Xin-Li formula attenuates heart failure induced by a combination of hyperlipidemia and myocardial infarction in rats via Treg immunomodulation and NLRP3 inflammasome inhibition

Fig. 1. Base peak intensity (BPI) chromatograms of XLF in the positive (A) and negative ion mode (B).

Fig. 2. Left ventricular function. Left ventricular function was measured by echocardiography. Representative M-mode echocardiograms are shown in (A). (B–G) LVEF, LVFS, LVESV, LVESD, LVEDV, LVEDD are shown as mean ± S.D (n = 6). ∗∗P < 0.01 versus Sham group, #P < 0.05 versus Model group. ∗∗P < 0.01 versus Sham group, #P < 0.05 versus Model group, ##P < 0.01 versus Model group.

Fig. 3. Left ventricular fibrosis, infarct size and cardiac index. Left ventricular fibrosis and infarct size were assessed by Masson staining. Representative images are shown in (A & B). Scale bar = 50 μm in (A). (C–D) The percentages of infarct size and quantitation of peri-infarct interstitial fibrosis (% area) are shown as mean ± S.D (n = 5). (E) Cardiac index for each rat was calculated using the following formula: heart weight/bodyweight × 100%. Data are shown as mean ± S.D. (n = 5–6). ∗∗P < 0.01 versus Sham group, #P < 0.05 versus Model group, ##P < 0.01 versus Model group.

Fig. 4. CD4+CD25+Foxp3+ Tregs frequencies in lymph node and spleen cells. Draining lymph node and spleen cells were isolated and CD4+CD25+Foxp3+ Tregs were detected using flow cytometric analysis. Representative images are shown in (A). (B–C) The percentages of CD4+CD25+Foxp3+ Tregs in LN and spleen are shown as mean ± S.D (n = 6). ∗P < 0.05 versus Sham group, ##P < 0.01 versus Model group.

Fig. 5. The expressions of mTOR, p-mTOR and Foxp3 in rat hearts. (A)The protein expressions of mTOR, p-mTOR and Foxp3 in heart tissues were determined by Western blot analysis. p-mTOR/mTOR ratio and Foxp3/GAPDH ratio were quantified as shown in (B) and (C). The distribution of Foxp3 in heart tissues were also assessed by immunohistochemistry (scale bar = 50 μm). Representative images are shown in (D) and quantitative assessment of Foxp3 positive areas are shown in (E). (F) The expressions of Foxp3 in mRNA level were analyzed by qPCR. Measurements were performed in three different samples and data are shown as mean ± S.D. ∗P < 0.05 versus Sham group, ∗∗P < 0.01 versus Sham group, #P < 0.05 versus Model group, ##P < 0.01 versus Model group.

Fig. 6. The protein expressions of NLRP3/caspase-1/IL-1β signaling pathway in rat hearts. (A) The protein expressions of NLRP3 and caspase-1 in heart tissues were determined by Western blot analysis. NLRP3/GAPDH ratio, pro-caspase-1/GAPDH ratio, caspase-1/GAPDH ratio, pro-IL-1β/GAPDH ratio and IL-1β/GAPDH ratio were quantified as shown in (B–F). Measurements were performed in three different samples and data are shown as mean ± S.D. (G) Serums were collected to evaluate the level of IL-1β. Data are shown as mean ± S.D (n = 6). ∗P < 0.05 versus Sham group, ∗∗P < 0.01 versus Sham group, #P < 0.05 versus Model group, ##P < 0.01 versus Model group.

Fig. 7. CD25+Foxp3+ Tregs frequencies and mTOR phosphorylation in CD4+ T cells. (A) CD4+ T cells were treated with indicated concentrations of XLF for 72 h, and cell viability was analyzed by CCK-8 kit. (B) CD25+Foxp3+ Tregs were detected using flow cytometric analysis. (C) The percentages of CD25+Foxp3+ Tregs in CD4+ T cells are shown as mean ± S.D. (D) The protein expressions of mTOR and p-mTOR in CD4+ T cells were determined by Western blot analysis. p-mTOR/mTOR ratio was quantified as shown in (E). Data were presented as mean ± SD (n = 3). ∗∗P < 0.01 versus untreated control, ##P < 0.01 versus XLF treatment.

Fig. 8. IL-1β and IL-18 levels and NLRP3 expression in LPS/ATP-induced THP-1 macrophages. (A) THP-1 macrophages were treated with indicated concentrations of XLF for 24 h, and cell viability was analyzed by CCK-8. (B–C) The supernatants of THP-1 macrophages were collected to evaluate the level of IL-1β and IL-18 after indicated treatment. (D) The protein expression levels of NLRP3 in THP-1 macrophages were analyzed by Western blotting. NLRP3/GAPDH ratio was quantified as shown in (E). Data were presented as mean ± SD (n = 3). ∗∗P < 0.01 versus untreated control, ^P < 0.05 versus LPS/ATP, ^^ P < 0.01 versus LPS/ATP, #P < 0.05 versus XLF treatment.