Gastrin attenuates sepsis-induced myocardial dysfunction by down-regulation of TLR4 expression in macrophages

Abstract

Myocardial dysfunction is the most serious complication of sepsis. Sepsis-induced myocardial dysfunction (SMD) is often associated with gastrointestinal dysfunction, but its pathophysiological significance remains unclear. The present study found that patients with SMD had higher plasma gastrin concentrations than those without SMD. In mice, knockdown of the gastrin receptor, cholecystokinin B receptor (Cckbr), aggravated lipopolysaccharide (LPS)-induced cardiac dysfunction and increased inflammation in the heart, whereas the intravenous administration of gastrin ameliorated SMD and cardiac injury. Macrophage infiltration plays a significant role in SMD because depletion of macrophages by the intravenous injection of clodronate liposomes, 48 h prior to LPS administration, alleviated LPS-induced cardiac injury in Cckbr-deficient mice. The intravenous injection of bone marrow macrophages (BMMs) overexpressing Cckbr reduced LPS-induced myocardial dysfunction. Furthermore, gastrin treatment inhibited toll-like receptor 4 (TLR4) expression through the peroxisome proliferator-activated receptor α (PPAR-α) signaling pathway in BMMs. Thus, our findings provide insights into the mechanism of the protective role of gastrin/CCKBR in SMD, which could be used to develop new treatment modalities for SMD.

Keywords: Cholecystokinin B receptor; Gastrin; Inflammation; Lipopolysaccharide; Macrophage; Peroxisome proliferators-activated receptor α; Sepsis-induced myocardial dysfunction; Toll-like receptor 4.

Graphical abstract

Figure 1

CCKBR deficiency aggravates LPS-induced cardiac dysfunction. (A) Plasma gastrin concentrations were measured using ELISA in patients with sepsis-induced myocardial dysfunction (SMD) and healthy controls (n = 10 per group). ^∗P < 0.05 vs. control. (B) Correlation between plasma gastrin concentrations and APACHE (the Acute Physiology and Chronic Health Evaluation) II scores in SMD patients. Each dot represents a single patient. The solid line is the regression plot. (C) Kaplan–Meier (K–M) analysis for the different experimental groups: WT (Cckbr^+/+) mice, gastrin receptor knock-out (Cckbr^−/−) mice, WT mice with LPS treatment (25 mg/kg, 200 μL, i.p., WT + LPS), Cckbr^−/− mice with LPS treatment (Cckbr^−/− +LPS). n = 10 per group; ^∗P < 0.05 vs. WT + LPS treatment. (D–H) Echocardiography of WT (Cckbr^+/+) and Cckbr^−/− mice with vehicle or LPS treatment (25 mg/kg, 200 μL, i.p.). Representative M-mode images 24 h after LPS treatment (D) and quantitative analysis of the left ventricular (LV) EF (E) and FS (F), LVEDD (G), and LVESD (H). n = 10 per group; ^∗P < 0.05 vs. WT + Vehicle; ^#P < 0.05 vs. WT + LPS treatment. (I) Representative H&E staining images of heart tissues from the different groups (I1) and quantitative analysis of myocardial injury (I2). n = 10 per group; ^∗P < 0.05 vs. WT + Vehicle; ^#P < 0.05 vs. WT + LPS treatment. (J) cTnT levels were quantified using ELISA in WT (Cckbr^+/+) and Cckbr^−/− mice with Vehicle or LPS treatment (25 mg/kg, i.p.). n = 10 per group; ^∗P < 0.05 vs. WT + Vehicle; ^#P < 0.05 vs. WT + LPS treatment.

Figure 2

Gastrin attenuates LPS-induced cardiac dysfunction. (A–E) Echocardiography of 129S2/SvPas mice treated with Vehicle or LPS (25 mg/kg, i.p.), followed with gastrin treatment. Gastrin (120 μg/kg) was injected into the tail vein immediately after LPS treatment. Representative M-mode images 24 h after treatment (A) and quantification of EF (B), FS (C), LVEDD (D), and LVESD (E). n = 10 per group; ^∗P < 0.05 vs. vehicle; ^#P < 0.05 vs. LPS. (F) Representative images of H&E staining (F1) and quantitative analysis (F2) of myocardial injury. n = 10; ^∗P < 0.05 vs. Vehicle treatment; ^#P < 0.05 vs. LPS treatment. (G) Plasma cTnT levels were quantified in 129S2/SvPas mice treated with vehicle alone or gastrin alone or also treated with LPS, as in (A). n = 10 per group; ^∗P < 0.05 vs. Vehicle; ^#P < 0.05 vs. LPS.

Figure 3

Gastrin/CCKBR ameliorates LPS-induced cardiac dysfunction by decreasing macrophage infiltration. (A) Represented images of F4/80 (macrophage marker, red) in the heart (A1) and quantification (A2) of F4/80⁺ staining cells per high power field (HPF). n = 10 per group; ^∗P < 0.05 vs. WT + Vehicle; ^#P < 0.05 vs. WT + LPS treatment. (B–D) TNF-α, IL-1β, and IL-6 mRNA expression in the heart of CCKBR wild type (WT) and Cckbr^−/− mice treated with CLS or vehicle (200 μL, i.v.) 2 days before LPS injection (25 mg/kg, 200 μL, i.p.). n = 5 per group; ^∗P < 0.05 vs. WT + LPS treatment; ^nsP > 0.05 vs. WT + LPS + CLS treatment. (E–I) Echocardiography of CCKBR wild type (WT) and Cckbr^−/− mice treated with vehicle or LPS. LPS and CLS treatments were the same as that in (B). Representative M-mode images and quantification of EF (F), FS (G), LVEDD (H), and LVESD (I). n = 5 per group; ^∗P < 0.05 vs. WT + LPS treatment; ^nsP > 0.05 vs. WT + LPS + CLS treatment.

