Research On Activating AMPKα1 To Inhibit MCP-1 In Perivascular Adipose Tissue And Alleviate Atherosclerosis

Objective: we use gene-editing technology to knock out and overexpress adenylate-activated protein kinase α1（AMP-activated protein kinase α1,AMPK α1）and investigate the role of activating AMPKα1 on inhibiting monocyte chemotaxis in perivascular adipose tissue,as well as its possible mechanisms in atherosclerosis.Methods: This research is launched in vivo and in vitro to observe the expression of MCP-1 in adipose tissue and its effect on atherosclerosis through regulating AMPKα1 in adipose tissue.（1）Plasmid construction: construct a vector plasmid for overexpression of AMPKα1.The cDNA was obtained from RAW264.7 cells,and RT-PCR amplified the target gene fragment of AMPKα1.The PLJM1-EGFP vector plasmid and the target fragment were digested with NHEI and AGEI restriction endonucleases and ligated with T4 DNA ligase to obtain AMPKα1.Overexpression vector plasmids.The small guide RNA of AMPKα1 was designed using the website of Zhang Feng’s laboratory,and the lenti CRISPRv2 plasmid was digested with Esp3 I restriction enzyme.The sgRNA was cloned into the vector plasmid using T4 DNA ligase.An AMPKα1 knockout plasmid was obtained.（2）Carotid fat transplantation experiment: In situ injection of blank,AMKPα1 knockout,and AMPKα1 overexpression plasmid-coated lentiviral fluid into epididymal fat of C57BL/6mice.After 14 days,the epididymal fat of each group was taken to detect the expression of AMPKα1 by Western Blot.The left carotid artery of ApoE^-/-mice was transplanted with blank,AMPKα1 knockout,and overexpressed epididymal fat.After one week of adaptation,the mice were fed a high-fat diet for eight weeks.Oil Red staining was used to observe the number and size of carotid plaques.（3）Cell co-culture experiment: The primary adipocytes were replaced by AMPKα1 blank,knockout,and overexpressed lentiviruses,respectively,and then induced.After co-culture,the abnormal proliferation of vascular smooth muscle cells and the expression of MCP-1 in macrophages were detected.Results:（1）The AMPKα1overexpression plasmid was successfully constructed.After HEK293 T cells were coated with lentivirus and infected RAW264.7 cells,the protein expression of AMPKα1 detected by WB was significantly higher than that of the blank Control group（P<0.05）;（2）AMPKα1 knockdown was successfully constructed.In addition to plasmids,after the HEK293 T cell-coated lentivirus infected RAW264.7 cells,the protein expression of AMPKα1 detected by the WB method was much lower than that of the blank Control group（P<0.05）;（3）After in situ injection of lentivirus in epididymal adipose tissue of C57BL/6 mice,the expression of AMPKα1 protein was detected.Compared with the Control group,the expression of AMPKα1 protein in the in situ injection of the AMPKα1 overexpression lentivirus group increased（P<0.05）.Compared with the Control group,the expression of AMPKα1 protein in the orthotopic injection of the AMPKα1 knockout lentivirus group was significantly decreased（P<0.05）;（4）The plaque changes in the carotid arteries of ApoE^-/-mice in each group were transplanted with fat.Oil Red O staining showed that compared with the right carotid artery without fat grafting,the left carotid artery with adipose tissue had plaques.The plaques in the group transplanted with AMPKα1 overexpressing adipose tissue were significantly less than those in the Control group（P<0.05）,and the plaques in the group transplanted with AMPKα1 knockout adipose tissue were significantly more than those in the Control group（P<0.05）;（5）The expression of AMPKα1 in the transplanted adipose tissue of each group was detected after sampling.Compared with the Control group,the expression of AMPKα1 protein in the orthotopic injection of AMPKα1 overexpressing lentivirus group increased（P<0.05）.Compared with the Control group,the expression of AMPKα1 protein in the orthotopic injection of the AMPKα1 knockout lentivirus group was significantly decreased（P<0.05）;（6）The expression of AMPKα1 protein and inflammatory factor MCP-1 in primary adipocytes treated with lentivirus was detected.Compared with the Control group,the expression of AMPKα1 protein in the AMPKα1overexpression lentivirus group was increased（P<0.05）.The expression of inflammatory factor MCP-1 was decreased（P<0.05）.Compared with the Control group,the expression of AMPKα1 in the AMPKα1 knockout lentivirus group was significantly decreased（P < 0.05）,while the expression of the inflammatory factor MCP-1 was increased（P < 0.05）;The serum was co-cultured with primary vascular smooth muscle cells.Compared with the Control group,the abnormal proliferation of vascular smooth muscle cells was inhibited in the overexpression group of AMPKα1,while the abnormal proliferation of vascular smooth muscle cells in the knockout group of AMPKα1 increased;（8）The adipocyte supernatant after lentivirus treatment was co-cultured with primary macrophages.Compared with the Control group,the expression of inflammatory factor MCP-1 in the macrophages in the overexpression AMPKα1 group was reduced.Compared with the Control group,the expression of the inflammatory factor MCP-1 in macrophages in the AMPKα1 knockout group was increased.Conclusion: Increasing the level of AMPKα1 in perivascular adipose tissue reduces the expression of inflammatory factors in adipose tissue,inhibit the abnormal proliferation of vascular smooth muscle,reduce the expression of inflammatory factors in macrophages,and alleviate the progression of atherosclerosis.Therefore,interfering with AMPKα1 levels in PVAT may serve as a potential target for anti-atherosclerosis from extravascular routes.