C-reactive protein/oxidised low-density lipoprotein/β2-glycoprotein I complex promotes atherosclerosis in diabetic BALB/c mice via p38mitogen-activated protein kinase signal pathway

Lipids in Health and Disease C-reactive protein/oxidised low-density lipoprotein/2-glycoprotein I complex promotes atherosclerosis in diabetic BALB/c mice via p38mitogen-activated protein kinase signal pathway Rui Zhang 0 Sai-Jun Zhou 0 Chun-Jun Li 0 Xiao-Nan Wang 1 Yun-Zhao Tang 0 Rui Chen 0 Lin Lv 0 Qian Zhao 0 Qiu-Ling Xing 0 De-Min Yu 0 Pei Yu 0 0 Department of Diabetic Nephropathy Hemodialysis, Key Laboratory of Hormones and Development (Ministry of Health), Metabolic Diseases Hospital & Tianjin Institute of Endocrinology Tianjin Medical University , Tongan Street, Heping District, Tianjin 300070 , China 1 Department of Hand Microsurgery, Tianjin Hospital , 300070, Tianjin , China Background: The aim of this study was to investigate the effect of C-reactive protein/oxidised low-density lipoprotein/2-glycoprotein I (CRP/oxLDL/2GPI) complex on atherosclerosis (AS) in diabetic BALB/c mice. Methods: BALB/c mice were fed high-fat and normal diet. Eight weeks later, the mice fed with high-fat diet were injected with streptozotocin (STZ) to induce diabetes. The diabetic mice were respectively injected twice monthly with 20 g oxLDL, 20 g 2GPI, 40 g oxLDL/2GPI complex, 44 g CRP/oxLDL/2GPI complex, and PBS. Aortas were stained with Sudan IV to investigate lipid plaque formation. The infiltration condition of smooth muscle cells (SMCs), macrophages, and T cells in the aortas were determined by immunohistochemistry (IH). The mRNA expressions of receptors associated with lipid metabolism were quantified by real-time PCR. The phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and MKK3/6 in aorta tissues were assessed by Western blot. The expression of inflammation cytokines was evaluated by protein chip. Results: The lipid plaques were more extensive, the lumen area was obviously narrower, the ratio of intima and media thickness were increased, and the normal internal elastic lamia structure and endothelial cell disappeared (P < 0.05) in the oxLDL and CRP/oxLDL/2GPI groups (P < 0.05). CRP/oxLDL/2GPI complex dramatically promoted infiltration of SMCs, macrophages, and T cells, improved the mRNA expression of ABCA1 and ABCG1, but reduced the mRNA expression of SR-BI and CD36 and increased the phosphorylation of p38MAPK and MKK3/6 (all P < 0.05). The highest expression levels of IL-1, IL-9, PF-4, bFGF, and IGF-II were detected in the CRP/oxLDL/2GPI group (P < 0.05). Conclusions: CRP/oxLDL/2GPI complex aggravated AS in diabetic BALB/c mice by increasing lipid uptake, the mechanism of which may be mediated by the p38MAPK signal pathway. Diabetes; Atherosclerosis; CRP/oxLDL/2GPI complex; p38MAPK; BALB/c mice - Background Atherosclerosis (AS) is the main etiology of diabetic macroangiopathy and the leading cause of disability and death of patients with diabetes mellitus [1]. Diabetic patients with cardiovascular diseases (CVD) possess more diffused AS lesions with more severe degrees compared with other CVD patients [2,3], suggesting that diabetic AS has a unique pathogenic mechanism. 2-glycoproteinI (2GPI), a highly glycosylated plasma protein with an approximate molecular weight of 50 kDa, was found to be the main antigen in the serum of anti-phospholipid syndrome (APS) patients [4]. 2GPI interacts with oxidised low density lipoprotein (oxLDL) via 7-ketocholesterol having an w-carboxyl acyl chain, producing stable and nondissociable oxLDL/2GPI complexes [5], which could further interacts with C-reactive protein (CRP), producing CRP/oxLDL/2GPI complex [6]. Recent studies have demonstrated that 2GPI complexes (oxLDL/2GPI and CRP/ oxLDL/2GPI) have a close relationship with AS [6,7]. Research has demonstrated that the levels of oxLDL/2GPI and CRP/oxLDL/2GPI complexes in the serum of diabetic patients were obviously higher than those of the normal control [6]. The amounts of these complexes have been found in AS plaque tissues using immunohistochemistry, indicating that 2GPI complexes have an important function in AS development [6,7]. In addition, Matsuura et al. discovered that the CRP/oxLDL/2GPI complex was significantly elevated only in diabetic AS patients [7], indicating that this complex is specific to diabetic AS but the pathogenic mechanism is not clear. Thus, this study further explored the effect of CRP/oxLDL/2GPI complex on AS occurrence and on the development of diabetic mice through in vivo research and the possible pathogenic mechanism. Results Blood glucose and body weight After STZ injection, the mice in the DM group all became diabetic, but no animal died until they were sacrificed. After the mice were fed with high-fat and sugar diet for eight weeks, the body weight of the DM group became noticeably higher, which was different from that of group NC (P<0.05). The DM group significantly lost weight after STZ injection for three weeks compared with the NC group (P<0.05), then gradually gained weight because of continuous subsistence on high-fat diet (Figure 1). The blood glucose of the DM group was three times higher than that of the NC group (P<0.01). Plasma lipid level of diabetic BALB/c mice Plasma triglycerol (TG) and total cholesterol (TC) levels were higher in DM group than in NC group. The TC levels of oxLDL, CRP/oxLDL/2GPI, and PBS groups were higher than those of NC group (P< 0.05), whereas the TG level did not exhibit significant difference in all the DM groups (Figure 2). The 2GPI and oxLDL/ 2GPI groups showed decreased levels of plasma TG and TC. Figure 1 Body weight of DM group and NC group. , DM group (n=120); , NC group (n=24). *P < 0.05, compared with group NC. Maintained on high-fat and sugar diet and standard chow diet, respectively, mice in DM group and NC group were weighed every week. Eight weeks later, DM group was intraperitoneally injected with 80 mg/kg 2% STZ three times for three consecutive days. The tail vein blood glucose was measured 72 h after the injection, and those with blood glucose 16.7 mmol/L were considered DM mice. The NC group was simultaneously injected with sodium citrate buffer. Two weeks later, the DM group was treated with oxLDL 20 g, 2GPI 20 g, oxLDL/2GPI complex 40 g, CRP/oxLDL/2GPI complex 44 g, and PBS, after which they were randomly divided into oxLDL group, 2GPI group, oxLDL/ 2GPI group, CRP/oxLDL/2GPI group, and PBS group, respectively (each group n=24). The same interventions were boosted four weeks later. NC group were injected with PBS at the same time. Four weeks later, blood was obtained via retro-orbital plexus and then sacrificed by cervical dislocation. Figure 2 Plasma lipid level of the DM group significantly increased. *P < 0.05, compared to group NC. CRP/oxLDL/2GPI promotes the formation of atherosclerotic plaque in aortas Pale yellow wax-like hills were spread along the aortic intima of the DM group, which were lined with lipid streak and atherosclerotic plaque. The entire aortic intima was uneven with a stiff wall. Tissues of the DM group seemed crisper and more difficult to exten (...truncated)