Protective effects of rolipram on endotoxic cardiac dysfunction via inhibition of the inflammatory response in cardiac fibroblasts

Figures

• 

  Fig. 1. Effects of LPS on the cardiac fibroblast inflammatory responses and DUSP1 expression. After cardiac fibroblasts were stimulated with 1 μg/ml LPS for 6 h, the mRNA levels and supernatant concentrations of inflammatory mediators were measured by qRT-PCR and ELISA, respectively. a-c and g, Relative fold change to control group of TNF-ɑ, IL-6, IL-1β and DUSP1 mRNA expression. d-f, Concentrations of TNF-ɑ, IL-6 and IL-1β in the supernatant. h, DUSP1 protein expression in cardiac fibroblasts (original, uncropped blot was shown in supplement Fig. S1A). I, Protein quantification of DUSP1. * represents P < 0.05 compared with the control group

• 

  Fig. 2. Effects of rolipram on the LPS-induced DUSP1 expression and inflammatory response in cardiac fibroblasts. Cells were pretreated with 10 μmol/L rolipram for 1 h before LPS stimulation. DUSP1 mRNA and protein expression, the mRNA expression and supernatant protein concentrations of inflammatory mediators in cardiac fibroblasts were measured after LPS stimuli for 6 h. For the RNAi experiment, cardiac fibroblasts were treated by small interfering RNA for 6 h, followed by normal culture for 48 h, then cells were pretreated with 10 μmol/L rolipram for 1 h before LPS stimulation. The expression of DUSP1 and the TNF-ɑ, IL-6 concentrations in the supernatant of cardiac fibroblasts were measured. a, Relative fold of changes to control of DUSP1 mRNA; b and c, DUSP1 expression detection and quantification (original, uncropped blot was shown in supplement Fig. S1B). d-f, Relative fold of changes to control of TNF-ɑ, IL-6 and IL-1β mRNA expression. g-i, Concentrations of TNF-ɑ, IL-6 and IL-1β in the supernatant. j. The expression of DUSP1 after RNAi (original, uncropped blot was shown in supplement Fig. S1C). k and l. The TNF-ɑ and IL-6 concentrations in the supernatant after the DUSP1…

• 

  Fig. 3. Effect of rolipram on the inflammatory response in the hearts of endotoxic mice. Endotoxic mice were established by intraperitoneal injection of 15 mg/kg LPS for 6 h. In the rolipram group, 10 mg/kg rolipram was injected intraperitoneally 1 h before LPS injection. The hearts of the mice were dissociated, and the mRNA and protein levels of proinflammatory molecules (TNF-ɑ, IL-6 and IL-1β) and DUSP1 were measured by qRT-PCR and western blotting assays. a, Relative fold of changes to control of DUSP1 mRNA expression in heart tissues. b, Representative image of western blots of DUSP1 expression (original blot was shown in supplement Fig. S1D). c-e, Relative fold of changes to control of TNF-ɑ, IL-6 and IL-1β mRNA expression in heart tissues. f-h, The concentration of TNF-ɑ, IL-6 and IL-1β in heart tissues. * represents P < 0.05, n = 5

• 

  Fig. 4. Effects of rolipram on inflammatory cell infiltration and cardiac function of the hearts of endotoxic mice. Inflammatory cell infiltration and heart function were examined on the endotoxic mice model by pathological analysis, flow cytometry and echocardiography, respectively. a. Representative pictures of HE staining of heart tissue from endotoxic mice (200×, the arrows showed the inflammatory cells). b. The inflammatory scores of the histopathological sections. c. The Cd45 positive cell rate in the single cells harvested from the heart tissue. d. The Ly6g positive cell rate in the Cd45 positive cells harvested from the heart tissue. e. Representative pictures of M-mode at the level of left ventricular short axis view. f. Analysis of left ventricular ejection fraction (EF) of the hearts of endotoxic mice. a, control + DMSO, b, control + rolipram, c, LPS + DMSO, d, LPS + rolipram. * represents P < 0.05, n = 5