Molecular Mechanisms Of Artemisinin Inhibiting Proliferation Of Vascular Smooth Muscle Cells By Regulation Of Small RNA

Objective:Vascular Smooth Muscle Cells are highly differentiated cells located in the middle layer of blood vessels.Once the blood vessels receive lesions,smooth muscle cells will proliferate and then repair the damage of blood vessels.During the formation of atherosclerotic plaques,vascular smooth muscle cells proliferate and migrate to the diseased site,resulting in intimal hypertrophy,narrowing of the vascular cavity,and insufficient blood supply to the tissue.Inhibition of vascular smooth muscle cell proliferation has become an important target for clinical prevention and treatment of atherosclerosis.Studies have found that mitogen-activated protein kinase 6(MAPK6)has the ability to promote the proliferation of vascular smooth muscle cells.Therefore,it is worth exploring whether it can inhibit the proliferation of smooth muscle by inhibiting the expression of MAPK6.Small RNAs are a class of RNA molecules less than 200 nt in length.Among them,miRNAs are a type that plays more physiological roles.More and more miRNAs have been found to be used as markers or important regulators of atherosclerosis.The let-7 family is the second miRNA found in humans,of which let-7g can reduce atherosclerosis in vivo and in vitro in mice.let-7g can inhibit the proliferation and migration of vascular smooth muscle through targeted binding to low-density lipoprotein receptors.Artemisinin is a drug extracted by Chinese scholar Tu Zhi from the plant Artemisia annua.Our research group put forward the hypothesis that artemisinin can be used in treating atherosclerosis and found that artemisinin can alleviate atherosclerosis plaque size in vivo.The main purpose of this study was to investigate whether artemisinin can inhibit the proliferation of vascular smooth muscle cells by promoting the expression of let-7g,reducing the expression of mitogen-activated protein kinase 6,and laying a foundation for the future clinical application of artemisinin to cardiovascular diseaseMethods:Mice aortic smooth muscle cells were cultured as a model and grouped for different treatments.Cell proliferation was observed using EdU staining under a fluorescent microscope.Western Blot was used to detect changes in the relative expression of MAPK6 protein.The expression level of let-7g was detected by PCR.The presence of a binding site between let-7g and 3'UTR of MAPK6 was detected using aluciferase reporter gene.Flow cell cytometry was used to determine the proliferation rate of smooth muscle cellsResults:(1)Compared with the control group,the cell proliferation rate of the PDGF group was significantly upregulated,and the cell proliferation rate of the ART group was significantly lower than the PDGF group(both p were less than 0.001).(2)The relative protein concentration of MAPK6 in PDGF group was significantly higher than that in Control group(p<0.001),and the relative RNA expression level of let-7g-5p was significantly lower than that in Control group(p<0.01).Compared with PDGF group,the relative concentration of MAPK6 protein in ART group decreased significantly(p<0.05),and the relative expression of let-7g-5p increased significantly(p<0.05).(3)The S/G2 phase ratio of let-7g-5p inhibitor group was significantly higher than that of inhibitor group(p<0.001),and the S/G2 phase ratio of let-7g-5p mimics group was significantly lower than that of mimics control group(p<0.001).The cell ratio of S/G2 phase of MAPK6 interference group was significantly lower than that of inhibitor group(p<0.01).(4)The relative protein expression of MAPK6 in the let-7g-5p inhibitor group was significantly higher than that in the inhibitor control group(p<0.0001).The relative protein expression of MAPK6 in let-7g-5p mimics group was significantly lower than that in mimics control group(p<0.01).Luciferase experiments showed that wild-type MAPK6(MAPK6 WT)significantly decreased fluorescence intensity compared with let-7g-5p co-transfection,confirming that let-7g-5p was bound to the 3'UTR of MAPK6(5)The ratio of S/G2 cells in ART group was significantly higher than that in Control group.Compared with the ART group,the S/G2 phase cell ratio in the ART+let-7g inhibitor group significantly increased(p<0.001)Conclusion:Artemisinin can inhibit the expression of MAPK6 by promoting the overexpression of let-7g,thereby inhibiting the proliferation of vascular smooth muscle in mice.