Expression Of Galectin-3 In Patients With Coronary Heart Disease And Its Mechanism Of Regulating Cholesterol Metabolism In Macrophages

Background:Atherosclerotic cardiovascular disease is a chronic inflammatory disease.Inflammatory cytokines are key mediators in accelerating the occurrence and development of atherosclerosis.Some cytokines can directly participate in the regulation of intracellular cholesterol metabolism to maintain cholesterol homeostasis.Galectin-3(Gal-3)is a pro-inflammatory cytokine that is mainly secreted by activated macrophages.It is the only unique chimaera-like galectin member of the vertebrate family.Gal-3 participates in and regulates many’ inflammatory response pathologies.In recent years,some studies have shown that Gal-3 is closely related to coronary atery disease,however,the relationship between Gal-3 and the occurrence,the stability and complexity of coronary heart diseaseare still lack of systematic reports,and whether Gal-3 is valuable in evaluating the prognosis of patients with coronary heart disease has not been fully clarified.As an inflammatory factor,whether Gal-3 is directly involved in the regulation of intracellular cholesterol metabolism is rarely reported.Latest research shows that Gal-3 can promote the expression of chemokine CCL2.Other report indicated that by binding to CCR2,CCL2 can activate the p38/Mitogenactivated Protein Kinase(p38/MAPK)pathway and inhibit the internalization of highdensity lipoprotein(HDL)and the efflux of cholesterol.It’s well confirmed that the Mitogen-activated protein-kinase(MAPK)signaling pathway can affect the outflow of cholesterol by regulating the expression of PPAR-γ and LXR-a.Based on these,we speculate that Gal-3 may also directly participate in the regulation of macrophage cholesterol metabolism to participate in the occurrence and development of atherosclerosis.The mechanism may be that Gal-3 promotes the expression of CCL2,activates the MAPK pathway,and thereby inhibits expression of PPAR-γ and LXR-a,reduce the outflow of cholesterol,and promote the occurrence and development of atherosclerosis.Objectives:In vivo,we aimed to explore the relationship between Gal-3 and coronary heart disease and its prognostic value in MACEs in patients with acute coronary syndrome.In vitro,we aimed to evaluate the effect of Gal-3 on cholesterol efflux rate and intracellular cholesterol concentration of macrophages in a RAW264.7 mice cell mode.We also determined the expression of CCL2,p38/MAPK and cholesterol metabolism related proteins such as PPAR-γ,LXR-a,ABCA1 and ABCG1 to explore the possible mechanism of Gal-3 regulating cholesterol metabolism.Methods and ResultsMethods:1.Patients who were diagnosed with acute myocardial infarction,unstable angina pectoris,stable angina pectoris,and no-coronary artery disease by coronary angiography were enrolled in this study from 1 January to 1 december 2018admitted to zhongshan people’s hospital.Expression of serum Gal-3 was detected by ELISA;2.Gal-3 stimulated RAW264.7 cells with different concentration gradients and time duration gradients to establish a model.Experimental methods such as HPLC,Elisa,RT-qPCR and Western Bolting to clarify the influence of Gal-3 on cholesterol efflux rate and cholesterol transporter expression in RAW264.7 cells;3.RT-qPCR and Western Bolting experiments Methods Determine the expression of CCL2 and P38/MAPK under the intervention of Gal-3;4.CCL2 and P38/MAPK inhibitors were invited respectively,and changes in the intracellular cholesterol concentration and cellular cholesterol efflux rate of RAW264.7 cells under the intervention of Gal-3 were compared accordingly;5.Sequentially introduce CCL2 and p38/MAPK inhibitors and the differences in CCL2 and p38/MAPK expression as well as cholesterol transport related proteins such as PPAR-γ LXR-a,ABCA1 and ABCG1 were measured and compared.Results:1.The serum Gal-3 in coronary heart disease is higher than that of noncoronary heart disease patients 2.Acute coronary syndrome is higher than patients with stable coronary heart disease and non-coronary artery diseasein serum Gal-3.Gal-3 is an independent risk factor for the occurrence of acute coronary syndrome.3.Serum Gal-3 positively correlates with Syntax Score and it is an independent predictor of high SyntaxScore.4.Acute coronary syndrome patients with serum Gal-3≥4.78ng/ml shows higher incidence of major adverse cardiac events(MACEs)during 1 year of follow up.5.With the increase of Gal-3 concentration gradients or stimulation time,the cholesterol efflux rate of RAW264.7 macrophages decreased and the intracellular cholesterol concentration increased.At the same time,the expressions of PPAR-γ and LXR-α were down regulated.The expressions of ABCA1 and ABCG1 were also decreased 6.Gal-3 stimulated RAW264.7 macrophages and upregulated the expression of CCL-2 and p38/MAPK.Inhibition of CCL-2 or p38/MAPK could relieve the inhibited expression of cholesterol transporter(PPAR-γ,LXR-a,ABCA1 and ABCG1)and restore the cholesterol transport rate of macrophages by Gal-3;7.Inhibition of CCL2 by bindarit could relieve the effect of Gal-3 on the expression of p38/MAPK,but the expression of LXR-α,PPAR-γ and their downstream ABCA1 and ABCG1 did not change significantly;8.After SB203580 was induced to inhibit p38/MAPK pathway,Gal-3 could still up regulate the expression of CCL2,but the expression of LXR-α,PPAR-γ and their downstream ABCA1 and ABCG1 had no significant change.Conclusion:In our study,we found that Gal-3 closelycorrelate with coronary heart disease and may be valuablein assessing the stability and complexity of coronary disease.It may be also useful in predicting the prognosis of acute coronary syndrome.Moreover,Gal-3 may promote the expression of chemokine CCL2 and activate the P38/MAPK signaling pathway,then inhibit the expression of LXR-α and PPAR-γ,and subsequently downregulate the expression of ABCA1 and ABCG1,leading to a decrease in the rate of macrophage cholesterol efflux and increases intracellular cholesterol concentration,and participates in the development of atherosclerosis.