Anti-fibrotic effects of pirfenidone and rapamycin in primary IPF fibroblasts and human alveolar epithelial cells

Fig. 1. Effect of TGF−β, pirfenidone and rapamycin on the viability of lung primary fibroblasts and A549 cells. Primary lung fibroblasts and bronchial epithelial human cells (A549) were incubated with pirfenidone (1 mg/ml) and rapamycin (1 μg/ml) in addition to the combination of the two agents with TGF−β (5 ng/ml) for 72 h, viability was measured with the WST reagent. No significant cytotoxic effect was observed for any combinatory treatments. The bars represent the mean values ± SEM of viability % related to control samples in three different experiments

Fig. 2. Pirfenidone and rapamycin effect in protein and gene expression of tenascin-c and fibronectin in human lung fibroblasts from IPF patients. Primary lung fibroblasts from IPF patients were treated with TGF−β (5 ng/ml), pirfenidone (1 mg/ml) and rapamycin (1 μg/ml) for 72 h. a The total cell lysates were subjected to immunoblot analysis for tenascin-c. The data shown are mean values ± SEM of four different experiments. b Results of tenascin-c transcript fold changes expressed as relative gene expression (RGE) analyzed by RT-PCR. c Fibronectin protein level. Density of protein bands were normalized against vinculin and are shown as ratios. d Fibronectin transcript fold changes expressed as relative gene expression (RGE). Charts represent mean values ± SEM of four different experiments. Levels of significance: *p < 0.05; ** p < 0.01; ***p < 0.001. * indicates the comparison between samples treated with TGF−β and all the other conditions: TGF−β versus untreated samples (control); rapamycin and pirfenidone (rapa/pirfe); and samples treated with TGF−β in combination with rapamycin (TGF−β/rapa); pirfenidone (TGF−β/pirfe) or both (TGF−β/rapa/pirfe). #p < 0.05; ## p < 0.01. # indicates the…

Fig. 3. Collagen I and III gene expression are decreased by pirfenidone and rapamycin, while only the protein expression level of collagen III is inhibited by pirfenidone and rapamycin. Primary lung fibroblasts from IPF patients were treated with TGF−β (5 ng/ml), pirfenidone (1 mg/ml) and rapamycin (1 μg/ml) for 72 h. a Densitometric analysis expressed as mean ± SEM and a representative blot showing the unchanged effect in COL1A1 levels by pirfenidone and rapamycin. b COL1A1 gene expression measured by RT-PCR was decreased by pirfenidone and rapamycin in IPF fibroblasts. Bars represent mean ± SEM values of the transcript fold changes expressed by relative gene expression (RGE) of four different experiments. c COL3A1 protein expression decreases with the combination treatment of rapamycin and pirfenidone. Bars expressed as mean ± SEM values. Treated samples with rapamycin and pirfenidone in the absence of TGF−β show a significant decrease of the protein compare to controls. d COL3A1 gene expression showing a significant decrease when cells were treated with rapamycin and pirfenidone combination treatment. Levels of significance: *p < 0.05; ** p < 0.01; ***p < 0.001. * indicates the…

Fig. 4. Fibroblast to myofibroblast transformation is inhibited by pirfenidone and rapamycin treatment. α-SMA marker was studied in primary lung fibroblasts from IPF patients treated with TGF−β (5 ng/ml), pirfenidone (1 mg/ml) and rapamycin (1 μg/ml) for 72 h. a Western blot analysis in cell lysates. Graphs represent means ± SEM of target/control ratios obtained by densitometric analysis of each experiment results. b The analysis of α-SMA gene expression after 72 h treatment showed differences between samples treated with TGF−β in the absence or presence of pirfenidone and rapamycin. α-SMA transcript fold changes expressed as relative gene expression (RGE) measured by RT-PCR in fibroblasts from IPF patients, mean ± SEM of four different experiments normalized using four different housekeeping genes as control (β-actin, GADPH, HPRT, and RNA18s). Levels of significance: ** p < 0.01; ***p < 0.001.* indicates the comparison between samples treated with TGF−β and all the other conditions: TGF−β versus untreated samples (control); rapamycin and pirfenidone (rapa/pirfe); and samples treated with TGF−β in combination with rapamycin (TGF−β/rapa); pirfenidone (TGF−β/pirfe) or both…

