| Background Atherosclerosis(atherosclerosis,AS)is the pathological basis of coronary heart disease.The dysfunction caused by vascular endothelial cell injury is the starting link of AS.At present,oxidized low density lipoprotein(ox-LDL)-induced damaged vascular endothelial cells are often used as cell models to investigate AS.It has been found that galectin-3(Gal-3)promotes the development of AS by regulating inflammatory factors,however,the role of Gal-3 in ox-LDL induced vascular endothelial cell injury remains unclear.Previous studies have shown that the biological behavior of Gal-3 is related to integrinβ1,Rho A and JNK,but it has not been reported in AS.We hypothesize that Gal-3 promotes ox-LDL-induced vascular endothelial cells by mediating inflammation through integrin β1-Rho A-JNK pathway.Our study is divided into two parts:(1)The effects of Gal-3 on ox-LDL-induced vascular endothelial cell injury;(2)The investigation of the signaling pathway of galectin-3 mediated inflammation promoting ox-LDL injury to vascular endothelial cells.Objective To investigate the effects and pathway of Gal-3 on ox-LDL-induced vascular endothelial cell injury.Methods 1.HUVECs were treated with ox-LDL at different concentrations(0,50,100 and 150μg/ml)and different duration(0,4,4 and 12h).The expression of Gal-3 was detected by real-time quantitative PCR and Western blotting(WB).2.Human umbilical vein endothelial cells(HUVECs)were divided into the following 9 groups: A.Control;B.ox-LDL;C.Gal-3;D.Gal-3+ox-LDL;E.control-si RNA+Gal-3+ox-LDL;F.integrin β1-si RNA+Gal-3+ox-LDL;G.Rho A inhibitor+ ox-LDL;H.Rho A inhibitor + Gal-3 + ox-LDL;I.JNK inhibitor + Gal-3 + ox-LDL.3.Effects of Gal-3 on ox-LDL-induced HUVECs: MTT was used to detect cell proliferation and flow cytometry was performed to detect apoptosis in group A,B,C and D;WB and ELISA were used to detect the expression of inflammatory related factors: NF-αB proteins(IKKα,IKKβ,p65)and their phosphorylated proteins(pIKKα,p-IKKβ,p-p65),adhesion molecules(ICAM-1,VCAM-1);proinflammatory factors(IL-6,IL-8,IL-1β)and chemokine(CXCL-1,CCL-2).4.Regulation of integrin β1-Rho A-JNK pathway by Gal-3 in ox-LDL-induced HUVECs 1)The effects of Gal-3 on the expression of integrin β1,Rho A and JNK protein: The expression of integrin β 1,Rho A and JNK protein in group A,B,C and D was detected by immunofluorescence or WB method.2)The effect of integrinβ 1-si RNA on the expression of Rho A and JNK: WB was used to detect the expression of Rho A,GTP-Rho A,JNK and p-JNK in group B,D,E and F.3)The expression of JNK and p-JNK protein in group B,D,G and H was detected by Western blotting.5.Gal-3 affects cell proliferation,apoptosis and expression of inflammatory factors through integrin β1-Rho A-JNK pathway in ox-LDL-induced HUVECs: Relevant indicators of group B,D,E,F and I were detected.1)MTT was used to detect proliferation level;2)Flow cytometry was used to detect apoptosis level;3)WB and ELISA were used to detect the expression of inflammatory related factors(Method 3).Results 1.Gal-3 expression was up-regulated in HUVECs after ox-LDL treatment;Gal-3 could inhibit the proliferation and promote apoptosis of HUVECs treated by oxLDL,and up-regulate the expression of inflammatory related factors(Method 3).2.Gal-3 could up-regulate the expression of integrin β 1,GTP-Rho A and p-JNK in HUVECs treated with ox-LDL.3.After integrin β1-si RNA,the expression of GTP-Rho A and p-JNK decreased;Rho A inhibitor down-regulated the expression of p-JNK,and the up-regulation of p-JNK by Gal-3 was inhibited when combined with Rho A inhibitor.4.Integrin β1-si RNA or JNK inhibitor inhibited the proliferation and inhanced the apoptosis of HUVECs induced by ox-LDL,and inhibit the expression of inflammatory related factors(Method 3).Conclusion 1.Gal-3 can inhibit cell proliferation,increase apoptosis and promote inflammatory response in ox-LDL-induced HUVECs;2.Gal-3 can promote ox-LDL-induced endothelial cell inflammation through integrin β 1-Rho A-JNK pathway and aggravate the cell injury effects. |
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