Non-alcoholic steatohepatitis aggravates nitric oxide synthase inhibition-induced arteriosclerosis in SHRSP5/Dmcr rat model

Abstract

Non-alcoholic steatohepatitis (NASH) is linked to increased cardiovascular risk, independent of the broad spectrum of metabolic syndrome risk factors. Stroke-prone (SP) spontaneously hypertensive rats (SHRSP5/Dmcr) fed a high-fat and high-cholesterol (HFC) diet developed hepatic lesions similar to those in human NASH pathology. These rats simultaneously developed lipid deposits in the mesenteric arteries, cardiac fibrosis, endothelial dysfunction and left ventricle (LV) diastolic dysfunction. However, the intermediary factors between NASH and cardiovascular disease are still unknown. We investigated whether NASH aggravates nitric oxide (NO) synthase inhibition-induced arteriosclerosis in SHRSP5/Dmcr rats. Wistar Kyoto and SHRSP5/Dmcr rats were divided into 4 groups of 5 and fed the stroke-prone (SP) or HFC diets for 8 weeks. To induce NO synthase inhibition, N^ω -nitro-L-arginine methyl ester hydrochloride (L-NAME) mixed with drinking water was administered in the final 2 weeks. The NASH+L-NAME group demonstrated the following characteristics related to arteriosclerosis and myocardial ischaemia: (a) LV systolic dysfunction with asynergy, (b) replacement fibrosis caused by the shedding of cardiomyocytes and (c) arterial lipid deposition and coronary occlusion secondary to endothelial dysfunction. These characteristics were not observed in the NASH or non-NASH+L-NAME groups. The SHRSP5/Dmcr rat model demonstrates that NASH significantly aggravates cardiovascular risk.

Keywords: SHRSP5/Dmcr; atherosclerosis; endothelial function; myocardial ischaemia; non-alcoholic steatohepatitis.

Figure 1

A, Changes in body weight (BW) and B, systolic blood pressure (SBP) at 9‐17 weeks of age. Four groups were evaluated: the control (CONT) group comprising WKY rats fed a spoke‐prone (SP) diet; the NASH group comprising SHRSP5/Dmcr rats fed a high‐fat and high‐cholesterol (HFC) diet; the non‐NASH+L‐NAME group comprising SHRSP5/Dmcr rats fed an SP diet and injected with N^ω‐nitro‐L‐arginine methyl ester hydrochloride (L‐NAME) at 25 mg/kg/day for 2 weeks; and the NASH+L‐NAME group comprising SHRSP5/Dmcr rats fed an HFC diet and injected with L‐NAME at 25 mg/kg/day. Data are presented as means ± standard error (SE); n = 5 in each group. *P < 0.05 vs. the CONT group, ^† P < 0.05 vs. the NASH group, ^‡ P < 0.05 vs. the non‐NASH+L‐NAME group

Figure 2

Pathological condition of the livers of the 4 groups is compared at 18 weeks of age. A, Macroscopic findings of the livers in the abdominal cavities. B, Masson's trichrome staining to detect hepatic fibrosis. Scale bars = 200 μm. C, D, Hepatic function in the blood biochemistry assay. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are measured. E, The liver weights are corrected by the BWs. Data are presented as means ± standard error (SE); n = 5 in each group. *P < 0.05 vs. the CONT group, ^† P < 0.05 vs. the NASH group, ^‡ P < 0.05 vs. the non‐NASH+L‐NAME group [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Evaluation of left ventricle (LV) hypertrophy in the 4 groups at 18 weeks of age. A‐E, Each organ weight is corrected according to its BW in the A, heart, B, atrium, C, ventricle, D, right ventricle (RV) and E, LV. F, Haematoxylin and eosin (HE) staining of LV myocardium cross sections. The area of myocardial fibrosis is selected in the NASH+L‐NAME group. Scale bars = 100 μm. Data are presented as means ± standard error (SE); n = 5 in each group. *P < 0.05 vs. the CONT group, ^† P < 0.05 vs. the NASH group, ^‡ P < 0.05 vs. the non‐NASH+L‐NAME group [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

The evaluation of myocardial fibrosis related to myocardial ischaemia in the 4 groups at 18 weeks of age. A, Collagen deposition revealed by picro‐sirius red (PSR) staining in whole LV myocardium. Scale bars = 1 mm. B, Collagen deposition revealed by Masson's trichrome staining in the perivascular and interstitial regions. Scale bars = 100 μm. C‐E, Myocardial infarction (MI)‐related serum biochemistry markers, including C, lactate dehydrogenase (LDH), D, creatine kinase (CK), E, and cardiac troponin T, were measured using routine laboratory methods. F, Interstitial fibrosis area was corrected according to total LV dimension in Figure 4A. G, Coronary smooth muscle cell (SMC) hypertrophy was evaluated by correcting SMCs by the dimension of the intravascular lumen. Data are presented as means ± standard error (SE); n = 5 in each group. *P < 0.05 vs. the CONT group, ^† P < 0.05 vs. the NASH group, ^‡ P < 0.05 vs. the non‐NASH+L‐NAME group [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5

Thickening and occlusion of the coronary artery adjacent to the fibrotic area in the NASH+L‐NAME group. The arterial lumen is narrowed with intimal cell proliferation and occluded with thrombus formation (A, EVG staining). Fibrin deposits are observed in the thickened intima and nearly occlude the small lumen (B, PTAH staining). Scale bars = 100 μm [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6

Immunohistochemical staining of MI‐related cells: A, myofibroblasts, B, macrophages, C, T cells, D, T‐helper cells, E, cytotoxic T cells and F, B cells in the MI area of the NASH+L‐NAME group at 18 weeks of age. Target antigenic markers are α‐smooth muscle actin (α‐SMA), CD68, CD3, CD4, CD8 and CD20 respectively. Scale bars = 200 μm [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 7

Echocardiography in the M‐mode on LV echograms in the 4 groups at 18 weeks of age. The non‐NASH+L‐NAME group represents severe LV hypertrophy, and the NASH+L‐NAME group represents depressed LV contractile function

Figure 8

Lipid deposition in the branches of the A, mesenteric artery B, and aorta in the 4 groups at 18 weeks of age. Isolated mesenteric arteries and the aorta were stained with oil red O to visualize lipid deposition. Scale bar = A, 2 mm, B, 1 mm [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 9

Endothelium‐dependent relaxation curve induced by acetylcholine (Ach) in the aortic rings. The statistical analysis among the 4 experimental groups was performed with ACh 10^−3.5 mol/L. All data are presented as means ± SE; n = 5 in each group. *P < 0.05 vs. the CONT group, ^† P < 0.05 vs. the NASH group, ^‡ P < 0.05 vs. the non‐NASH+L‐NAME group