Effects Of Ezrin Knockdown On Ox-LDL And Angiotensin Ⅱ-induced Calpain Activation,ABCA1 Degradation And Intracellular Cholesterol Contents

Objective:The cytoskeleton protein Ezrin,a member of ERM,is a ubiquitous protein.Studies have shown that Ezrin is closely related to the activity of calpain which can mediate the degradation of ABCA1 protein,a central protein in reverse Cholesterol transport.It is not clear whether Ezrin plays a role in the formation of atherosclerosis.The aim of this study was to investigate the expression of Ezrin gene in atherosclerosis,and then to study the effect of down regulation of Ezrin expression on the Calpain-1 activity of macrophages,the expression of ABCA1 protein and the effect of intracellular cholesterol content on the intervention of atherosclerotic factors in order to clarify the role of Ezrin in the formation of atherosclerosis.Methods: 1.The atherosclerosis model was established in ApoE-/-mice fed with a high-fat diet and confirmed by HE staining.Immunofluorescence and immunohistochemistry were used to detect the expression of Ezrin at the plaque.Effects of different concentrations and times of Ox-LDL and Ang Ⅱ on Ezrin gene expression in RAW264.7 cells were tested by Western blotting.2.RAW264.7 cells transfected with small interfering RNA(siRNA)targeting Ezrin were subjected to real time PCR and Western blot to detect the inhibition ratio.And then,to examine the effect of Ezrin siRNA on the expression of Ezrin induced by Ox-LDL and Ang Ⅱ.3.Western blotting was used to detect different degradation fragments of calpain protein in RAW264.7 cells after knockdown of Ezrin gene and analyze the calpain activity.4.Different concentrations of Ox-LDL acted on RAW264.7 macrophages to observe the formation of foam cells.The expression of ABCA1 protein was examined by Western blot after the treatment with Ezrin and Calpain-1.5.Furthermore,the effect of Ezrin gene expression change on intracellular cholesterol concentration detected by enzyme-linked high performance liquid chromatography(HPLC).Results:1.The atherosclerotic plaque was found in the aorta of high-fat diet group.The immunofluorescence results showed that the expression of Ezrin in plaque was higher than that in the normal diet group(P<0.05).Western blotting showed that both Ox-LDL and Ang Ⅱcould up-regulate Ezrin gene protein level in RAW264.7 cells(P<0.05),and showed a significant time and concentration dependence.2.Ezrin mRNA(0.25±0.29,0.26±0.40)and protein expression(0.19±0.28,0.17±0.12)in Ezrin siRNA groups were inhibited(P<0.05).3.Western blotting showed that Ox-LDL and Ang Ⅱ could up-regulate the activity of Calpain-1(P<0.05).However,Ezrin siRNA reversed both results(P<0.05).4.The results of oil red O staining of RAW264.7 cells showed that with the increasing of Ox-LDL concentration,lipid particles in foam cells showd a gradually increasing phenomenon.The results of Western blotting showed Ox-LDL up-regulated ABCA1 protein expression,compared with Ox-LDL group ABCA1 protein expression in Ox-LDL + Ezrin siRNA group and Ox-LDL + ALLN group were up-regulated.In Ox-LDL + Ezrin siRNA + ALLN group ABCA1 protein expression was further up-regulated(P<0.05).And ABCA1 protein expression was down-regulated after Ang Ⅱ treatment(P<0.05),which was reversed by Ezrin siRNA(P <0.05),while the inhibition of calpains inhibitor ALLN failed to further decrease the ABCA1 protein level(P>0.05).5.The intracellular cholesterol concentrations revealed that TC,FC,CE and CE/TC were up-regulated in Ox-LDL group compared with the control group(P<0.05),and the TC,FC,CE and CE/TC ratios of Ox-LDL+ siRNA group also increased.However,compared with Ox-LDL group,the concentrations of TC,FC and CE were down-regulated(P<0.05),and there was no significant difference in CE/TC ratio(P>0.05).Compared with the control group,TC and CE were up-regulated in Ang Ⅱgroup(P<0.05),and CE was also increased in Ang Ⅱ+siRNA group.TC,FC,and CE were down-regulated in Ang Ⅱ+siRNA group compared with Ang Ⅱgroup.(P<0.05).Conclusion: The expression of Ezrin was elevated in atherosclerotic plaques and Ox-LDL,Ang Ⅱ interventions.Ezrin knockdown reduced calpain activity,inhibited ABCA1 degradation and decreased intracellular cholesterol content.The results suggest that high expression of Ezrin in atherosclerotic plaque may be involved in cholesterol metabolism and atherosclerotic plaque formation through the calpain/ABCA1 pathway.Ezrin may be An important therapeutic target for anti-atherosclerotic plaques.