PLK1 Promotes Atherosclerosis By Regulating Macrophage Foam Cell Formation

Objective:The study used Apo E^-/-mouse AS model,macrophage foam model,PA/OA induced steatosis model in hepatocyte and PLK1 inhibitor Volasertib,in order to investigate the correlation between PLK1 and atherosclerosis（AS）and the molecular mechanism of PLK1regulating the pathogenesis of AS.Methods:Thirty-two male Apo E^-/-mice（7 weeks old）with C57BL/6J background were acclimatized and fed for one week,and randomly divided into four groups:control group,AS group,Volasertib low dose group（VL group）and Volasertib high dose group（VH group）,8mice in each group.Control group was fed with normal diet,AS,VL and VH groups were fed with high-fat diet for 16 weeks.control and AS groups were administered saline.VL（5mg/kg）and VH（10mg/kg）groups were administered volasertib by tail vein injection twice a week at the last 4 weeks.After 16 weeks,serum total cholesterol（TC）,triglycerides（TG）,low-density lipoprotein cholesterol（LDL-C）,glutamic oxaloacetic transaminase（GOT）,and glutamic alanine transaminase（GPT）were measured;Oil red O staining,HE staining and Masson staining were used to observe aortic valve and aortic tree plaque size and analyze the proportion of lipid droplets and collagen;HE staining and oil red O staining were used to observe the pathological damage of mouse liver tissue and analyze the proportion of lipid droplets.Ox-LDL was used to induce the foam model of Bone marrow derived macrophages（BMDM）,different doses of Volasertib were added,oil red O staining was used to observe the content of lipid droplets in cells,the levels of total cholesterol（TC）and triglyceride（TG）in cells were detected.Using PA/OA induced HepG2 cells,different doses of Volasertib were added,the content of intracellular lipid droplets was observed by oil red O staining,and the levels of total cholesterol（TC）and triglycerides（TG）in the cells were measured;Real time fluorescence quantitative PCR（q PCR）was used to detect the gene expression level of PLK1;Western blot was used to detect the expression level of PLK1 protein.Using PA/OA induced primary liver cells,treated with different doses of Volasertib,and oil red O staining was used to observe the content of intracellular lipid droplets.Results:1.PLK1 expression is positively correlated with the development of atherosclerosisPLK1 expression was found to be higher in the aorta and liver of AS model mice than in the control group.2.PLK1 inhibitor ameliorated atherosclerosis in Apo E^-/-mice2.1 No significant effect of PLK1 inhibitor on body weight of Apo E^-/-mice during the experimentAfter PLK1 inhibitor treatment,it was found that VL and VH group decreased body weight compared to AS group,but there was no statistical difference（p>0.05）.PLK1 inhibitor Volasertib did not affect body weight of mice.2.2 PLK1 inhibitor decreased serum lipid levels in AS miceCompared with the control group,the blood lipid TC and LDL-C levels were significantly increased in the AS group（p<0.001）,and the blood lipid TG was increased in the AS group（p<0.05）;compared with the AS group,the TC,TG and LDL-C levels were significantly decreased in the VH group（p<0.05）;the TC,TG and LDL-C levels were decreased in the VL group,but there was no statistical significance（p>0.05）.2.3 No significant effect of PLK1 inhibitor on serum glutathione and glutamate transaminase in AS miceCompared with the AS group,glutathione and ghrelin levels in VH and VL groups were unchanged.PLK1 inhibitor had no effect on the liver function of AS mice.2.4 PLK1 inhibitor reduced atherosclerotic plaque formation in AS miceCompared with the AS group,aortic root plaques were significantly reduced in the VH group（p<0.05）.Oil red O staining analysis showed a decrease in lipid droplets in the inner wall of the aortic root in the VH group,and in the VL group.HE staining analysis showed a reduction in the thickness of lipid plaques in the aortic root.Masson staining analysis showed an increase in collagen fibers within the aortic root plaques in mice.2.5 PLK1 inhibitor reduced liver lipid levels in AS miceCompared with the control group,liver lipid TG and TC levels were significantly increased in the AS group（p<0.0001）;compared with the AS group,TG and TC levels were significantly decreased in the VH group（p<0.001）.The liver HE staining results showed that the swelling of hepatocytes in the VH group was reduced and the area of fat vacuoles in the cytoplasm was decreased compared with that in the AS group.The results of liver oil red O staining showed that the liver lipid droplet content was significantly higher in the AS group compared with the control group（p<0.001）;and significantly lower in the VH group compared with the AS group（p<0.001）.3.PLK1 inhibitor reduced ox-LDL-induced macrophage foaminess（1）Cellular lipid droplet content was elevated in the ox-LDL group compared to the control group;addition of PLK1 inhibitor Volasertib co-incubated with ox-LDL（100μg/ml）for 24/48h decreased cellular lipid droplet content with increasing dose of Volasertib compared to the ox-LDL group.（2）Compared with the control group,the TG content of cells in the ox-LDL group increased（p<0.05）and TC content increased（p<0.001）;the addition of Volasertib（200 ng/ml）co-incubated with ox-LDL for 48 h,compared with the ox-LDL group,the Volasertib（200 ng/ml）treatment,TG,TC content was reduced（p<0.05）.4.No significant effect of PLK1 inhibitors on lipid droplet content in PA/OA induced steatosis model in hepatocyte4.1 No significant effect of PLK1 inhibitor on lipid droplet content of PA/OA-treated HepG2cellsCompared with the control group,the lipid droplet content of cells in the PA/OA group was increased;the addition of Volasertib and PA/OA were incubated together for 24/48 h.Compared with the PA/OA group,the lipid droplet content of cells in the Volasertib group did not change significantly.Compared with the control group,the TG and TC contents of cells in the PA/OA group were increased（p<0.05）;the addition of Volasertib（40 ng/ml）and PA/OA were co-incubated for24/48 h.Compared with the PA/OA group,Volasertib（40 ng/m L）treatment,there was no significant change in TG,TC content.4.2 No significant effect of PLK1 inhibitor on lipid droplet content of PA/OA-treated mouse primary hepatocytesWhen Volasertib was added and co-incubated with PA/OA for 24 h,compared with the PA/OA group,the lipid droplet content of cells in the Volasertib group did not significant changes.Conclusions:1.PLK1 expression was positively correlated with AS pathogenesis.2.Inhibition of PLK1 activity improves the onset of atherosclerosis in AS mice.（1）Inhibition of PLK1 activity reduced aortic plaque area and decreased lipid levels in serum and liver of AS mice.（2）Inhibition of PLK1 activity can inhibit foam cell formation and anti-atherosclerosis by reducing lipid deposition in BMDM.