Xiao-Qing-Long Tang Prevents Cardiomyocyte Hypertrophy, Fibrosis, and the Development of Heart Failure with Preserved Ejection Faction in Rats by Modulating the Composition of the Gut Microbiota

Figures

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  Figure 1. Study design. The preparation of the microbiota (a) is described in Sections 3.1–3.6 and in the second paragraph of the Discussion. Fecal microbiota transplantation (b) is described in Sections 3.7to 3.8 and in the third paragraph of the Discussion. The prepared gut microbiota was transplanted into the corresponding groups (c) as described in the Materials and Methods.

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  Figure 2. XQLT reduces systolic blood pressure and increases body weight in rats with heart failure with preserved ejection fraction (HFpEF). The effects of XQLT treatment on (a) systolic blood pressure and (b) body weight are shown. The data was presented as the mean±SD. (n=6 rats per group) ∗ p < 0.05 and ∗∗ p < 0.01 compared with the NS group; # p <0.05 and ## p <0.01 compared with the HS group.

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  Figure 3. XQLT improves echocardiographic indexes, protects cardiac function, and ameliorates organ weight changes. Echocardiographic indexes such as the (a) wall thickness (WT), (b) left ventricular mass (LV mass), (c) left ventricular diastolic diameter (LVDd), and (d) left ventricular ejection fraction (LVEF) were assessed using an M5 Vet Veterinary Ultrasound system. The (e) heart weight/body weight and (f) lung weight/body weight ratios are shown. (g) NT-proBNP was assessed by ELISA. The data are presented as the mean±SD (n=6 rats per group) ∗ p < 0.05 and ∗∗ p < 0.01 compared with the NS group; # p <0.05 and ## p <0.01 compared with the HS group.

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  Figure 4. Treatment with XQLT prevents fibrosis in rats with heart failure with preserved ejection fraction (HFpEF). (a) Masson staining of the left ventricle is shown; the % area of fibrosis was assessed by analysis of the Masson staining. (b) The fibrosis-associated protein expression of collagen I and collagen III in the left ventricle was assessed using western blot analysis and was compared with that in the NS group. The data are presented as the mean±SD (n=6 rats per group). ∗ p < 0.05 and ∗∗ p < 0.01 compared with the NS group; # p <0.05 and ## p <0.01 compared with the HS group.

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  Figure 5. Treatment with XQLT prevented inflammation in rats with heart failure with preserved ejection fraction (HFpEF). (a) The inflammation-associated protein expression of MCP-1, TNF-α, and IL-6 in the left ventricle was assessed using western blot analysis and was compared with that in the NS group. (b) HE staining of the left ventricle is shown; inflammatory cell infiltration was assessed by analysis of the HE staining. The data are presented as the mean±SD (n=6 rats per group). ∗ p < 0.05 and ∗∗ p < 0.01 compared with the NS group; # p <0.05 and ## p <0.01 compared with the HS group.

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  Figure 6. The beneficial effects of XQLT on the small intestine and on endotoxemia. (a) HE staining of the small intestine is shown. (b) ZO-1 mRNA expression in the intestinal mucosa was assessed using qRT-PCR and was compared with that in the NS group. (c) The levels of serum endotoxin are shown. The data are presented as the mean±SD (n=6 rats per group). ∗ p < 0.05 and ∗∗ p < 0.01 compared with the NS group; # p <0.05 and ## p <0.01 compared with the HS group.

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  Figure 7. Effect of XQLT on the gut microbiota. (a) Nonmetric multidimensional scaling (NMDS), (b) principal component analysis (PCA), and (c) principal coordinate analysis (PCoA) were performed between the NS group, the HS group, and the XQLT group. (d) Composition of the bacteria at the genus level. (e) LEfSe analysis of the bacterial taxa between the HS group and the XQLT group. (f, g, h) The levels of (f) acetate, (g) propionate, and (h) butyrate in feces. The data are presented as the mean± SD (n=6 rats per group). ∗ p < 0.05 and ∗∗ p < 0.01 compared with the NS group; # p <0.05 and ## p <0.01 compared with the HS group.

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  Figure 8. Effects of fecal transplantation from XQLT-treated rats on systolic blood pressure and body weight. Compared to that from rats in the FMT HS group, fecal transfer from rats in the NS group or the XQLT group reduced the increase in (a) systolic blood pressure and delayed the loss of (b) body weight. The data are presented as the mean± SD (n=5 rats per group). ∗ p < 0.05 and ∗∗ p < 0.01 compared with the FMT NS group; # p <0.05 and ## p <0.01 compared with the FMT HS group.

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  Figure 9. Effects of fecal transplantation from XQLT-treated rats on echocardiography measurements, organ weights, and blood test results. Seven-week-old specific pathogen-free male Dahl salt-sensitive rats were fed 8% NaCl chow for 11 weeks and colonized with fecal samples from different groups for 4 weeks. Then, the (a) wall thickness (WT) (b) left ventricular mass (LV mass), (c) left ventricular diastolic diameter (LVDd), (d) left ventricular ejection fraction (LVEF), (e) heart weight/body weight ratios, (f) lung weight/body weight ratios, and (g) NT-proBNP levels were measured. The data are presented as the mean± SD (n=5 rats per group). ∗ p < 0.05 and ∗∗ p < 0.01 compared with the FMT NS group; # p <0.05 and ## p <0.01 compared with the FMT HS group.

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  Figure 10. Analysis of fibrosis following transplantation from XQLT-treated rats. (a) Masson staining of the left ventricle and the area of fibrosis. (b) The fibrosis-associated protein expression of collagen I and collagen III in the left ventricle was assessed using western blot analysis and was compared with that in the FMT NS group. The data are presented as the mean± SD (n=5 rats per group). ∗ p < 0.05 and ∗∗ p < 0.01 compared with the FMT NS group; # p <0.05 and ## p <0.01 compared with the FMT HS group.

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  Figure 11. Effects of fecal transposition from XQLT-treated rats on proinflammatory cytokines and inflammatory cell infiltration. (a) The inflammation-associated protein expression of MCP-1, TNF-α, and IL-6 in the left ventricle was assessed using western blot analysis and was compared with that in the FMT NS group. (b) HE staining of the left ventricle is shown; inflammatory cell infiltration was assessed by analysis of the HE staining. The data are presented as the mean± SD (n=5 rats per group). ∗ p < 0.05 and ∗∗ p < 0.01 compared with the FMT NS group; # p <0.05 and ## p <0.01 compared with the FMT HS group.

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  Figure 12. Effect of fecal transposition from XQLT-treated rats on the small intestine. (a) HE staining of the small intestine. (b) The expression of ZO-1 in the intestinal mucosa was assessed using qRT-PCR and was compared with that in the FMT NS group. (c) The levels of serum endotoxin. The data are presented as the mean± SD (n=5 rats per group). ∗ p < 0.05 and ∗∗ p < 0.01 compared with the FMT NS group; # p <0.05 and ## p <0.01 compared with the FMT HS group.