TLR4 antagonist attenuates atherogenesis in LDL receptor-deficient mice with diet-induced type 2 diabetes

Abstract

Although a large number of studies have well documented a key role of toll-like receptor (TLR)4 in atherosclerosis, it remains undetermined if TLR4 antagonist attenuates atherogenesis in mouse model for type 2 diabetes. In this study, we induced type 2 diabetes in low-density lipoprotein receptor-deficient (LDLR(-/-)) mice by high-fat diet (HFD). At 8 weeks old, 20 mice were fed HFD and 20 mice fed regular chow (RC) for 24 weeks. In the last 10 weeks, half HFD-fed mice and half RC-fed mice were treated with Rhodobacter sphaeroides lipopolysaccharide (Rs-LPS), an established TLR4 antagonist. After the treatment, atherosclerotic lesions in aortas were analyzed. Results showed that the HFD significantly increased bodyweight, glucose, lipids including total cholesterol, triglycerides and free fatty acids, and insulin resistance, indicating that the HFD induced type 2 diabetes in LDLR(-/-) mice. Results also showed that Rs-LPS had no effect on HFD-increased metabolic parameters in both nondiabetic and diabetic mice. Lipid staining of aortas and histological analysis of cross-sections of aortic roots showed that diabetes increased atherosclerotic lesions, but Rs-LPS attenuated atherogenesis in diabetic mice. Furthermore, immunohistochemical studies showed that Rs-LPS reduced infiltration of monocytes/macrophages and expression of interleukin (IL)-6 and matrix metalloproteinase-9 in atherosclerotic lesions of diabetic mice. Finally, the antagonistic effect of Rs-LPS on TLR4 was demonstrated by our in vitro studies showing that Rs-LPS inhibited IL-6 secretion from macrophages and endothelial cells stimulated by LPS or LPS plus saturated fatty acid palmitate. Taken together, our study demonstrated that TLR4 antagonist was capable of attenuating vascular inflammation and atherogenesis in mice with HFD-induced type 2 diabetes.

Keywords: Atherosclerosis; Diabetes; Inflammation; Toll-like receptor 4.

Figure 1

The effects of regular chow (RC) or high-fat diet (HFD) and Rs-LPS on bodyweight, glucose, insulin and cholesterol. A and B: The changes of bodyweight and fasting blood glucose during 24 weeks. C and D: Fasting plasma insulin and total cholesterol levels at the end of the study. The lines reflect the means of all mice in each group. HOMA-IR: homeostasis model assessment of insulin resistance (see calculation formula in Materials and Methods). Data are mean +/− SD (n=10). **p<0.05 vs *.

Figure 2

Reduction of atherosclerotic lesions in LDLR^−/− mice by Rs-LPS. A: Sudan IV staining of en face aortas. B: Quantification of atherosclerotic lesion area. Data show mean ± SD (n=10). C: Representative images from histological analysis of atherosclerotic lesions in aortic roots. The arrows point to the intimal lesions. D: Quantification of intimal lesion area of atherosclerotic plaques. Data show mean ± SD (n=10). RC: regular chow. HFD: high-fat diet. Bar = 500 μm.

Figure 3

Inhibition by Rs-LPS of monocyte and macrophage accumulation in atherosclerotic lesions of LDLR^−/− mice. A: Immunohistochemical staining of CD68. B: Quantification of lesion area with positive CD68 immunohistochemical staining. Data show mean ± SD (n=10). RC: regular chow. HFD: high-fat diet. Bar = 200 μm.

Figure 4

Inhibition of IL-6 and MMP-9 expression in atherosclerotic lesions of LDLR^−/− mice by Rs-LPS. A and C: Immunohistochemical staining of IL-6 (A) and MMP-9 (C). B and D: Quantification of IL-6 (B) and MMP-9 protein (D). Data show mean ± SD (n=10). RC: regular chow. HFD: high-fat diet. Bar = 200 μm.

Figure 5

Inhibition of collagen accumulation in atherosclerotic lesions of diabetic LDLR^−/− mice by Rs-LPS. A: Sirius red staining of collagen in atherosclerotic lesions. B: Quantification of collagen (pixels per aorta). Data show mean ± SD (n=10). RC: regular chow. HFD: high-fat diet.

Figure 6

Diabetes, but not Rs-LPS, changes serum inflammatory cytokine levels. The serum levels of IL-1β (A) and TNFα (B) in mice of 4 groups were quantified using ELISA. RC: regular chow. HFD: high-fat diet.

Figure 7

Inhibition of IL-6 secretion from macrophages (A) and vascular endothelial cells (B) by Rs-LPS. Human macrophages and aortic endothelial cells were treated with 100 ng/ml of LPS, 100 μM of palmitic acid (PA) or LPS plus PA in the absence or presence of 100 ng/ml of Rs-LPS for 24 h. After the treatment, IL-6 in culture medium was quantified using ELISA.