DBZ (Danshensu Bingpian Zhi), a Novel Natural Compound Derivative, Attenuates Atherosclerosis in Apolipoprotein E-Deficient Mice

Abstract

Background: DBZ (Danshensu Bingpian Zhi), a synthetic derivative of a natural compound found in traditional Chinese medicine, has been reported to suppress lipopolysaccharide-induced macrophage activation and lipid accumulation in vitro. The aim of this study was to assess whether DBZ could attenuate atherosclerosis at early and advanced stages.

Methods and results: The effects of DBZ on the development of atherosclerosis were studied using apolipoprotein E-deficient (apoE^-/-) mice. For early treatment, 5-week-old apoE^-/- mice were fed a Western diet and treated daily by oral gavage with or without DBZ or atorvastatin for 10 weeks. For advanced treatment, 5-week-old apoE^-/- mice were fed a Western diet for 10 weeks to induce atherosclerosis, and then they were randomly divided into 4 groups and subjected to the treatment of vehicle, 20 mg/kg per day DBZ, 40 mg/kg per day DBZ, or 10 mg/kg per day atorvastatin for the subsequent 10 weeks. We showed that early treatment of apoE^-/- mice with DBZ markedly reduced atherosclerotic lesion formation by inhibiting inflammation and decreasing macrophage infiltration into the vessel wall. Treatment with DBZ also attenuated the progression of preestablished diet-induced atherosclerotic plaques in apoE^-/- mice. In addition, we showed that DBZ may affect LXR (liver X receptor) function and that treatment of macrophages with DBZ suppressed lipopolysaccharide-stimulated cell migration and oxidized low-density lipoprotein-induced foam cell formation.

Conclusions: DBZ potentially has antiatherosclerotic effects that involve the inhibition of inflammation, macrophage migration, leukocyte adhesion, and foam cell formation. These results suggest that DBZ may be used as a therapeutic agent for the prevention and treatment of atherosclerosis.

Keywords: LXR; atherosclerosis; foam cell; inflammation.

Figure 1

DBZ reduces atherosclerotic plaque formation in apoE^−/− mice. Five‐week‐old male apoE^−/− mice were fed a Western diet and were administered DBZ (20 mg/kg per day [DBZ 20] or 40 mg/kg per day [DBZ 40]) or atorvastatin (10 mg/kg per day) daily for 10 weeks by gavage. A, Schematic of the experimental procedures. B, Representative images and quantification of Oil Red O–stained en face aortic preparations (n=5 per group). Aortic root section staining with Oil Red O (C) and MOMA‐2 (D) and quantification (scale=200 μm; n=6–10). E, Gene expression analysis of the indicated macrophage phenotypes, M1 (proinflammatory) and M2 (anti‐inflammatory), in the aortic arch with plaques of mice (n=5 per group). Data are presented as the mean±SEM. *P<0.05; **P<0.01. Arg 1 indicates arginase 1; Atorva, atorvastatin; COX‐2, cyclooxygenase 2; DBZ, Danshensu Bingpian Zhi; IL‐6, interleukin 6; MOMA‐2, monocyte macrophages 2; Mrc1, mannose receptor 1; NF‐κB, nuclear factor κB; TNFα, tumor necrosis factor α; TGF‐β, transforming growth factor β.

Figure 2

Treatment with DBZ inhibits the development of preestablished atherosclerotic plaques in apolipoprotein E–deficient (apoE^−/−) mice. A, Schematic of the experimental procedure for late DBZ intervention. B, Aortic valve (AV) peak velocity was measured using Doppler ultrasonography in mice. ^# P<0.05 vs WT; *P<0.05 vs vehicle. n=6 per group. C, Representative in situ photographs of the aortic arch and iliac artery regions with white plaques. D, Representative images and quantification of Oil Red O–stained en face aortic preparations (n=5 per group). Representative cross‐sections and quantification of (E) Oil Red O staining (scale bar=200 μm), (F) immunostaining for MOMA‐2 (scale bar=200 μm), (G) α‐SMA staining for smooth muscle actin (scale bar=100 μm), and (H) Masson's Trichrome staining (scale bar=100 μm) in the aortic sinus of the mice. All data are presented as the mean±SEM. *P<0.05; **P<0.01. α‐SMA indicates α‐smooth muscle actin; Atorva, atorvastatin; DBZ, Danshensu Bingpian Zhi; DBZ 20, DBZ 20 mg/kg per day; DBZ 40, DBZ 40 mg/kg per day; MOMA‐2, metallophilic macrophages 2; WT, wild type.

