| Objective: Atherosclerosis(AS),a chronic inflammatory disease,is the pathological basis of numerous cardiovascular diseases,leading to aortic aneurysms,coronary artery disease,cerebral insufficiency,cerebral infarction,intractable hypertension,and lower limb artery stenosis,which seriously affect the quality of life of patients.The mechanism of AS formation is complex,and macrophage-derived foam cells are the typical pathological feature and basis of AS formation.Our group has demonstrated that low-molecular-weight fucoidan sulfate(LMWF)has great antiAS effect,but its specific mechanism is still unclear.The study aims to investigate the molecular mechanism of LMWF to slow down the development of AS by regulating foam cells and related cytokines based on the inhibitory effect of LMWF on AS.Methods: A stable foam cell model was constructed by human myeloid leukemia mononuclear cells(THP-1)incubated with phorbol 12-myristate13-acetate(PMA)and oxidized low-density lipoprotein(ox-LDL).The effect of different concentrations of LMWF on the activity of THP-1 cells was detected by CCK-8,and the accumulation of lipids in foam cells after different treatments was compared by oil red O staining.Flow Cyto Metry(FCM)detected apoptosis of foam cells and immunofluorescence examined the effect of LMWF on key factors of foam cell formation.Macrophage scavenger receptor A1(SR-A1)RNAi and adenosine triphosphate-binding cassette transporter A1(ABCA1)RNAi cell models were constructed by lentiviral transfection.Western blot(WB),real-time quantitative polymerase chain reaction(RT-qPCR)and FCM were used to detect the cell transfection efficiency.Dil-ox-LDL uptake assay and cholesterol efflux assay were used to evaluate the effects of different treatment groups on lipid uptake and cholesterol efflux.WB detected the expression of phosphorylated p38 mitogen-activated protein kinase(p-p38MAPK),SR-A1 and ABCA1 in different treatment groups,and RT-qPCR detected the m RNA levels of the downstream targets of p38 MAPK to further explore the possible mechanisms.Results:(1)CCK-8 and Oil Red O results showed that 50 μg/m L LMWF had no significant effect on cell activity(P>0.05)and could effectively reduce lipid accumulation and inhibit foam cell formation(P< 0.001),and FCM results suggested that LMWF promoted foam cell apoptosis(P<0.001),and the inhibitory effect of LMWF on foam cells was significantly better than that of simvastatin(P< 0.001).The FCM results suggested that LMWF promoted apoptosis of foam cells(P < 0.001),and the inhibitory effect of LMWF on foam cells was significantly better than that of simvastatin(P < 0.001).(2)Immunofluorescence staining observed that LMWF significantly inhibited the expression level of SR-A1 and increased the expression level of ABCA1,while did not obviously affect the expression of CD36 and SR-BI.(3)WB,RT-qPCR and FCM results suggested that SR-A1 RNAi and ABCA1 RNAi cell models were successfully established and transfected with high efficiency(P < 0.01),and the results of Dil-ox-LDL uptake and cholesterol efflux suggested that LMWF inhibited the formation and development of foam by suppressing SR-A1-mediated lipid uptake and promoting ABCA1-mediated cholesterol efflux cell formation(P < 0.001).(4)WB results suggested that LMWF could inhibit p38 MAPK phosphorylation and thus down-regulate SR-A1 protein expression and up-regulate ABCA1 protein expression(P< 0.001),inhibiting lipid uptake and accelerating cholesterol efflux.(5)RT-qPCR results suggested that LMWF down-regulated the m RNA levels of Stat1,Elk-1 and Myc,the downstream targets of p38MAPK(P< 0.01),these downstream targets might be involved in the regulation of SR-A1 and ABCA1 by LMWF.Conclusion: LMWF could achieve bidirectionally regulate of SR-A1 and ABCA1 by inhibiting p38 MAPK phosphorylation,which slowed down the accumulation of foam cells and prevented the development of atherosclerotic plaque.Stat1,Elk-1and Myc might be involved in the regulation of SR-A1 and ABCA1 by LMWF. |
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