Toll-Like Receptor 4 Activation Prevents Rat Cardiac Fibroblast Death Induced by Simulated Ischemia/Reperfusion

Figures

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  Figure 1. Protocol of experimental design. Representative diagram of the experimental groups in the biological assays.

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  Figure 2. Lipopolysaccharide (LPS) increases the viability of cardiac fibroblasts exposed to simulated ischemia/reperfusion (sI/R). Cardiac fibroblasts were subjected to 8 h of ischemia and 16 h of reperfusion (sI/R). Cell viability was determined using the trypan blue exclusion test (A,C,E,G) or the MTT assay (B,D,F,H). (A,B) Effects of preconditioning (PC) with LPS. Cardiac fibroblasts were pretreated with LPS (1 μg/ml) for 24 and 16 h before performing sI/R. (C,D) Effect of LPS only during ischemia. Cardiac fibroblasts were treated with LPS (1 μg/ml) during the 8 h of ischemia and then were perfused (16 h). (E,F) Effect of LPS only during reperfusion. Cardiac fibroblasts were treated with LPS (1 μg/ml) only during the 16 h of reperfusion. (G,H) Effect exerted by LPS during ischemia and reperfusion. Cardiac fibroblasts were treated with LPS (1 μg/ml) during the 8 h of ischemia and the 16 h of reperfusion. Results are expressed as mean ± SEM of six independent experiments. ***p < 0.001 vs. control; #p < 0.05 and ###p < 0.001 vs. sI/R.

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  Figure 3. Lipopolysaccharide (LPS) reduces apoptosis induced by simulated ischemia/reperfusion (sI/R) in cardiac fibroblasts. Cardiac fibroblasts were exposed to 8 h of ischemia, followed by 16 h of reperfusion (sI/R). Cells were treated with LPS (1 μg/ml) at the onset of ischemia and at the start of reperfusion. (A) The percentage (%) of necrosis was quantified by flow cytometry using propidium iodide (left), with representative histograms of each experimental group and the graphical analysis on the right. (B) The percentage (%) of the sub-G1 population (apoptosis) was quantified by flow cytometry using propidium iodide (middle), with representative histograms of each experimental group and the (C) graphical analysis on the right panel. The results are expressed as mean ± SEM of five independent experiments. *p < 0.01 and **p < 0.01 vs. each population control, ##p < 0.01 vs. apoptosis population in sI/R.

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  Figure 4. Blockade of TLR4 increases the loss of cardiac fibroblasts viability induced by ischemia or ischemia/reperfusion (I/R). Cardiac fibroblasts were treated with the specific TLR4 inhibitor TAK-242 (4 μM) during the 8 h of ischemia (A) or during the 8 h of ischemia and the 16 h of reperfusion (B). Cell viability was determined using the MTT assay. Results are expressed as mean ± SEM of six independent experiments. ***p < 0.001 vs. control; #p < 0.05 vs. ischemia; ##p < 0.01 vs. sI/R.

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  Figure 5. Effect of TLR4 blockade on p-ERK1/2 and p-AKT levels after treatment of cardiac fibroblasts with lipopolysaccharide (LPS) during ischemia and during ischemia and reperfusion. Cardiac fibroblasts were treated with LPS (1 μg/ml) in the presence of the TAK-242 (4 μM) inhibitor during the 8 h of ischemia (A,B) and during the 8 h of ischemia and the 16 h of reperfusion (sI/R) (C,D). The expression levels of p-ERK1/2 and p-AKT were determined by Western blot. GAPDH was used as a loading control (we use the same blot for A,B). Results are expressed as mean ± SEM of four independent experiments. ***p < 0.001 vs. control; **p < 0.01 vs. control; %p < 0.05 and %%p < 0.01 vs. ischemia. #p < 0.05 and ##p < 0.01 vs. sI/R.

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  Figure 6. Effect of TLR4 blockade, ERK1/2, and AKT inhibition on the loss of viability induced by ischemia/reperfusion (I/R) in cardiac fibroblasts treated with lipopolysaccharide (LPS). Cardiac fibroblasts were treated with LPS (1 μg/ml) in the presence of (A) TAK-242 (4 μM), (B) PD98059 (25 μM), or (C) LY294002 (10 μM). Cell viability was determined by the MTT assay. Results are expressed as mean ± SEM of six independent experiments. ***p < 0.001 and **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. sI/R; $p < 0.05 vs. sI/R + LPS.