Study On The Mechanism Of Periostin On AngⅡ Mediated Cholesterol Reversal In ApoE^-/- Mice

Objective:Previous studies have confirmed that angiotensin II（AngⅡ）may promote atherosclerosis by up-regulating periostin protein expression and inhibiting cholesterol reversal transport.This study aims to explore the regulatory mechanism of periostin on RCT in AngⅡ-mediated Apo E^-/-mouse AS model,and to provide experimental evidence,so as to provide a new potential target for AS prevention and treatment.Methods:（1）A total of 50 Apo E^-/-male mice with C57BL/6J genetic background aged 6-8weeks were selected.They were divided into ordinary diet group（ND group）,high fat diet group（HFD group）,high fat diet+AngⅡ group（HFD+AngⅡ group）,periostin silence group（HFD+Periostin-Mus-769+AngⅡ group）,and periostin over-expression group（HFD+Periostin-mus+AngⅡ group）,10 animals per group,were fed in SPF grade animal house for 12 weeks.（2）After 12 weeks of feeding on ordinary diet or high-fat diet,mice in HFD+AngⅡ group were injected with normal saline through tail vein,and mice in HFD+Periostin-Mus-769+AngⅡ group were injected with Periostin-Mus-769 recombinant lentivirus to inhibit the expression of Periostin.Mice in HFD+Periostin-mus+AngⅡ group were injected with the Periostin-mus recombinant lentivirus to enhance the expression of Periostin in the tail vein.24-48h later,mice in the above 3 groups were injected with AngⅡ continuously with micropump implanted subcutaneously at the back of the neck.To study the effect of periostin on AngⅡ-mediated Apo E^-/-mice AS lesions,and further explore the mechanism of effect of periostin on AS.（3）Normal saline or lentivirus was injected into the tail vein,micropump was inserted subcutaneously,and the mice were killed after 4 weeks of feeding.Blood samples were collected and frozen in a-20℃refrigerator.The aorta tissues were sent to the Department of Pathology,the Second Hospital of Shanxi Medical University for pathological sections and oil red O staining.Liver tissues were stored in liquid nitrogen tank for further Western-blotting and RT-PCR.（4）Oil red O staining of aortic tissue was observed to evaluate the formation of atherosclerotic plaques and the status of vascular lesions.（5）The contents of TC,TG,HDL-C and LDL-C in serum of mice were detected by automatic biochemical analyzer to evaluate the degree of dyslipidemia.（6）Western-blotting and RT-PCR were used to detect the expression of related proteins and genes in liver tissues of mice in each group.Results:1.Modeling results of mice:Oil red O staining of mouse aortic slices showed that compared with ND group,significant plaque was formed in the aorta of HFD group,with a large amount of lipid formation in it,and the intima was significantly thickened and protruded into the lumen.The degree of lipid deposition was significantly greater than that of ND group（P<0.05）.The AS model was successfully established.2.Comparison of serum lipid related indexes in each group:2.1 Compared with ND group,TC,TG and LDL-C values were increased in HFD group,while HDL-C values were decreased（P<0.05）,indicating that high-fat diet can cause lipid metabolism disorders in Apo E^-/-mice.2.2 Compared with HFD group,TC,TG and LDL-C values were increased in HFD+AngⅡ group,while HDL-C values were decreased（P<0.05）,indicating that AngⅡ could inhibit lipid metabolism and promote AS process in AS mice.2.3 Compared with HFD+AngⅡ group,TC,TG and LDL-C value were decreased,while HDL-C value was increased（P<0.05）.The results indicated that caudal intravenous injection of Periostin-mus-769 lentivirus,namely the silence of periostin gene expression in AS mice reduced the degree of dyslipidemia in AS mice.2.4 Compared with HFD+AngⅡ group,TC,TG and LDL-C value were increased,while HDL-C value was decreased（P<0.05）.The results indicated that caudal injection of Periostin-mus lentivirus,namely the expression of periostin gene in AS mice increased the degree of dyslipidemia in AS mice.3.Comparison of expression of related proteins and m RNA in liver tissues of mice in each group:3.1 Compared with ND group,the expression of RCT-related factors ABCA1 and ABCG1 in liver tissue decreased in HFD group.Compared with HFD group:the expression of periostin protein increased,and the expression of RCT-related factors ABCA1 and ABCG1 protein decreased in HFD+AngⅡ group,the above differences were statistically significant（P<0.05）.It was found that high-fat diet inhibited the progression of RCT,and AngⅡ was further inhibited by subcutaneous micropump pumping,indicating that AngⅡ could promote the development of AS by inhibiting the progression of RCT in Apo E^-/-mice.3.2 Compared with the HFD+AngⅡ group,the expression of periostin decreased,the expression of TIGAR and CYP27A1 increased,and the expression of ABCA1 and ABCG1 increased in the HFD+periostin-mus-769+AngⅡ group,the RCT process was promoted.The above differences were statistically significant（P<0.05）.The results showed that silent periostin promoted the expression of TIGAR and CYP27A1,and further promoted the RCT process.3.3 Compared with the HFD+AngⅡ group,the expression of periostin increased,the expression of TIGAR and CYP27A1 decreased,the expression of ABCA1 and ABCG1decreased in the HFD+Periostin-mus+AngⅡ group,the RCT process was inhibited.The above differences were statistically significant（P<0.05）.The results showed that over-expression of periostin inhibited the expression of TIGAR and CYP27A1,and further inhibited the RCT process.Conclusion:AngⅡ promotes AS progression by up-regulating the expression of periostin to inhibit the RCT process.In AngⅡ-induced Apo E^-/-mouse AS model,silent periostin up-regulated the expression of TIGAR and CYP27A1 and the expression of RCT-related regulatory factors ABCA1 and ABCG1,overexpressed periostin,Inhibit the expression of TIGAR and CYP27A1 and the expression of RCT-related regulatory factors ABCA1and ABCG1.It is suggested that periostin may regulate RCT through TIGAR/CYP27A1pathway,thus affecting the occurrence and development of AS...