Effect Of Antimicrobial Peptides On Mice Macrophage Foaming Induced By Oxidized Low Density Lipoprotein(ox-LDL)

Atherosclerosis(AS)is one of the most important pathological bases of cardiovascular and cerebrovascular diseases,is the leading cause of death and disability.It is currently well accepted that AS is a chronic inflammatory disease within vascular walls.Macrophages play an important role in all stages of lipid accumulation,atheroma plaque formation,and development of AS in the blood vessel wall.Macrophages produce pro-inflammatory responses induced by oxidized low-density lipoprotein(ox-LDL)and then uptake ox-LDL thereby transforming into foam macrophages.Foaming of macrophages is a hallmark of early atherosclerotic lesions.The mechanism of macrophage foaming is complex,involving macrophage activation,oxidative stress,NF-κB activation,cytokine secretion,and upregulation of scavengers and cell adhesion receptors/ligands.At present,research on anti-inflammatory drugs has become a hot spot for the treatment of AS.Temporin-1CEa is a natural antimicrobial peptide isolated and purified from the skin secretion of Rana chensinensis in our lab.It contains three net positive charges and adopts anα-helical structure in the membrane environment.LK2(6)was designed and synthesized by replaced L-Asp and L-Gly residues at position 3 and 16 were with L-Lys in Temporin-1CEa,and LK2(6)A(L)was designed and synthesized by replaced L-Ala at position 8with L-Leu in LK2(6).The net positive charge number of LK2(6)and LK2(6)A(L)has increased to seven,and the hydrophobicity is also greatly improved.Previous studies in our laboratory found that Temporin-1CEa and its two modified peptides have high anti-bacterial activity against Gram-positive and negative bacteria,and anti-tumor activity,with little side effects on normal cells.In addition,the three antimicrobial peptides exhibited anti-inflammatory activity on mice macrophage by inhibiting NF-κB and MAPK signal pathway.So in this study,the effect of Temporin-1CEa,LK2(6)and LK2(6)A(L)on mice macrophage foaming induced by ox-LDL was studied.First,an model of mouse macrophage foaming induced by ox-LDLwas established.Different concentration of ox-LDL was incubated with mice macrophage RAW264.7 cells for24 h.The cholesterol content was analyzed by using Oil red O test and cholesterol enzymatic assay.The results showed that when the concentration of ox-LDL was 100 μg/mL,the total cholesterol(TC)and free cholesterol(FC)the cholesterol ester(CE)was 92.58 nmol/mg and35.53 nmol/mg respectively,cholesterol ester(CE)specific gravity was greater than 50%.The oil red O assay futhurly confirmed that a large amount of lipid droplets existed in the cytoplasm of macrophage when ox-LDL was 100μg/mL,suggesting that mice macrophage foaming model was succeedly established by ox-LDL at a concentration of 100 μg/mL.The antimicrobial peptides Temporin-1CEa,LK2(6)and LK2(6)A(L)significantly inhibited the foamed mouse macrophage RAW264.7.Antimicrobial peptides Temporin-1CEa,LK2(6)and LK2(6)A(L)cholesterol enzymatic assays at concentrations of 3.75 μM,1.875μM and 0.937 μM compared to ox-LDL,Temporin-1 CEa allows total intracellular foam cells Cholesterol content decreased by 33%,cholesterol ester content decreased by 50%;LK2(6)decreased total cholesterol content in foam cells by 36%,cholesterol ester content decreased by 54%;LK2(6)A(L)caused total cholesterol in foam cells The decrease was 44% and the cholesterol ester content decreased by 66%.Among them,LK2(6)A(L)has the best inhibitory effect on cholesterol in foamed macrophages,and the inhibition rate can reach 44%.The results of the oil red O experimental observation also showed that the three antimicrobial peptides significantly reduced the cholesterol lipid droplets in the foamed mouse macrophages.The above experimental results show that the three antimicrobial peptides have an inhibitory effect on the formation of foam cells.CD36 and SR-AI are membrane receptor proteins that take up ox-LDL on macrophage membranes.ABCA1 and ABCG1 are cholesterol efflux proteins,which are the major membrane proteins that regulate cholesterol metabolism.Western Blotting assay was used to detect the effects of Temporin-1CEa,LK2(6)and LK2(6)A(L)on CD36 and SR-AI and ABCA1 and ABCG1 protein expression.The results showed that the three antimicrobial peptides inhibited the expression of CD36 protein in a concentration-dependent manner.The inhibitory rates of CD36 expression in Temporin-1CEa,LK2(6)and LK2(6)A(L)at 3.75 μM were 44%,34% and 55%,respectively,of which LK2(6)A(L)The inhibitory effect was the best;there was no significant inhibition on the expression of SR-AI;no inhibition on the expression of ABCA1 and ABCG1.It is suggested that the inhibition of mouse macrophage foaming by three antimicrobial peptides may occur in the uptake of ox-LDL by cells,rather than the efflux of cholesterol.The antimicrobial peptide LK2(6)A(L)was selected to pretreat mouse macrophage RAW264.7 for 24 h,and 100 μg/mL ox-LDL was added to induce cell foaming.The oil red O experiment and cholesterol content showed that Compared with non-pretreatment,LK2(6)A(L)pretreatment showed no significant difference in TC and FC content in ox-LDL-induced foam cells;Western Blotting results showed LK2(6)A(L)pre-preparation There was no significant difference in the expression of CD36 and SR-AI protein in ox-LDL-induced foam cells compared with non-pretreatment.It is furthersuggested that the antimicrobial peptide may inhibit mouse macrophage foaming by inhibiting the uptake of ox-LDL by mouse macrophage RAW264.7.Three antimicrobial peptides such as Temporin-1CEa can reduce the release of proinflammatory cytokines TNF-α and IL-6 from foaming macrophages RAW264.7 cells.The best inhibitory effect is LK2(6)A(L).At a concentration of 3.75 μM,the inhibition of TNF-α and IL-6 was 50% and 52%,respectively.RT-qPCR and Western Blotting experiments showed that LK2(6)A(L)can significantly inhibit the phosphorylation of IκB and NF-κB in ox-LDL-induced foaming mouse macrophages and significantly inhibit the MAPK pathway proteins JNK and ERK.And expression of p38 phosphorylation levels.In conclusion,the antimicrobial peptides Temporin-1CEa,LK2(6)and LK2(6)A(L)inhibit NF-κB and MAPK inflammatory pathway signaling protein phosphorylation during ox-LDL-induced mouse macrophage foaming.Reduce the release of pro-inflammatory factors TNF-α and IL-6.