Qing-Xin-Jie-Yu Granule attenuates myocardial infarction-induced inflammatory response by regulating the MK2/TTP pathway

Abstract

Context: Qing-Xin-Jie-Yu Granule (QXJYG) has shown promise in the treatment of myocardial infarction. However, the mechanism of action of QXJYG underlying its anti-inflammation remain unknown.

Objective: The study aimed to evaluate the effectiveness and mechanism of QXJYG in a mouse model of myocardial infarction and hypoxia-induced H9C2 cells.

Materials and methods: Myocardial infarction was induced in mice via left anterior descending coronary artery ligation, and hypoxia-induced H9C2 cells was served as the in vitro model. The cardiac function was evaluated by echocardiography, while myocardial tissue pathology was examined using HE and Masson's trichrome staining. Changes in serum markers of cardiac injury were measured using ELISA kits. The levels of inflammatory cytokines in both the serum and cardiac tissue were quantified using the Bio-Plex Pro Mouse Chemokine assay, and hypoxia-induced inflammatory factors in H9C2 cells were assessed by RT-qPCR. Additionally, western blot analysis was conducted to evaluate the expression of proteins related to the MK2/TTP signaling pathway both in vivo and in vitro experiments.

Results: QXJYG significantly enhanced cardiac function in mice with myocardial infarction, as evidenced by improved myocardial tissue structure, reduced collagen fiber deposition, and lowered serum levels of creatine kinase isoenzyme MB (CK-MB), cardiac Troponin T (cTnT), and brain Natriuretic Peptide (BNP). QXJYG may reduce the expression of inflammatory factors in both the heart and serum of myocardial infarction-induced mice and attenuate hypoxia-induced levels of inflammatory factors in cardiomyocytes by decreasing the ratio of p-MK2/MK2 and increasing the protein expression of TTP.

Discussion and conclusions: QXJYG improved cardiac function and reduced injury, fibrosis, and inflammation after myocardial infarction, likely through modulation of the MK2/TTP signaling pathway.

Keywords: Ischemic heart disease; inflammation-induced injury; traditional Chinese medicine formula.

Figure 1.

QXJYG improves cardiac function in mice after myocardial infarction surgery. (A and B) Representative images of echocardiographic in each group were presented. (C) The heart weight index (heart weight/body weight) was calculated and presented as a histogram (n = 6). (D–G) The echocardiography measurement of EF, FS, LVIDd, LVIDs was shown (n = 6). (H) The echocardiography measurement of MPI was shown (n = 5). ***P < 0.001,vs. Sham group. ^#P < 0.05; ^##P < 0.01; ^###P < 0.001,vs. Model group. EF: ejection fraction; FS: fractional shortening; LVIDd: ventricular internal diameter in diastole; LVIDs: left ventricular internal diameter in systole; MPI: myocardial performance index.

Figure 2.

QXJYG improves pathological changes of heart and the markers of myocardial injury in mice after myocardial infarction surgery. (A) Representative images of HE staining of each group (scale bar = 50 μm) were displayed; The area indicated by the black arrow showed inflammatory cell infiltration; Magnification, ×400. (B and C) Masson’s trichrome–stained histological pictures and quantitation of the area of fibrosis (blue) in cardiac tissues of mice from each group (scale bar = 50 μm) were displayed. Magnification, ×400. (D–F) CK-MB, cTnT and BNP levels in the serum of each group were determined by ELISA kits, respectively (n = 5). **P < 0.01;***P < 0.001,vs. Sham group. ^#P < 0.05; ^##P < 0.01; ^###P < 0.001,vs. Model group. CK-MB: creatine kinase isoenzyme MB; cTnT: cardiac troponin T; BNP: brain natriuretic peptide.

Figure 3.

QXJYG alleviates inflammatory cytokine levels both in the serum and cardiac tissue of mice after myocardial infarction surgery. (A and B) Levels of pro-inflammatory factors IL-1β, TNF-α, IFN-γ, MCP-1, MIP-1α, and IL-12(p40) in the serum and cardiac tissue of each group were performed via Bio-Plex pro mouse chemokine assay, respectively. **P < 0.01; ***P < 0.001,vs. Sham group. ^#P < 0.05; ^##P < 0.01; ^###P < 0.001,vs. Model group. IL: interleukin; TNF: tumor necrosis factor; IFN: interferon; MCP: monocyte chemoattractant protein; MIP: macrophage inflammatory protein.

Figure 4.

QXJYG regulates the MK2/TTP sgnaling pathway in the cardiac tissues of mice after myocardial infarction surgery. (A-C) The protein levels of p-MK2, MK2, TTP and Vinculin were measured via western blot analysis (n = 3); Vinculin was used as the internal control. **P < 0.01; ***P < 0.001,vs. Sham group. ^#P < 0.05,vs. Model group. MK2: Mitogen-activated protein kinase-activated protein kinase 2; TTP: tristetraprolin.

Figure 5.

QXJYG reduces inflammatory cytokine content in hypoxia-induced H9C2 cells via the MK2/TTP pathway suppression. (A and B) The optimal concentrations of QXJYG and ISMN intervention in H9C2 cells was evaluated by CCK-8 assay, respectively. (C–E) The mRNA levels of pro-inflammatory factors IL-6, IL-1β and TNF-α were measured via RT-qPCR analysis (n = 3). (F–H) The protein levels of p-MK2, MK2, TTP and Vinculin were measured via western blot analysis (n = 3); Vinculin was used as the internal control. *P < 0.05; ***P < 0.001,vs. Control group. ^# P< 0.05; ^##P < 0.01; ^###P < 0.001,vs. Model group. IL: interleukin; TNF: tumor necrosis factor; MK2: mitogen-activated protein kinase-activated protein kinase 2; TTP: tristetraprolin.