Qishen Granule (QSG) Inhibits Monocytes Released From the Spleen and Protect Myocardial Function via the TLR4-MyD88-NF-κB p65 Pathway in Heart Failure Mice

Abstract

Preliminary clinical and basic researches have proved that Qishen granule (QSG) is an effective prescription for treating heart failure (HF) in China, with a characteristic of regulating the ratio of M1/M2 macrophage in the myocardium. However, the regulative mechanism of monocytes targeting the cardio-splenic axis has not been fully elucidated. This study aimed to investigate the effects and mechanism of QSG inhibiting the release of splenic monocytes and the recruitment of myocardial tissue both in vivo and in vitro. Experiments in mice with acute myocardial infarction (AMI)-induced HF demonstrated that QSG could exert anti-inflammatory effects by inhibiting splenic monocytes release and phenotypic changes. Moreover, in vitro experiments indicated QSG could inhibit LPS-stimulated macrophage-conditioned medium (CM)-induced H9C2 cardiomyocyte injury by upregulating the key proteins in TLR4-MyD88-NF-κB p65 pathway. In addition, knockdown or overexpression of TLR4 in H9C2 cells further confirmed that QSG could attenuate inflammatory injury in cardiomyocytes via the TLR4-MyD88-NF-κB p65 pathway. Overall, these data suggested that QSG could improve cardiac function and reduce the inflammatory response in AMI-induced HF by inhibiting splenic monocytes release, and protecting myocardial function via the TLR4-MyD88-NF-κB pathway in heart failure mice.

Keywords: heart failure; inflammation; macrophages; qishen granule; splenic monocytes.

FIGURE 1

QSG improved cardiac function and reduced pathological changes in HF mice. (A) Experimental protocol for QSG studies in AMI-induced HF mice. (B) Representative M-mode echocardiographic images of mice in each group. (C) Evaluation of LVEF, LVFS, LVID; d, LVID; s, LVAW; d, LVAW; s, LVPW; d, LVPW; s levels in each group by echocardiography. N = 6 per group. (D) Representative images of HE staining in each group. Scale bar = 20 µm. (E) The level of CK-MB and LDH in serum from each group. N = 5 per group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. model group.

FIGURE 2

QSG inhibited the pathological changes of the spleen and the release of splenic monocytes in HF mice. (A) Representative photomicrograph of the spleen morphology and histogram of length statistics. N = 6 per group. (B) Representative spleen images of HE staining in each group. Scale bar = 50 µm. (C) Representative spleen tissue images of CD11b immunofluorescence from different groups. Scale bar = 20 µm. (D) Flow cytometry for CD11b monocytes counts and statistical analysis in the spleen. N = 3 per group. (E) Flow cytometry to detect the number of Ly6C^high and Ly6C^low monocytes in the spleen and statistical analysis. N = 3 per group. **p < 0.05, ***p < 0.001 vs. model group.

FIGURE 3

QSG exerted an anti-myocardial inflammation effect through the TLR4-MyD88-NF-κB p65 pathway in HF mice. (A) Flow cytometry detection of CD11b monocytes in blood and statistical analysis. N = 3 per group. (B) Flow cytometry detection of monocytes/macrophages in heart and statistical analysis. N = 3 per group. Representative western blot images and quantification of three experiments of TLR4 (C), MyD88 (D), p-IκBα (E), and p-NF-κB p65 (F) in myocardial tissue. GAPDH was used as a loading control. N = 6 per group. **p < 0.01, ***p < 0.001 vs. model group.

FIGURE 4

QSG might attenuate inflammatory injury in cardiomyocytes by inhibiting the TLR4-MyD88-NF-κB p65 pathway. (A) The CCK8 assay showed that QSG treatment for 24 h had no cytotoxic effect on RAW264.7 macrophages below 1,500 μg/ml, N = 6 per group. (B) The level of NO in the supernatant of RAW264.7 macrophages was evaluated by NO kit determination, N = 6 per group. (C) The CCK8 assay showed that QSG treatment for 24 h had no cytotoxic effect on H9C2 cells below 1,500 μg/ml, N = 6 per group. (D) The effective concentrations of QSG in CM-stimulated H9C2 cells were analyzed by CCK8 assay, N = 6 per group. (E) The knockdown efficiency of TLR4 siRNA in H9C2 cells was checked by western blot analysis, N = 3 per group. (F) TLR4 siRNA treatment increased the viability of CM-stimulated H9C2 cells. N = 6 per group. Representative western blot images and quantification of TLR4 (G), MyD88 (H), p-IκBα and IκBα (I), p-NF-κB p65 and NF-κB p65 (J) in H9C2 cells from different groups, N = 3. GAPDH was used as a loading control. #p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. control group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. model group.

FIGURE 5

Validation of the regulatory effect by QSG on the TLR4-MyD88-NF-κB p65 pathway. (A) The structure of rTlr4 pcDNA3.1-T2A-DsRed plasmid. Representative western blot images and quantification of TLR4 (B), MyD88 (C), p-NF-κB p65 and NF-κB p65 (D) in H9C2 cells, N = 3 per group. GAPDH was used as a loading control. *p < 0.05, **p < 0.01, ***p < 0.001 vs. rTlr4 pcDNA3.1-T2A-DsRed transfected group. (E) Representative immunofluorescence staining images of p-IκBα and DsRed from different groups. H9C2 cells were transfected with rTlr4 pcDNA3.1-T2A-DsRed to check the overexpression of TLR4. pcDNA3.1-T2A-DsRed transfected cells were used as a negative control. *p < 0.05, **p < 0.01, ***p < 0.001 vs. rTlr4 pcDNA3.1-T2A-DsRed transfected group.

FIGURE 6

The underlying mechanism of QSG in the treatment of heart failure is mediated by TLR4-MyD88-NF-κB p65 pathway.