| Objective:To observe the effects of periostin on the related indexes of reverse cholesterol transport induced by angiotensinⅡ（AngⅡ）in atherosclerosis,and to explore whether periostin promotes atherosclerosis by regulating the reverse cholesterol transport induced by AngⅡ.Methods:1.To observe the changes of periostin、ABCA1、ABCG1 and LXRαprotein expression levels after different concentrations of AngⅡintervention foam cells:primary peritoneal macrophages of C57 male mice were cultured in vitro and induced into foam cells by 50mg/L oxidized low density lipoprotein（Ox-LDL）.The foam cell model was identified by oil red O staining.Foam cells were randomly divided into five groups and treated with different concentrations of AngⅡ:0mmol/L,10-5mmol/L,10-4mmol/L,10-3mmol/L,10-2mmol/L for24 hours.The lipid content of foam cells was measured by oil red O staining.The expression levels of periostin,ABCA1,ABCG1 and LXRαin foam cells were measured by Western blotting.2.To observe the effects of silencing periostin gene on the protein expression levels of ABCA1,ABCG1 and LXRαin foam cells:foam cells were randomly divided into 6 groups:（1）Blank group:no treatment,（2）AngⅡgroup:no transfection,only treated with optimal concentration of AngⅡfor 24 hours.（3）LV3-NC group:48 hours after LV3 empty vector lentivirus transfection,without AngⅡ;（4）LV3-NC+AngⅡgroup:48 hours after LV3 empty vector lentivirus transfection,followed by AngⅡintervention for 24 hours;（5）LV3 group:48 hours after LV3 silencing lentivirus transfection,without AngⅡ;（6）LV3+AngⅡgroup:48 hours after LV3 silencing lentivirus,followed by AngⅡintervention for 24 hours.The transfection effect was evaluated by fluorescence inverted phase contrast microscope.The lipid content of foam cells was measured by oil red O staining and the expression of periostin,ABCA1,ABCG1 and LXRαwas measured by Western blotting.3.The effects of periostin gene on the protein levels of ABCA1、ABCG1、LXRαin liver tissue were observed in animal experiments:after 8 weeks of high fat diet,male Apo E—/<sub>mice aged 6-8 weeks were randomly divided into different groups:（1）Blank group:no treatment;（2）AngⅡgroup:tail vein injection of normal saline,subcutaneous implantation of osmotic mini pump containing AngⅡ（1000ng/kg/min）;（3）LV3-NC+AngⅡgroup:tail vein injection of LV3 empty vector lentivirus,subcutaneous implantation of osmotic mini pump containing AngⅡ.（4）LV3+AngⅡgroup:LV3 was injected into tail vein to silence lentivirus and subcutaneously implanted into osmotic mini pump containing AngⅡ;（5）LV5-NC+AngⅡgroup:LV5 empty vector lentivirus was injected into tail vein and osmotic mini pump containing AngⅡwas implanted subcutaneously;（6）LV5+AngⅡgroup:LV5overexpressed lentivirus was injected into tail vein and osmotic mini pump containing AngⅡwas implanted subcutaneously.The mice were kept on a high-fat diet for 4 weeks and then killed.Western blotting to determine the expression of periostin,ABCA1,ABCG1 and LXRαin liver tissue.Results:1.AngⅡpromoted the expression of periostin in foam cells and inhibited the reverse cholesterol transport in foam cells:（1）at the same time（24h）,the higher the concentration of AngⅡ,the more obvious the lipid deposition of foam cells（P<0.05）.（2）the expression level of periostin in foam cells increased with the increase of AngⅡconcentration at the same time （24h）.When the concentration of AngⅡwas 10-3mmol/L,the expression level of periostin in foam cells was the highest （1.765±0.153,P<0.05）.（3）at the same time （24h）,the concentration of AngⅡwas inversely proportional to the expression of ABCA1,ABCG1 and LXRα（P<0.05）.2.Silencing periostin gene could promote the reverse cholesterol transport induced by AngⅡand reduce lipid deposition in foam cells:（1）After silencing periostin gene,the lipid deposition degree of foam cells in empty carrier group has no significant difference compared with blank group（P>0.05）,but the lipid deposition in the silent group was lower than that in the empty vector group（P<0.05）.（2）After silencing periostin gene,there was no significant difference in the expression of periositn between the blank group and the empty vector group（P>0.05）.The expression level of periostin protein in foam cells was lower than that in the empty vector group（P<0.05）.（3）After silencing periostin gene,there was no significant difference in the expression of ABCA1,ABCG1 and LXRαbetween the blank group and the empty vector group（P>0.05）.The expression of ABCA1,ABCG1 and LXRαin foam cells induced by AngⅡin the silent group was higher than that in the non-transfection group and the transfection group（P<0.05）.3.Periostin is involved in the reverse cholesterol transport induced by AngⅡin Apo E—/<sub>mice:（1）Compared with the blank group,the expression of periostin in AngⅡgroup increased,while the expression of ABCA1,ABCG1,LXRαdecreased in AngⅡgroup（P<0.05）.Compared with AngⅡgroup,the expression levels of periostin,ABCA1,ABCG1 and LXRαin LV3-NC+AngⅡgroup and LV5-NC+AngⅡgroup had no significant difference（P>0.05）;（3）Silencing periostin gene could promote the expression of ABCA1,ABCG1 and LXRαcompared with LV3-NC+AngⅡgroup（P<0.05）;（4）Compared with LV5-NC+AngⅡgroup,overexpression of periostin could inhibit the expression of ABCA1,ABCG1 and LXRα（P<0.05）.Conclusion:1.AngⅡcould promote the expression of periostin in foam cells,which is proportional to the concentration of AngⅡ.AngⅡcould promote lipid deposition and inhibit the expression of cholesterol reverse transport related proteins ABCA1,ABCG1 and LXRαin foam cells.2.Silencing periostin gene could promote the expression of cholesterol reverse transport related proteins ABCA1,ABCG1 and LXRαin foam cells,and reduce lipid deposition.3.Silencing periostin gene could promote the expression of ABCA1,ABCG1 and LXRαin atherosclerotic mice induced by AngⅡ,while overexpression of periostin gene could inhibit the expression of ABCA1,ABCG1 and LXRα.4.AngⅡcan regulate RCT through periostin-LXRα-ABCA1/ABCG1 pathways,thus accelerate the progression of AS. |
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