The Mechanism Of ER Stress Protein CHOP Pathway Involved In The Occurrence And Development Of Atherosclerosis

Background:Atherosclerosis(AS)is a chronic inflammatory disease characterized by the deposition of atherosclerotic plaques on the walls of arteries,which can cause stenosis of the corresponding arteries.It is a myocardial infarction,stroke,and other ischemic heart and brain the basis of vascular disease.Vascular smooth muscle cells(Vascular smooth muscle cells,VSMCs)are one of the important components of the blood vessel wall.They are mainly distributed circularly in the media of the blood vessel wall and are responsible for the contraction of blood vessels.They are closely related to the compliance and elastic retraction of blood vessels.VSMCs have played different roles in the development of AS.Recent studies have found that VSMCs can also transform into different cell types such as macrophages,and participate in vascular local inflammation and vascular calcification.At the same time,VSMCs can also ingest the lipid components deposited in the tube wall to form foam cells,which can form plaque necrotic cores due to delayed clearance after apoptosis.The endoplasmic reticulum is an important organelle involved in protein synthesis,processing and modification by eukaryotic cells,and is of great significance for maintaining the homeostasis of the intracellular environment.Under the stimulation of various causes of atherosclerosis,the protein folding ability of the endoplasmic reticulum of the cell cannot meet the needs of the cell,which leads to a large number of unfolded protein misfolding,causing the unfolded protein response(UPR),and then triggering Endoplasmic reticulum stress(ERS).The promoter of CCAAT/enhancer binding protein homologous protein(CHOP)gene also contains multiple binding sites for important ERS-related genes,and is a transcription factor that plays a key role in the ERS process.When the CHOP protein is activated,it can play its own transcription factor to regulate the expression of a series of downstream genes and affect the physiological functions of cell apoptosis,proliferation and autophagy.Studies have found that CHOP expression increases significantly during the progression of AS,but its role in AS and its specific mechanism are still unclear.Micro RNA(miRNA)is a type of single-stranded non-coding RNA molecule with a length of 18-23 nucleotides.It mainly participates in the physiological activities of cells by specifically binding to the 3'untranslated region of the targeted messenger RNA to form an RNA-induced silencing complex(RISC)to promote its degradation.A considerable number of miRNAs have been found to be related to the progression of AS.Among them,miR-208,which is significantly upregulated in a high-fat diet,has been found to be closely related to coronary heart disease.However,its relationship with atherosclerosis is still unclear.Objective:This study verified the expression of endoplasmic reticulum stress protein CHOP and the role of CHOP/TRIB3/miR-208/TIMP3 regulation pathway in atherosclerosis by constructing a mouse atherosclerosis model and an in vitro vascular smooth muscle cell model.The prevention of atherosclerosis and future targeted therapy provide data support and theoretical basis.Method:This study first induced atherosclerosis in mice with apolipoprotein E(ApoE)gene deficiency by feeding them with a high-fat diet for 12 weeks.The modeling was verified by oil red O staining,hematoxylin and eosin(HE)staining,serological index determination,gross anatomy and other methods.At the same time,the stability of the plaque was evaluated by detecting the content of VSMCs and type I collagen in the plaque.The high expression of miR-208 in animal models was verified by real-time fluorescence quantitative polymerase chain reaction(q PCR),fluorescence in situ hybridization,immunofluorescence staining and other methods.Flow cytometric screening technology sorts out the vascular smooth muscle cells that are positive for ACTA2,positive for TAGLN,and negative for PECAM.The interaction between miR-208 and TIMP3 is predicted by bioinformatics methods and verified by the dual luciferase reporter experiment.By constructing a series of lentiviral vectors,Antagomir,miR-mimics,miR-inhibitor and other expression regulators to interfere with the expression of miR-208 and CHOP,TRIB3,TIMP3 and other target molecules in animal models and cells,and by q RT-PCR and Western blot.The effect of the intervention is verified.After successful treatment,Ed U cell proliferation test and scratch test were used to evaluate the changes of VSMCs proliferation and migration.At the same time,gross anatomy,pathological smears,and serum index levels were used to evaluate the progression of AS in animal models under different treatments.At the same time,the proportion of VSMCs and type I collagen in the plaque was detected to evaluate the stability of the plaque.Result:The ApoE gene-deficient mice developed atherosclerosis after 12 weeks of highfat diet.The carotid artery wall lipid deposition,the proportion of damaged area,and the damaged area of the aortic sinus of AS mice increased significantly(P<0.01),the blood lipid indexes of AS mice are significantly different from those of the control group(P<0.05).These results indicate that the model of this study was successfully constructed.After further testing the miR-208 in AS mice,it was found that its expression increased significantly(P<0.05),and its location basically overlapped with the VSMCs marker molecule ?-SMA,all located in the vascular wall.After downregulating the expression of miR-208,total cholesterol,total triglycerides,and lowdensity lipoprotein cholesterol are improved,carotid artery lipid deposition and damaged areas are significantly reduced,and VSMCs and type I collagen in plaques are significantly increased,suggesting plaque stability Improve(P<0.05).ACAT2+,TAGLN+,PECAM-VSMCs were selected by flow cytometry technology to carry out subsequent cell experiments.The dual luciferase reporter experiment verified the mutual targeting relationship between miR-208 and TIMP3.After further upregulating the expression of miR-208 in VSMCs,the expression of TIMP3 was inhibited,and the proliferation and migration ability of VSMCs was also enhanced.This phenomenon can be reversed by overexpression of TIMP3(P<0.05).QPCR and Western blot tests verified that the expression of TRIB3 in atherosclerotic mice and VSMCs was significantly increased(P<0.05).After down-regulating its expression,the proliferation and migration of VSMCs were inhibited,and the expression of miR-208 was also significantly decreased(P<0.05).After inhibiting the expression of TRIB3,the blood lipid index,carotid artery lipid deposition,the proportion of damaged area,and the damaged area of aortic sinus were all significantly decreased(P<0.05).In addition,the expression of CHOP in disease animal models and VSMCs was significantly up-regulated.After inhibiting its expression,the expression of TRIB3 and miR-208 decreased correspondingly,while the expression of TIMP3increased(P<0.05).The proliferation and migration of VSMCs decreased after downregulation of CHOP(P<0.05).This result indicates that CHOP can inhibit the formation of atherosclerotic plaques.In AS mice after inhibiting the expression of CHOP,significant reductions in blood lipid indexes,carotid lipid deposition,percentage of damaged area,and damaged area of aortic sinus were also observed(P<0.05).The proportion of ?-SMA positive cells in the plaque,the proportion of type I collagen,and the content of TIMP3 protein all increased significantly(P<0.05),suggesting that the plaque became more stable after CHOP was inhibited.Further research found that the changes in cells and animal models after inhibiting the expression of CHOP were reversed by the overexpression of TRIB3.Conclusion:The results of this study show that down-regulating the expression of CHOP can delay the progression of atherosclerosis and enhance the stability of atherosclerosis,and it also inhibits the proliferation and migration of vascular smooth muscle cells.After further verification,it was found that this series of regulatory effects were partly achieved by down-regulating the expression of TRIB3-dependent miR-208 and then increasing the expression of TIMP3.This study explores for the first time that the mechanism of CHOP/TRIB3/miR-208/TIMP3 pathway involved in the occurrence and development of atherosclerosis.This pathway has been effectively confirmed in vivo and in vitro,and its pathway may become a new target for atherosclerosis treatment.