The Mechanism Of ASGR1 Promoting Vascular Inflammation By Regulating Macrophage Function

Background and Objective:Atherosclerosis(AS)characterized by vascular wall lipid deposition and vascular inflammation,is the common pathogenesis of various cardiovascular diseases.Blocked reverse cholesterol transport and M1 polarization of macrophages play a vital role in vascular wall lipid deposition and vascular inflammation.A large genome-wide association analysis published in the NEJM has discovered the reduced serum LDL-c levels and a 34% reduction of coronary heart disease risk in individuals with asialoglycoprotein receptor(ASGR1)loss-of-function mutations.However,the mechanism is still unclear.In this study,we will construct the hypercholesterolemia mouse model to investigate the effect of ASGR1 on serum cholesterol levels,aortic lipid deposition,and vascular inflammation and its underlying mechanisms.Methods:(1)ASGR1 knockout mice were fed with high cholesterol diet to construct the hypercholesterolemia mouse model.The effects of ASGR1 knockout on serum cholesterol level,aortic lipid deposition and vascular inflammation were measured by ELISA and oil red O staining.(2)Primary macrophages were isolated from ASGR1 knockout mice.And overexpression of ASGR1 with adenovirus was performed in primary macrophages from wild-type mice.Then the macrophages were induced by ox-LDL to detect the effects of ASGR1 on foam cell formation,cholesterol uptake,cholesterol uptake and efflux related proteins.(3)Primary macrophages were isolated from ASGR1 knockout mice,and ASGR1 was overexpressed in primary macrophages from wild-type mice,followed by ox-LDL stimulation.The impact of ASGR1 on ox-LDL-induced macrophage M1 polarization,inflammatory factor secretion and p38 MAPK and NF-κB inflammatory signaling pathway were evaluated.(4)ASGR1 knockout primary macrophages were intervened by gene silencing,agonists and inhibitors to elucidate the mechanism of ASGR1 regulating the expression of cholesterol efflux-related factor SR-BI through PPARγ/LXRα,as well as the downstream of SR-BI modulating the P38 MAPK and NF-κB signaling pathways.Results:(1)ASGR1 knockout did not affect serum cholesterol levels in hypercholesterolemic mice,but inhibited lipid deposition in the aortic intima.Compared with the wild-type primary macrophages,ASGR1 knockout reduced macrophage foaming,decreased intracellular cholesterol level,and up-regulated expression of cholesterol efflux-related factor SR-BI induced by ox-LDL.Overexpression of ASGR1 in the wild-type primary macrophages produced the opposite effect compared to the ASGR1 knockout mice.(2)ASGR1 knockout reduced aortic inflammatory cell infiltration and decreased serum IL-6,IL-8,IL-1β and TNF-α levels in hypercholesterolemic mice.Compared with the primary macrophages of wild-type mice,macrophage M1 polarization,inflammatory cytokine secretion and ROS production induced by ox-LDL were inhibited in primary macrophages from ASGR1 knockout mice,while overexpression of ASGR1 in the primary macrophages of wild-type mice produced the opposite effect.(3)Upon oxldl stimulation,ASGR1 knockout up-regulated PPARγ/LXRα protein expression compared with the wild-type primary macrophages.Overexpression of ASGR1 in the primary macrophages of wild-type mice produced the opposite effect.Administration of the inhibitor of PPARγ/LXRα and si RNA could reduce SR-BI expression in ASGR1 knockout primary macrophages,while PPARγ/LXRα agonists increased SR-BI expression in ASGR1 knockout primary macrophages.(4)Compared with wild-type primary macrophages,ASGR1 knockout inhibited P38 and NF-κB signaling pathways induced by ox-LDL,while ASGR1 overexpression promoted P38 and NF-κB signaling pathways induced by ox-LDL in primary macrophages.Upon ox-LDL stimulation,silencing SR-BI reversed ASGR1 knockout-induced inactivation of p38 and NF-κB signaling pathways and inhibition of M1 polarization.Conclusion:This study demonstrates that ASGR1 knockout can reduce aortic lipid deposition and vascular inflammation without affecting serum cholesterol levels;The possible mechanism that ASGR1 contributes to macrophage cholesterol efflux may be ASGR1 regulating SR-BI expression through PPARγ/LXRα signaling pathway.And SR-BI-P38/NF-κB pathway is involved in ASGR1-mediated macrophage polarization.The elucidation of these mechanisms provides a theoretical basis for identifying potential therapeutic targets of atherosclerosis.