Guanxinning Tablet Attenuates Coronary Atherosclerosis via Regulating the Gut Microbiota and Their Metabolites in Tibetan Minipigs Induced by a High-Fat Diet

Abstract

Coronary atherosclerosis (CA) is a chronic and evolving inflammatory disease characterized by the build-up of atherosclerotic plaque in the wall of coronary arteries. Guanxinning tablet (GXNT) is a novel Chinese medicine formula, which has been clinically used to treat coronary heart disease for many years. However, the potential mechanism for treating CA remains unclear. Thus, the study was aimed at investigating the therapeutic effect of GXNT on CA and further explore the underlying mechanisms from the perspective of gut microbiota. Following the establishment of a CA model in Tibetan minipigs, GXNT was orally administrated. We simultaneously detected blood lipid levels, observed ventricular function using ultrasound examination, measured platelet aggregation, and checked changes in inflammatory factors, oxidative stress factors, and vascular endothelial injury-related indexes applying ELISA assays. Histopathological changes of coronary artery tissue were subsequently evaluated using Sudan IV staining, HE staining, Oil red "O" staining, and immunohistochemistry assays. Finally, alterations of the gut microbiota and microbial metabolites were detected using metagenomic sequencing and targeted metabolomics, respectively. The results have suggested that GXNT could regulate dyslipidemia, improve heart function, and inhibit the levels of ox-LDL, CRP, TNF-α, IL-1β, SOD, MDA, vWF, and ET-1, as well as platelet aggregation. Additionally, histopathological findings revealed that GXNT could reduce lipid deposition, alleviate AS lesions, and restrain the expressions of NF-κB, TNF-α, and MMP-9. Furthermore, the composition of the gut microbiota was altered. Specifically, GXNT could upregulate the relative abundance of Prevotellaceae and Prevotella and downregulate the abundance of Proteobacteria, Enterobacteriaceae, and Escherichia. As for microbial metabolites, GXNT could increase fecal propionic acid, butyric acid, and LCA-3S and decrease fecal TMA-related metabolites, CDCA, and serum TMAO. In sum, the results showed that GXNT had a satisfactory anti-CA effect, and the mechanism was closely associated with modulating gut microbiota and related metabolites.

Figure 1

Effects of GXNT on physical signs of the CA model. (a) Representative pictures of habitus. (b) Habitus index. (c) Total fat weight. (d) Abdominal fat weight. (e) Backfat thickness. n = 6 in each group. Data are presented as the means ± SE. ^##P < 0.01 vs. the control group; ^∗P < 0.05 or ^∗∗P < 0.01 vs. the model group.

Figure 2

Effects of GXNT on blood lipids of the CA model. Changes of (a) TG, (b) TC, (c) LDL-C, and (d) HDL-C levels and (e) AI during 12 weeks of GXNT administration. n = 6 in each group. Data are presented as the means ± SE. ^#P < 0.05 or ^##P < 0.01 vs. the control group; ^∗P < 0.05 or ^∗∗P < 0.01 vs. the model group.

Figure 3

Effects of GXNT on serum inflammatory factors of the CA model. (a) ox-LDL, (b) CRP, (c) TNF-α, and (d) IL-1β after 12 weeks of GXNT treatment. n = 6 in each group. Data are presented as the means ± SE. ^#P < 0.05 or ^##P < 0.01 vs. the control group; ^∗P < 0.05 or ^∗∗P < 0.01 vs. the model group.

Figure 4

Effects of GXNT on serum SOD and MDA of the CA model. (a) SOD activity. (b) MDA content. n = 6 in each group. Data are presented as the means ± SE. ^##P < 0.01 vs. the control group; ^∗∗P < 0.01 vs. the model group.

Figure 5

Effects of GXNT on serum ET-1 and vWF of the CA model. The contents of (a) ET-1 and (b) vWF after GXNT treatment for 12 weeks. n = 6 in each group. Data are presented as the means ± SE. ^##P < 0.01 vs. the control group; ^∗P < 0.05 or ^∗∗P < 0.01 vs. the model group.

Figure 6

Effects of GXNT on left ventricular structure and function of the CA model induced by HF diet. (a) LVd mass. (b) IVSd. (c) LVPWd. (d) EF. (e) FS. n = 6 in each group. Data are presented as the means ± SE. ^#P < 0.05 or ^##P < 0.01 vs. the control group; ^∗P < 0.05 vs. the model group.

Figure 7

Effects of GXNT on lipid deposition in aortic vessels of the CA model. (a) The representative figures of Sudan IV staining of aortic vessels. (b) Quantitative results of lipid deposition percentage in aortic vessels. n = 6 in each group. Data are presented as the means ± SE. ^##P < 0.01 vs. the control group; ^∗∗P < 0.01 vs. the model group.