Figure 4

Adoptive transfer of CCKBR-overexpressed BMMs ameliorates LPS-induced cardiac dysfunction. (A–E) Echocardiography of 129S2/SvPas mice without (BMM-Vector) or with CCKBR-overexpressed BMMs (BMM-CCKBR) treated with vehicle or LPS (25 mg/kg, i.p.). Representative M-mode images (A) and quantification of EF (B), FS (C), LVEDD (D), and LVESD (E). n = 10 per group; ^∗P < 0.05 vs. Vehicle + BMM-Vector; ^#P < 0.05 vs. LPS + BMM-Vector. (F) Representative images of H&E staining (F1) and quantitative analysis (F2) of myocardial injury in 129S2/SvPas mice without (BBM-Vector) or with CCKBR-overexpressed BMMs (BBM-CCKBR) and Vehicle or LPS treatment. n = 10 per group; ^∗P < 0.05 vs. vehicle + BMM-Vector; ^#P < 0.05 vs. LPS + BMM-Vector. (G) Plasma cTnT levels in 129S2/SvPas mice with or without CCKBR-overexpressed BMMs and LPS treatment. n = 10 per group; ^∗P < 0.05 vs. Vehicle + BMM-Vector treatment; ^#P < 0.05 vs. LPS + BMM-vector treatment.

Figure 5

CCKBR inhibits the expression of TLR4 and its downstream activation in BMMs. (A) TLR4 mRNA expression in BMMs from Cckbr^−/− or WT mice treated with Vehicle or LPS (100 ng/mL) for 24 h n = 5 per group; ^∗P < 0.05 vs. WT + Vehicle; ^#P < 0.05 vs. WT + LPS. (B) TLR4 mRNA expression in BMMs from 129S2/SvPas mice. BMMs treated with vehicle or LPS (100 ng/mL) for 24 h after CCKBR overexpression (CCKBR-OE) by transfection with pEnCMV-CCKBR. n = 5 per group; ^∗P < 0.05 vs. vector; ^#P < 0.05 vs. Vector + LPS treatment. (C) Represented immunofluorescence images with double staining of P65 (red) and DAPI (blue) in BMMs from 129S2/SvPas mice. The BMMs not over expressing (Vector) or overexpressing CCKBR (CCKBR-OR) by transfection with pEnCMV-CCKBR were treated with Vehicle (no LPS) or LPS. (D) Western blots of cytoplasmic and nuclear P65 in BMMs from 129S2/SvPas mice. The BMMs were treated with Vehicle or LPS as in (B) and (C). n = 5 per group. (E–G) TNF-α, IL-1β, and IL-6 mRNA expression in BMMs treated with LPS in BBMs not overexpressing CCKBR (Vector) or overexpressing CCKBR by transfection with pEnCMV-CCKBR (CCKBR-OE) as that in (B). TNF-α (E), IL-1β (F), and IL-6 (G) levels. n = 5 per group; ^∗P < 0.05 vs. Vector only; ^#P < 0.05 vs. Vector + LPS treatment. (H–J) Proinflammatory cytokines mRNA expression in BMMs from WT and Cckbr^−/− mice treated with LPS and TLR4 inhibitor TAK-242 (100 nmol/L): TNF-α (H), IL-1β (I), and IL-6 (J) levels. n = 5 per group; ^∗P < 0.05 vs. WT + LPS treatment; ^nsP > 0.05 vs. WT + LPS + TAK-242 treatment.

Figure 6

CCKBR/gastrin reduces LPS-induced inflammation in BMMs through PPAR-α. (A) Representative immunofluorescence images with double staining of CCKBR (red) and DAPI (blue) in BMMs treated with Vehicle, Gastrin (10⁻⁷ mol/L) alone, LPS (100 ng/mL) alone, or LPS + Gastrin for 24 h. The representative fluorescent images (left) and line intensity profiles (right) were acquired from the white arrows; the abscissa value is the distance from the detection point to the tail of the arrow. (B) Western blot analyses of nuclear and cytoplasmic distribution of CCKBR in BMMs treated with Vehicle, Gastrin (10⁻⁷ mol/L) alone, LPS (100 ng/mL) alone, or LPS + Gastrin for 24 h. (C) TLR4 mRNA expression in BMMs without CCKBR overexpression (Vector) or CCKBR overexpression (CCKBR-OE) with or without shPPAR-α and LPS treatment (100 ng/mL). n = 6 per group; ^nsP > 0.05 Vector + LPS + shPPAR-α vs. Vector + LPS treatment; ^∗P < 0.05 CCKBR-OE + LPS treatment vs. Vector + LPS treatment; ^#P < 0.05 vs. CCKBR-OE + LPS treatment. (D–F) Proinflammatory cytokines mRNA expressions in BMMs treated as that in (C). TNF-α (D), IL-1β (E), and IL-6 (F) levels. n = 6; ^nsP > 0.05 vs. Vector + LPS treatment; ^∗P < 0.05 vs. Vector + LPS treatment; ^#P < 0.05 vs. CCKBR-OE + LPS treatment.