The Cordyceps militaris-Derived Polysaccharide CM1 Alleviates Atherosclerosis in LDLR(-/-) Mice by Improving Hyperlipidemia

Abstract

Atherosclerotic cardiovascular disease has a high mortality worldwide. Our lab previously purified a polysaccharide designated as CM1 with (1→4)-β-D-Glcp and (1→2)-α-D-Manp glycosyls as the backbone. In this study, we investigated the anti-atherosclerosis effect of CM1 and the underlying mechanisms of action in a low-density lipoprotein receptor knockout (LDLR^(-/-) mouse model. It was found that CM1 significantly decreased the formation of atherosclerotic plaques. Mechanistically, CM1 enhanced plasma level of apolipoprotein A-I and decreased the plasma levels of triglyceride, apolipoprotein B, and total cholesterol. In the absence of LDLR, CM1 elevated the expression of very low-density lipoprotein receptor for liver uptake of plasma apolipoprotein B-containing particles and reduced hepatic triglyceride synthesis by inhibiting sterol regulatory element binding protein 1c. CM1 improved lipids excretion by increasing the liver X receptor α/ATP-binding cassette G5 pathway in small intestine. CM1 reduced lipogenesis and lipolysis by inhibiting peroxisome proliferator-activated receptor γ and adipose triglyceride lipase in epididymal fat. Furthermore, CM1 improved lipid profile in C57BL/6J mice. Collectively, CM1 can modulate lipid metabolism by multiple pathways, contributing to reduced plasma lipid level and formation of atherosclerotic plaques in LDLR^(-/-) mice. This molecule could be explored as a potential compound for prevention and treatment of hyperlipidemia and atherosclerosis.

Keywords: atherosclerosis therapy; bioactive polysaccharide; hyperlipidemia; lipid homeostasis; pcsk9.

FIGURE 1

Structure and effect of polysaccharide CM1 on the formation of atherosclerotic plaques in the LDLR^(-/-) mice (n ≥ 6). (A), structure of CM1; (B), elution curve of CM1 on a TOSOH TSKgel G5000PWXL column (7.8 mm × 300 mm); (C), representative images of the aortic tree; (D), representative images of whole aorta after Oil Red O staining; (E), statistical analysis of the lesion/lumen ratio in the aorta root; (F), representative cross-sectional lesions of the aorta root. Data are presented as mean ± SD. Blank: chow diet group; Model: high-fat and–cholesterol diet group; Simv.: simvastatin; CM1-L: LDLR^(-/-) mice treated with CM1 at the dose of 25 mg/kg/d; CM1-H: LDLR^(-/-) mice treated with CM1 at the dose of 100 mg/kg/d. ^*means p < 0.05 vs. Model, and ^**means p < 0.01 vs. Model. All the abbreviations are applied for the rest of the figures.

FIGURE 2

Effect of CM1 on the body weight, fat pad index, plasma lipid profiles and expression of apoAI and apoB (n ≥ 4). (A), body weight of the mice in each group; (B), fat pad index of the mice in each group. Fat pad index means the percentage of fat pad to body weight; (C), plasma TC concentrations; (D), plasma TG concentrations; (E), plasma TC profiles of the lipoproteins after ÄKTA-FPLC separation; (F), plasma TG profiles of the lipoproteins after ÄKTA-FPLC separation; (G), plasma apoAI expression and densitometric quantification; (H), plasma apoB expression and densitometric quantification. ^*means p < 0.05 vs. Model, and ^**means p < 0.01 vs. Model.

FIGURE 3

Effect of CM1 on TC metabolism related proteins in the liver and plasma of the LDLR^(-/-)mice (n ≥ 4). Protein expression and densitometric quantification (A), SR-B1 in the liver; (B), SREBP-2 in the liver, precursor (∼125 kDa) and the cleaved mature form (∼68 kDa); (C), PCSK9 in the liver; (D), PCSK9 in the plasma; (E), VLDLR in the liver; (F), LXRα in the liver; (G), ABCG5 in the liver; (H), ABCG8 in the liver. ^*means p < 0.05 vs. Model, and ^**means p < 0.01 vs. Model.

FIGURE 4

Effect of CM1 on TG metabolism related proteins in the liver and plasma of the LDLR^(-/-) mice (n ≥ 4). Protein expression and densitometric quantification. (A), SREBP-1c, precursor (∼125 kDa) and cleaved mature form (∼68 kDa); (B), apoB; (C), PPARα; (D), PPARγ; (E), LPL; (F), LPL activity in the plasma. ^*means p < 0.05 vs. Model.

FIGURE 5

Effect of CM1 on the mRNA expression of lipid metabolism related genes in the liver of the LDLR^(-/-) mice (n = 3). (A), SR-BI; (B), SREBP-2; (C), LXRα; (D), ABCG5; (E), PPARα; (F), PPARγ; (G), SCD-1; (H), FAS; (I), ACC-1. ^*means p < 0.05 vs. Model, and ^**means p < 0.01 vs. Model; ^&&means p < 0.01 vs. Simvastatin.

FIGURE 6

Effect of CM1 on the expression of lipid metabolism related genes and proteins in the small intestine of the LDLR^(-/-) mice (n ≥ 4). Protein expression and densitometric quantification. (A), NPC1L1; (B), SREBP-2; (C), SREBP-1c; (D), LXRα; (E), ABCG5; (F), ABCG8; (G), mRNA expression of LXRα; (H), mRNA expression of ABCG5. ^*means p < 0.05 vs. Model, and ^**means p < 0.01 vs. Model.

FIGURE 7

Effect of CM1 on the expression of lipid metabolism related genes and proteins in the epididymal fat of the LDLR^(-/-) mice (n ≥ 4). Protein expression and densitometric quantification. (A), PPARα; (B), PPARγ; (C), ATGL; (D), mRNA expression of PPARγ. ^*means p < 0.05 vs. Model, and ^**means p < 0.01 vs. Model.

FIGURE 8

Effect of CM1 on the lipid profile and protein levels in the plasma of the C57BL/6J mice (n ≥ 4). (A), body weight of the mice; (B), fat pad index of the mice; (C), plasma TC concentrations; (D), plasma TG concentrations; (E), plasma apoAI expression and densitometric quantification; (F), plasma apoB expression and densitometric quantification; (G), plasma PCSK9 expression and densitometric quantification. ^#means p < 0.05 vs. Blank; ^##means p < 0.01 vs. Blank; ^*means p < 0.05 vs. Model, and ^**means p < 0.01 vs. Model.

FIGURE 9

Effect of CM1 on the lipid metabolism related proteins in the liver of the C57BL/6J mice (n ≥ 3). Protein expression and densitometric quantification. (A), SR-BI; (B), SREBP-2; (C), LDLR; (D), PCSK9; (E), HNF1α; (F), PPARα; (G), mRNA expression of FAS; (H), mRNA expression of ACC-1. ^#means p < 0.05 vs. Blank; ^##means p < 0.01 vs. Blank; ^*means p < 0.05 vs. Model, and ^**means p < 0.01 vs. Model.

FIGURE 10

The proposed mechanisms of action of CM1 in mice.