Research On The Role And Mechanism Of HMGB1 In Atherosclerosis Progression Via Aggravating Inflammation

Objective:Inflammation of atherosclerosis is a critical factor to accelerate plaque progression and aggravating plaque instability. High mobility group box protein 1 (High Mobility Group Box 1, HMGB1) contribute to the development of inflammation through multiple pathways. This study aimed to investigating the role and mechanism of HMGB1 in atherosclerosis progression via aggravating inflammation.Methods:(1) Twelve 8-week-old SPF C57BL/6 male mice and twelve C57BL/6 ApoE-/-male mice were used as control group and experimental group, each group was divided into two subgroups, the early and medial stage group (10 weeks high fat diet) and late stage group (20 weeks high fat diet). After feeding for 10 weeks and 20 weeks, the mice were sacrificed. Aortic sinus frozen sections for oil red O staining was used to assess plaque size, macrophage immunohistochemical staining for localization of macrophages and aortic sinus paraffin sections for immunohistochemical staining to detect the expression of HMGB1. (2) 14 male ApoE-/-mice were randomly divided into 2 groups. One group of mice were fed with high-fat diet and received intraperitoneal injection of Ethyl pyruvate (EP,50 mg/kg) for 12 weeks (n=7). The other group was fed with high-fat diet and received intraperitoneal injection of equal volumes physiological saline for 12 weeks as a control. After 12 weeks high fat diet, the mice were sacrificed. Serum were collected for lipid levels measured. Aortic sinus frozen sections were used to Oil Red O staining to assess plaque size. Immunohistochemical staining used to assess macrophage content. The expression of HMGB1 was detected by immonohistochemical and western blot. RT-qPCR were used to investigate the mRNA levels of TLR2, TLR4, MCP-1 and TNF-a. (3) THP-1 cells were differentiated into macrophages by treated with PMA (100 ng/mL) and incubated with oxLDL only or added with EP (5mM) for 24 hour. Cell climbing film were stained with Oil red O to assess lipid uptake. Immunohistochemistry were used to assess HMGB1 expression. RT-qPCR were used to investigate the mRNA levels of inflammatory cytokines IL-1?.Results:(1) HMGB1 in normal mice aorta mainly expressed in endothelial cells nucleus, while in aorta atherosclerotic plaques of ApoE-/-mice it mainly expressed in nucleus and cytoplasm of endothelial cells and macrophages, and also in smooth muscle cell nuclei. HMGB1 expression in early and medial stage atherosclerosis was higher than advanced atherosclerosis (0.05±0.007 vs 0.02±0.005, P<0.05). Atherosclerotic plaque area ratio in advanced stage was higher than early stage (0.40±0.03 vs 0.25±0.02, P<0.05). (2) EP reduced the HMGB1 expression nearly by 85%. EP markedly reduced aortic sinus atherosclerosis lesions (289845±26450?m2 vs.499247±45880?m2, P<0.05) and plaque area ratio (22.7%±2.1% vs.39.1%±3%, P<0.05) with no influence to serum lipid levels. EP decreased macrophages content (11.3%±8% vs.39.24%±13.85%, p<0.05). The levels of TLR2, TLR4, TNF-a, MCP-1 in atherosclerosis plaque were decreased nearly by 59%,39%,45%,47% respectively. (3) oxLDL can induce the expression of IL-1? and HMGB1 in macrophage. EP can suppressed oxLDL-induced HMGB1 and IL-1? expression And decreased lipid uptake in macrophage (4.89% ± 0.8% vs.8.44%±1.2%,p<0.05).Conclusions:(1) HMGB1 in atherosclerotic plaques mainly expresses in endothelial cells and macrophages. The expression of HMGB1 in atherosclerosis is higher in early and medial stage than advanced stage. (2) HMGB1 aggravated atherosclerosis progression via promote the inflammation of plaque. (3) HMGB1 is high expression in foam cells. Inhibited of HMGB1 by EP can decreased inflammatory response and lipid uptake induced by oxLDL in macrophage.