Lubiprostone as a potential therapeutic agent to improve intestinal permeability and prevent the development of atherosclerosis in apolipoprotein E-deficient mice

Abstract

The interaction between atherosclerosis and commensal microbes through leaky gut syndrome (LGS), which is characterized by impaired intestinal permeability and the introduction of undesired pathogens into the body, has not been fully elucidated. Our aim was to investigate the potential role of a ClC-2 chloride channel activator, lubiprostone, which is reported to have beneficial effects on LGS, in the development of atherosclerosis in apolipoprotein E-deficient (ApoE-/-) mice. After a 15-week feeding period of a Western diet (WD), ApoE-/- mice were treated with a Western-type diet (WD) alone or WD with oral supplementation of lubiprostone for 10 weeks. This feeding protocol was followed by experimental evaluation of LGS and atherosclerotic lesions in the aorta. In mice with lubiprostone, in vivo translocation of orally administered 4-kDa FITC-dextran was significantly improved, and RNA expression of the epithelial tight junction proteins, Zo-1 and occludin, was significantly up-regulated in the ileum, compared to the WD alone group, suggesting a possible reversal of WD-induced intestinal barrier dysfunction. As a result, WD-induced exacerbation of atherosclerotic lesion formation was reduced by 69% in longitudinally opened aortas and 26% in aortic root regions. In addition, there was a significant decrease in circulating immunoglobulin level, followed by an attenuation of inflammatory responses in the perivascular adipose tissue, as evidenced by reduced expression of pro-inflammatory cytokines and chemokines. Lubiprostone attenuates atherosclerosis by ameliorating LGS-induced inflammation through the restoration of the intestinal barrier. These findings raise the possibility of targeting LGS for the treatment of atherosclerosis.

Fig 1. Experimental design.

ND; normal diet, WD; western diet, Mg; magnesium hydroxide, Sen; sennoside, Lub; lubiprostone. FITC-Dx; fluorescein isothiocyanate-dextran.

Fig 2. Suppression of the development of atherosclerotic lesions by lubiprostone in longitudinally opened aortas.

(A) Representative images of an en-faced aorta, from the aortic arch to the common iliac arteries, visualized using staining Oil Red O. The (B) percentage (%) area of the entire aorta comprising the lesion section and (C) size of atherosclerotic lesions (mm²) were analyzed using ImageJ software. Data are presented as the mean±standard error of the mean (SEM), with 5 animals in each group. Global significance among multiple groups was determined by one-way ANOVA. ND; normal diet, WD; western diet, Mg; magnesium hydroxide, Sen; sennoside, Lub; lubiprostone. #:p<0.001, ✽:p = 0.001, ※:p<0.01, compared to the 25-week WD group.

Fig 3. Suppressive effects of lubiprostone on the development of atherosclerotic lesions, quantified on cross-sectional analysis of the aortic root.

Representative images of (A) mice in the 25-week WD group and (B) 25-week WD combined with lubiprostone group, analyzed using hematoxylin and eosin staining. The expression of macrophage antigen (F4/80) in each atherosclerotic lesion was detected and visualized by immunohistochemical staining (C and D); higher magnification images are shown for the highlighted areas. Scale bar = 300 μm. (E) Plaque size (μm²) and (F) the percentage of plaque-to-vessel area, analyzed using ImageJ software. (G and H) Macrophage infiltration and T cell infiltration were also assessed as the percentage of the F4/80-positive area and CD3-positive area to vessel area, respectively. Data are presented as the mean±standard error of the mean (SEM), with 5 animals in each group. Global significance among multiple groups was determined by one-way ANOVA. HE; hematoxylin and eosin staining. ND; normal diet, WD; western diet, Mg; magnesium hydroxide, Sen; sennoside, Lub; lubiprostone. HE; hematoxylin and eosin staining. ✽:p<0.01, #:p<0.05, compared to the 25-week WD group.

Fig 4. Lubiprostone improved intestinal barrier dysfunction.

(A) In vivo intestinal permeability, determined by the measurement of serum concentrations of FITC- dextran, 1 h after oral gavage. (B-C) mRNA levels of ZO-1 and occludin, extracted from the ileum, were determined using qPCR. (D–K) Representative images of the terminal ilea in the 25-week WD group (D and E) and 25-week WD combined with lubiprostone group (H and I), analyzed using hematoxylin and eosin staining and Masson’s trichrome. The expression of tight junction proteins such as ZO-1 (F and J) and Occludin (G and K) in serial sections was detected and visualized by immunohistochemical staining. Scale bar = 100 μm. Global significance among multiple groups was determined by one-way ANOVA. HE; hematoxylin and eosin staining, MT; Masson’s trichrome staining, ND; normal diet, WD; western diet, Mg; magnesium hydroxide, Sen; sennoside, Lub; lubiprostone. ✽:p<0.01, #:p<0.05, compared to the 25-week WD group.

Fig 5. Relative mRNA expression in PVAT was quantified using qPCR and normalized to that of Gapdh.

The relative ratio is shown compared to mRNA extracted from WD mice. IL1β; interleukin 1 beta, TNFα; tumor necrosis factor-α, IL1r1; interleukin 1 receptor, type 1, TLR5; toll-like receptor 5. Data are presented as the mean±standard error of the mean (SEM), with n = 5 in each group. Global significance among multiple groups was determined by one-way ANOVA. ND; normal diet, WD; western diet, Mg; magnesium hydroxide, Sen; sennoside, Lub; lubiprostone. ✽:p<0.01, #:p<0.05, compared to the 25-week WD group.

Fig 6. Relative mRNA expression in the spleen, quantified using qPCR and normalized to that of Gapdh.

The relative ratio is shown compared to the mRNA extracted from WD mice. IL1β; interleukin 1 beta, TNFα; tumor necrosis factor-α, IL1r1; interleukin 1 receptor, type 1, TLR5; toll-like receptor 5. Data are presented as the mean±standard error of the mean (SEM), with n = 5 in each group. Global significance among multiple groups was determined by one-way ANOVA. ND; normal diet, WD; western diet, Mg; magnesium hydroxide, Sen; sennoside, Lub; lubiprostone. ✽:p<0.01, #:p<0.05, compared to the 25-week WD group.

Fig 7. Lubiprostone suppressed the production of immunoglobulin through a lipid-independent pathway.

(A and B) Concentration of total IgG and IgG3, assessed using ELISA. (C–E) Serum levels of low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), and triglyceride (TG). Data are presented as the mean±standard error of the mean (SEM). n = 7 for A and B, n = 5 for all other observations. Global significance among multiple groups was determined by one-way ANOVA. ND; normal diet, WD; western diet, Mg; magnesium hydroxide, Sen; sennoside, Lub; lubiprostone. FITC-Dx; fluorescein isothiocyanate-dextran. ✽:p<0.01, #:p<0.05, compared to the 25-week WD group.