Figure 3

DBZ treatment inhibits systemic inflammation and LPS‐induced cell migration in RAW 264.7 macrophages. A, Quantification of the chemokines TNFα, IL‐6, and MCP‐1 in mouse serum by ELISA, as indicated in Figure 1 (n=6–10). RAW 264.7 cells pretreated with various concentrations of DBZ for 24 h were seeded onto Transwell chambers and stimulated with or without LPS (1 μg/mL) for 6 h in 3 independent experiments. B, Culture medium concentrations of TNFα, IL‐6, and MCP‐1 were assayed by ELISA. C, Representative images and quantification of migrated cells. D, Western blot analysis of MMP9 protein expression in RAW 264.7 macrophages. E, Relative VCAM and ICAM mRNA expression in the aortic arch was measured by quantitative reverse transcriptase polymerase chain reaction and normalized to cyclophilin, as indicated in Figure 1 (n=5 per group). F, VCAM‐1 protein expression in the aorta was assessed by Western blot analysis and normalized to GAPDH (n=3 per group). G, Representative images and quantification of VCAM‐1 staining in the intima layer at the aortic root (scale=100 μm; n=5 per group). Data are presented as the mean±SEM. ^### P<0.001 vs control group, *P<0.05; **P<0.01; and ***P<0.001 vs LPS group or vehicle. Atorva indicates atorvastatin; Con, control; DBZ, Danshensu Bingpian Zhi; DBZ 5, DBZ 5 μmol/L; DBZ 10, DBZ 10 μmol/L; DBZ 20, DBZ 20(B,C,D) 20 μmol/L, DBZ 20(A,E,F,G) 20 mg/kg/day; DBZ 40, DBZ 40 mg/kg/day; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; ICAM‐1, intercellular adhesion molecule 1; IL‐6: interleukin 6; LPS, lipopolysaccharide; MCP‐1, monocyte chemoattractant protein 1; MMP9, matrix metallopeptidase 9; TNFα, tumor necrosis factor α; VCAM‐1, vascular cell adhesion molecule 1.

Figure 4

DBZ attenuates ox‐LDL–induced foam cell formation and promotes cholesterol efflux in macrophages. A, THP‐1 macrophages were cotreated with the indicated concentrations of DBZ (5, 10, and 20 μmol/L) and OxLDL (50 μg/mL) for 48 h. Lipid accumulation was observed by Oil Red O staining (scale=20 μm). B, Intracellular cholesteryl ester levels were normalized to cellular protein content in THP‐1 macrophages. Macrophages were treated with DBZ and NBD cholesterol. Cholesterol efflux to apoA‐1 (C) and HDL (D) from THP‐1 macrophages. Cholesterol efflux was expressed as the percentage fluorescence in the medium relative to total fluorescence. E, THP‐1 macrophages were treated with the indicated concentrations of DBZ for 24 h. The relative protein levels of ABCA1 and ABCG1 were determined by Western blot analysis. T1317 (1 μmol/L) was used as a positive control. F, The transcriptional activity of LXRα was assessed by the transactivation reporter assay in 293T cells. G and H, The model structure of the complex formed by the LXRα ligand‐binding pocket and DBZ by molecular docking. I, THP‐1 macrophages were pretreated with GGPP (10 μmol/L) for 2 h and then treated with DBZ (20 μmol/L) for 24 h. The relative protein level of ABCA1 was determined by Western blot analysis. Prior to treatment with DBZ (20 μmol/L), THP‐1 macrophages were treated with GGPP for 2 h and then incubated with 50 μg/mL OxLDL for 48 h. J, Representative Oil Red O staining of THP‐1 macrophage foam cells (scale=20 μm). K, Intracellular cholesteryl ester levels that were normalized to cellular protein content in THP‐1 macrophages. Values are presented as the mean±SEM of at least 3 experiments (^### P<0.001, *P<0.05, and **P<0.01). ABCA1 indicates ATP binding cassette subfamily A member 1; ABCG1, ATP binding cassette subfamily G member 1; apoA‐1, apolipoprotein A1; Ctrl, control; DBZ, Danshensu Bingpian Zhi; DBZ 5, DBZ 5 μmol/L; DBZ 10, DBZ 10 μmol/L; DBZ 20, DBZ 20 μmol/L; GGPP, geranylgeranyl pyrophosphate; HDL, high‐density lipoprotein; LXRα, liver X receptor α; OxLDL, oxidized low‐density lipoprotein.

Figure 5

DBZ treatment affects cholesterol efflux gene expression in preestablished atherosclerotic plaque apolipoprotein E–deficient mice. A, Aortic, (B and C) hepatic, and intestinal mRNA levels of genes involved in the metabolism and transport of cholesterol as determined by real‐time polymerase chain reaction and normalized to cyclophilin in the mice, as in Figure 2 (n=6 per group). Data are expressed as the fold change plus or minus SEM (*P<0.05; **P<0.01). ABCA indicates ATP binding cassette subfamily A; ABCG, ATP‐binding cassette subfamily G; Atorva, atorvastatin; CD36, cluster of differentiation 36; DBZ, Danshensu Bingpian Zhi; DBZ 20, DBZ 20 mg/kg per day; DBZ 40, DBZ 40 mg/kg per day; HMGCoAR, 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase; HMGCoAS, 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A synthase; SR‐A, scavenger receptor class A.

Figure 6

Schematic presentation of the process of atherosclerosis attenuation by DBZ. DBZ suppresses NF‐κB activation and decreases pro‐inflammatory cytokine release and macrophage migration. Meanwhile, DBZ promotes cholesterol efflux and inhibits foam cell formation by activating the LXRα‐ABCA1 signaling pathway. ABCA1 indicates ATP binding cassette subfamily A member 1; apoA‐1, apolipoprotein A‐1; DBZ, Danshensu Bingpian Zhi; LXRα, liver X receptor α; NF‐κB, nuclear factor κB.