A Simplified Herbal Formula Improves Cardiac Function and Reduces Inflammation in Mice Through the TLR-Mediated NF-κB Signaling Pathway

Figures

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  FIGURE 1. NXK improves the cardiac function of mice with heart failure. (A) Representative M-mode images via echocardiography. (B) Representative cardiac function (n = 10 per group). LVDD (mm), left ventricular end-diastolic dimension; LVDS (mm), left ventricular end-systolic dimension; LVEF (%), left ventricular ejection fraction. (C) mRNA expression levels of ANP and BNP were detected using RT-PCR. Quantification was performed after normalization to β-actin (n = 10 per group). Values are shown as means ± SD, <0.05, compared to the sham group *p < 0.05, **p < 0.01, ***p < 0.001, compared to the Tac group #p < 0.05, ##p < 0.01, ###p < 0.001.

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  FIGURE 2. The effect of NXK on myocardial fibrosis in mice with heart failure. (A) Myocardial fibrosis was evaluated through Masson’s Trichrome staining (n = 10 per group). The fibrotic area is shown in blue. (B) Quantification of fibrosis area in the sham, TAC, low-, mid- and high-dose NXK groups. (C) mRNA expression levels of α-SMA and TGF-β quantified by RT-PCR (n = 10 per group). Quantification was performed after normalization to β-actin. (D) mRNA expression levels of Col1, Col3, and MMP9 quantified by RT-PCR (n = 6). Quantification was performed after normalization to GAPDH. Values are shown as means ± SD, compared to the sham group *p < 0.05, **p < 0.01, ***p < 0.001, compared to the Tac group #p < 0.05, ##p < 0.01, ###p < 0.001.

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  FIGURE 3. NXK reduces the inflammation due to heart failure by inhibiting the infiltration of pro-inflammatory monocytes/macrophages. (A) Representative images of heart tissue from the sham, TAC, perindopril, and NXK groups. DAPI staining (blue), CD11b (green), and Ly6c (red) are indicated (n = 6 per group). (B) mRNA expression levels of IL-1, IL-6, TNF-α, ICAM-1, and VCAM-1 quantified by RT-PCR (n = 10 per group). Values are shown as means ± SD, compared to the sham group *p < 0.05, **p < 0.01, ***p < 0.001, compared to the Tac group #p < 0.05, ##p < 0.01, ###p < 0.001.

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  FIGURE 4. Western blot analysis was used to measure the protein level of TLR4/NF-κB pathway proteins. (A) Expression of TLR4, MyD88, NF-κB p65 proteins in different mice groups (n = 10 per group). (B) Quantification was performed after normalization to Tubulin. Values are shown as means ± SD, <0.05, compared to the sham group *p < 0.05, **p < 0.01, ***p < 0.001, compared to the Tac group #p < 0.05, ##p < 0.01, ###p < 0.001.

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  FIGURE 5. Western blot analysis was used to measure the protein levels of IκBα and P-IκBα. Expression levels of IκBα and P-IκBα proteins in different mice groups (n = 6 per group). Quantification was performed after normalization to Tubulin. Values are shown as means ± SD, compared to the sham group *p < 0.05, **p < 0.01, ***p < 0.001, compared to the Tac group #p < 0.05, ##p < 0.01, ###p < 0.001.

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  FIGURE 6. Western blot analysis was used to measure the protein levels of NF-κB p65 and P-IκBα, treated by NXK (M), TAK242, and LPS. (A) Expression of NF-κB p65 protein in different mice groups. (B) Expression levels of NF-κB p65 and P-IκBα proteins in different groups (n = 6 per group). Quantification was performed after normalization to Tubulin. Values are shown as means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, vs. LPS group.

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  FIGURE 7. Western blot analysis was used to measure the protein levels of TLR2, TLR7, and TLR9 pathway proteins. (A) Expression levels of TLR2, TLR7, and TLR9 proteins in different mice groups and quantification was performed after normalization to GAPDH (n = 6 per group). (B) Values are shown as means ± SD, compared to the sham group *p < 0.05, **p < 0.01, ***p < 0.001, compared to the Tac group #p < 0.05, ##p < 0.01, ###p < 0.001.