The atheroprotective roles of heart-protecting musk pills against atherosclerosis development in apolipoprotein E-deficient mice

Abstract

Background: Heart-protecting musk pill (HMP), derived from Chinese herbal medicines, has been found to possess protective roles against atherosclerosis-related cardiovascular diseases (CVDs), however, the anti-atherosclerotic mechanisms of HMP are still unclear. Here, we investigated the effects of HMP on alleviating atherosclerotic lesion severity in mice and explored the molecular mechanisms.

Methods: Apolipoprotein E-deficient mice were fed western-type diet supplemented with HMP (25 mg/kg/day) or normal saline gavage for 20 weeks. Then histopathological staining was performed to assess the atheromatous plaque burden. Biochemical kits were used to detect levels of lipid profiles. Moreover, effector factors associated with lipid metabolism in liver and intestinal tissues were investigated by western blot and real-time PCR assays. Levels of signal molecules participating in the mitochondrial-mediated apoptosis pathway were detected by Western blot.

Results: We found that HMP notably reduced atherosclerotic lesion size (P<0.05) and improved plaque stability (P<0.05). HMP treatment decreased circulating TC (P<0.01), LDL-C (P<0.01) and TG (P<0.05) levels and increased HDL-C (P<0.05) content. HMP was found to suppress SREBP2, HMGCR and PCSK9 expressions (P<0.05), yet promote LDLR expression (P<0.05) in hepatocytes. Moreover, HMP was discovered to activate PPARα/CPT-1A cascade (P<0.05) and inhibit contents of SREBP1c and the lipogenic genes FAS and ACCα (P<0.05). The LBK1/AMPK cascade was also activated after HMP administration (P<0.05). Additionally, HMP was found to facilitate transintestinal cholesterol excretion by increasing ABCG5 and ABCG8 levels and reducing NPC1L1 content (P<0.05). In terms of vasoprotective activities, we observed that HMP decreased cleaved caspase-3 content (P<0.05) in the vascular intima, which might be due to inhibition of mitochondrial-related signaling pathway.

Conclusions: Altogether, our study indicates that HMP plays anti-atherosclerotic roles via regulating lipid metabolism and improving vascular intimal injury.

Keywords: Heart-protecting musk pill (HMP); atherosclerosis; lipid metabolism; vascular intimal injury.

Figure 1

The schematic experimental procedure of this study. HMP, heart-protecting musk pill.

Figure 2

Heart-protecting musk pill (HMP) administration attenuated the progression of atherosclerosis in apoE^−/− mice. (A) Representative images of plaque lesions in the area of aortic inner surface stained with Oil Red O. The arrows indicated the plaques in the aortic inner surface. (B) H&E staining assessed the extent of plaque-induced luminal stenosis and the degree of lesion hyperplasia in three segments of the entire aorta. The arrows showed the plaques in the cross-section. Data were expressed as the mean ± SD, n=5. *P<0.05, **P<0.01 vs. WTD group. Scale bar =200 µm.

Figure 3

The effects of heart-protecting musk pill (HMP) on the stability of atherosclerotic lesions in the aortic root. (A) The size of necrotic core in the plaque area was determined by H&E staining. The arrows showed the necrotic core in the plaques. (B) The collagen content was detected with Masson’s Trichrome staining. The arrows indicated the area of collagen. (D) Immunostaining for MOMA-2 (C) and α-SMA (D) in the lesion area. The arrows indicated the representative area. Results were presented as the mean ± SD, n = 5. *P<0.05, **P<0.01 vs. WTD group. Scale bar =200 µm.

Figure 4

Heart-protecting musk pill (HMP) treatment played effective roles in the regulation of lipid profiles in the bloodstream and the liver. (A) The concentrations of serum TG, TC, LDL-C and HDL-C. (B) Representative micrographs of liver sections stained with H&E and Oil Red O, respectively. (C) The contents of liver TG and TC. Data were expressed as the mean ± SD, n=6. *P<0.05, **P<0.01 vs. WTD group. Scale bar =200 µm.

Figure 5

Heart-protecting musk pill (HMP) affected the expressions and activities of molecules participating in hepatic lipid metabolism. (A) Protein levels of SREBP2 and downstream effectors involved in cholesterol biosynthesis and uptake. (B) Signal transduction of Raf/ERK1/2 pathway was examined by western blot. The protein expression of CYP7A1 (C), PPARα and CPT-1A (D). (E) Protein levels of SREBP1c and downstream factors involved in de novo lipogenesis. (F) Total protein content and corresponding phosphorylated level of AMPK, LKB1 and CaMKKβ. (G) The mRNA expressions of effector genes associated with cholesterol (left part) and fatty acid (right part) metabolism. Results were presented as the mean ± SD, n=6. *P<0.05, **P<0.01 vs. WTD group.

Figure 6

The regulatory roles of heart-protecting musk pill (HMP) in intestinal lipid metabolism. (A) The contents of TC and TG in the fecal particles. (B) The food intake and feces weight of apoE^−/− mice per week. (C) Representative images of intestine sections stained with H&E. The arrows showed the features of the intestine tissue. The protein (D) and mRNA (E) levels of ABCG5, ABCG8 and NPC1L1. Data were expressed as the mean ± SD, n=6. *P<0.05, **P<0.01 vs. WTD group. Scale bar =50 µm.

Figure 7

Oral gavage of heart-protecting musk pill (HMP) displayed protective effects against vascular wall injury. (A) Concentrations of serum TNF-α, IL-1β, IL-6 and CRP were detected by ELISA kits. (B) The expression of cleaved caspase-3. The arrows showed the positive area of cleaved caspase-3. (C) Protein levels of Bcl-2 family members in the aortic tissue. (D) The contents of signal proteins associated with mitochondrial-dependent apoptosis pathway. (E) The concentration of serum NO. Data were expressed as the mean ± SD, n=6 for (A) and (E), n=4 for (B-D). *P<0.05, **P<0.01 vs. WTD group. Scale bar =50 µm.

Figure 8

The schematic illustration of the potential molecular mechanisms by which heart-protecting musk pill (HMP) alleviated atherosclerosis development.