Figure 8

Effects of GXNT on the pathological morphology of coronary artery tissue of the CA Model. (a) Representative images of HE staining of the coronary vessels. Left: lower magnification (scale bars, 500 μm); right: higher magnification (scale bars, 100 μm) of the black-boxed area. (b) IMT. (c) NIA. (d) NIA/MA. (e) NIA/IELA. IMT: intima-media thickness; NIA: neointimal area; NIA/MA: neointimal area/media area; NIA/IELA: neointimal area/internal elastic layer area. n = 6 in each group. Data are presented as the means ± SE. ^##P < 0.01 vs. the control group; ^∗P < 0.05 or ^∗∗P < 0.01 vs. the model group.

Figure 9

Effects of GXNT on lipid deposition of coronary artery tissue of the CA model. (a) Representative images of oil red “O” staining of coronary vessels. Top: lower magnification (scale bars, 500 μm); bottom: higher magnification (scale bars, 100 μm) of the black-boxed area. (b) Quantitative analysis of coronary vascular lipid deposition. n = 6 in each group. Data are presented as the means ± SE. ^##P < 0.01 vs. the control group; ^∗P < 0.05 vs. the model group.

Figure 10

Effects of GXNT on the expressions of NF-κB, TNF-ɑ, and MMP-9 in coronary artery tissue of the CA model. The protein expressions of (a) NF-κB, (b) TNF-ɑ, and (c) MMP-9 by immunohistochemical staining. Left: lower magnification (scale bars, 500 μm); right: higher magnification (scale bars, 100 μm) of the black-boxed area. The positive expression rates of (d) NF-κB, (e) TNF-ɑ, and (f) MMP-9 by quantitative analysis. n = 6 in each group. Data are presented as the means ± SE. ^##P < 0.01 vs. the control group; ^∗P < 0.05 or ^∗∗P < 0.01 vs. the model group.

Figure 11

Effects of GXNT on the composition of gut microbiota in the CA model. (a) Effect of GXNT on the diversity of gut microbiota in the CA Model. (b, d, g) Effects of GXNT on the relative abundance distribution of gut microbiota at the level of phylum (top10), family (top20), and genus (top20) in the CA model. (c, e, f, h, i) Effects of GXNT on the relative abundance of Proteobacteria, Enterobacteriaceae, Prevotellaceae, Escherichia, and Prevotella. n = 6 in both the control and GXNT groups, n = 5 in the model group. Data are presented as the means ± SE. ^#P < 0.05 vs. the control group; ^∗P < 0.05 vs. the model group.

Figure 12

Effects of GXNT on the module function and pathway of gut microbiota in the CA model. Effects of GXNT on the relative abundance of (a) Entner-Doudoroff pathway-related microbiota, (b) pentose phosphate cycle-related microbiota, (c) hydroxypropionate pathway-related microbiota, (d) lipopolysaccharide production-related microbiota, (e) phosphoacetyltransferase-acetate kinase pathway-related microbiota, (f) choline production-related microbiota, (g) MAPK pathway-related microbiota, and (h) AMPK pathway-related microbiota. M00008: Entner-Doudoroff pathway, glucose-6P=>glyceraldehyde-3P+pyruvate. M00167: reductive pentose phosphate cycle, glyceraldehyde-3P=>ribulose-5P. M00376: 3-hydroxypropionate bi-cycle. M00060: lipopolysaccharide biosynthesis, KDO2-lipid A. M00579: phosphate acetyltransferase-acetate kinase pathway, acetyl-CoA=>acetate. M00555: betaine biosynthesis, choline=>betaine. Map04016: MAPK signaling pathway-plant. Map04152: AMPK signaling pathway. n = 6 in both the control and GXNT groups, n = 5 in the model group. ^#P < 0.05 or ^##P < 0.01 vs. the control group; ^∗P < 0.05 or ^∗∗P < 0.01 vs. the model group.

Figure 13

Effects of GXNT on fecal SCFAs in the CA model. n = 6 in each group. ^#P < 0.05 vs. the control group; ^∗∗P < 0.01 vs. the model group.

Figure 14

Effects of GXNT on TMA-related metabolites in the CA model. Effects of GXNT on (a) fecal total TMA-related metabolites, (b) fecal choline, and (c) serum TMAO. n = 6 in each group. ^#P < 0.05 or ^##P < 0.01 vs. the control group; ^∗P < 0.05 vs. the model group.

Figure 15

Effects of GXNT on fecal BAs in the CA model. The contents of metabolite BAs in the (a) control group, (b) model group, and (c) GXNT group were detected using LC-MS. Effects of GXNT on fecal (d) CDCA and (e) LCA-3S. n = 6 in each group. ^#P < 0.05 or ^##P < 0.01 vs. the control group; ^∗P < 0.05 vs. the model group.