Lipoxin A4 Exerts Anti-atherosclerotic Effect Through Keap1/Nrf2/ARE Signaling Pathway

Background:Atherosclerosis(AS)is the most common disease in the cardiovascular system.At present,the principles of atherosclerosis treatment are lipid-lowering,anticoagulation and thrombolysis,but the effect is not good.Inflammation and oxidative stress are the two key mechanisms of the pathogenesis of AS.The two coexist,are mutually regulated and inseparable.During the long-term response to reactive oxygen species(ROS)damage,the body gradually formed a complex system of anti-oxidative stress.Keap1/Nrf2/ARE is the most important endogenous anti-oxidation signal pathway in the body,negatively regulating oxidative stress and inflammation.Lipoxin A4(LXA4)is a metabolite of arachidonic acid,which can inhibit the expression of inflammation-related genes and promote the regression of inflammation.With the in-depth study of LXA4,it was found that it is not only an important anti-inflammatory and pro-resolving substance in the body,but also has important biological effects in cardiovascular diseases.Studies have shown that LXA4 can inhibit foam cell formation,reduce ox-LDL-induced inflammation,inhibit macrophage apoptosis signaling and promote inflammation regression,thereby exerting anti-AS effects.However,the specific mechanism of LXA4 anti-AS is still unclear.Therefore,we speculate that LXA4 may play an anti-AS role by activating the Keap1/Nrf2/ARE pathway,reducing inflammation and oxidative stress damage during AS lesions.Objective:To explore the role of Keap1/Nrf2/ARE signaling pathway in LXA4 against AS injury.Methods:1.Model establishment and group processingHigh-fat diet was used to replicate the AS rat model;SD rats(n=40)were randomly divided into Control group,HF(High fat diet)group,HF+BML-111 group,HF+BML-111+Boc-2 group.There were 10 animals in each group.After the model was established successfully,LXA4 analog BML-111 and ALX blocker Boc-2 were injected intraperitoneally.The drug was given 3 times,and the drug was given every other day.2.Assess the degree of AS(1)Automatic biochemical analyzer detects cholesterol(TC),triglyceride(TG),low density lipoprotein(LDL),high density lipoprotein(HDL)and lipid peroxidation index superoxide dismutase(SOD)content in each group,biochemical kit to detect malondialdehyde(MDA)content;(2)The aortic gross oil red O staining was used to compare the degree of AS lesions in each group of rats.3.Observe the effect of BML-111 on Keap1/Nrf2/ARE signaling pathway at the overall levelImmunohistochemistry and immunofluorescence were used to observe the nuclear translocation of Nrf2 and the expression levels of Keap1,HO-1 and NQO1.4.Establish foam cell model and group(1)Phophorate(PMA)induces THP-1 monocytes to differentiate into M0 macrophages,causing them to foam up oxidized low density lipoprotein(ox-LDL)to form macrophage-derived foam cells,oil red O Staining method to identify the success of the model;observe the effect of different concentrations of LXA4 on the formation of foam cells;(2)Divide the successfully induced foam cells into three groups: Control group,LXA4 group,Boc-2+LXA4 group,and oil red O staining to observe the effect of LXA4 on foam cell formation.5.Observe the effect of LXA4 on Keap1/Nrf2/ARE signaling pathway at the cellular levelWestern Blot method and immunofluorescence method were used to observe the Nrf2 nuclear translocation of foam cells and the expression levels of Keap1,HO-1and NQO1.Result:1.Biochemical test result showed that: HF group plasma TC,TG,LDL and MDA content increased,HDL and SOD content decreased;BML-111 can increase plasma HDL content,reduce MDA content,failed to change TC,TG,LDL and SOD content;Boc-2 could not reverse the effect of BML-111;the whole aortic oil red O staining showed: lipid infiltration occurred in the arterial wall of HF group,BML-111 reduced the degree of lipid infiltration in arterial wall,while there was no significant difference in aortic disease between the Boc-2 group and the BML-111 group;2.The result of immunohistochemistry and immunofluorescence showed that BML-111 down-regulated Keap1 expression,promoted Nrf2 nuclear translocation,and increased the expression levels of HO-1 and NQO1;Boc-2 could not reverse the effect of BML-111.3.Cell oil red O staining result showed that: LXA4 can effectively inhibit the formation of foam cells,and the inhibition effect is the best at 100 n M;4.Western Blotting and immunofluorescence results of foam cells showed that LXA4 down-regulates Keap1 expression,promotes Nrf2 nuclear translocation,and increases the expression levels of HO-1 and NQO1;Boc-2 cannot reverse the effect of LXA4.Conclusion:Lipoxin A4 and its analog BML-111 exert an anti-AS effect through the Keap1/Nrf2/ARE signaling pathway.This effect does not depend on the LXA4 receptor.