellular CLIC1 and chloride ion concentration(P<0.05).3.Compared with HUVECs,the levels of ROS,MDA and TNF-α,IL-6 and ICAM-1,VCAM-1 in CLIC1-/-cells were lower.Compared with HUVECs treated with H2O2,the oxidative index,inflammatory factors and adhesion molecules were significantly decreased in H2O2-treated CLIC1-/-cells(P<0.01).There was no significant difference in ROS,MDA content and SOD activity between H2O2-treated CLIC1-/-cells and CLIC1-/-control cells,while TNF-α,IL-6 and ICAM-1 were significantly increased(P<0.05),VCAM-1 decreased.What is more,After MSTS treatment in CLIC1-/-cells,expression of TNF-α,ICAM-1 and VCAM-1 was decreased significantly(P<0.05)compared with H2O2-treated CLIC1-/-cells,but no significant changes observed in IL-6.Conclusions:1.The expression of CLIC1 mRNA and protein was increased in atherosclerotic mice.2.After induced by H2O2,oxidative stress and inflammation were observed in HUVECs,and over-expression of CLIC1 was also found,there was a tendency of CLIC1 membrane transfer.After inhibiting CLIC1,oxidative injury and inflammatory response were decreased,indicating CLIC1 was associated with endothelial cell oxidative damage and inflammation3.STS could reduce endothelial cell oxidative damage and inflammatory response by down-regulating CLIC1 expression and membrane transfer,so as to play an anti-atherosclerotic role. |
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