Fig. 5. Pirfenidone and rapamycin inhibited the TGF−β-induced migration of human IPF lung fibroblasts. Cell migration assay were performed in fibroblasts from IPF patients incubated with TGF−β (5 ng/ml), rapamycin (1 μg/ml) and pirfenidone (1 mg/ml). Images show that cell migration occurred within 72 h from culture scratch in medium containing 2% (a) and 10% (b) fetal bovine serum (FBS) in the presence of TGF−β, combination of TGF−β and rapamycin, TGF−β and pirfenidone, and the combination of TGF−β, rapamycin and pirfenidone. The uncovered area has been quantified by the ImageJ Software at each time point and represented in the graphs as mean values ± SEM of three different experiments. Magnification, 100X. Levels of significance; * indicates the statistical significance between all conditions assayed at 72 h time point. # indicates the significance between scratch and 24 and 72 h time points for each treatment conditions

Fig. 6. Inhibitory effect of pirfenidone and rapamycin on tenascin-c and fibronectin gene and protein expression in bronchial epithelial human cells. A549 cells were incubated with TGF−β (5 ng/ml), pirfenidone (1 mg/ml) and rapamycin (1 μg/ml). Protein and gene expression were determined after the 72 h incubation period. a Representative immune blot showing the expression of tenascin-c in A549 cells. The bar chart summarizes the densitometric analysis of total protein expression normalized to β-actin. b Analysis of tenascin-c gene expression normalized with four different housekeeping genes (β-actin, GADPH, HPRT, and RNA18s). The bars represent the mean values ± SEM of the transcript fold changes expressed as relative gene expression (RGE) in at least three different experiments. c Fibronectin protein expression measured in cell lysates by western blot in at least three different experiments. The bar chart represents the densitometric analysis using α-tubulin as loading control. d Fibronectin gene expression was normalized using four different housekeeping genes as control (β-actin, GADPH, HPRT, and RNA18s). Bars describe the mean values ± SEM of the transcript fold changes, expressed as…

Fig. 7. Pirfenidone and rapamycin combined treatment inhibit the expression of collagen type I and III induced by TGF−β in alveolar human epithelial cells. A549 cells were incubated with TGF−β (5 ng/ml), pirfenidone (1 mg/ml) and rapamycin (1 μg/ml) for 72 h. a and c The expression of COL1A1 and COL3A1 were measured by western blot. Representative immunoblot analysis of at least three different experiments normalized by α-tubulin. The combination of rapamycin and pirfenidone treatment inhibits the protein expression of collagen type III. Gene expression b and d were analyzed in A549 cells after the drug treatment by RT-PCR. Reported values are means ± SEM of the transcript fold changes expressed as relative gene expression (RGE) normalized by four housekeeping genes and calculated on the level of untreated (control) cells. Only collagen type III gene expression is decreased by the treatment of rapamycin and pirfenidone. Levels of significance; *p < 0.05; **p < 0.01 * indicates the comparison between samples treated with TGF−β and all the other conditions: TGF−β versus untreated samples (control); rapamycin and pirfenidone (rapa/pirfe); and samples treated with TGF−β in combination with…

Fig. 8. Pirfenidone and rapamycin treatment in the presence of TGF−β has no effect on EMT pathway. A549 cells were incubated with TGF−β (5 ng/ml), pirfenidone (1 mg/ml) and rapamycin (1 μg/ml) for 72 h. a E-cadherin protein expression measured by western blot in A549 cells. Representative blots of one out of three separate experiments. Expression of α-tubulin was used to normalize sample loading. b E-cadherin transcript fold changes expressed as relative gene expression (RGE) was analyzed by RT-PCR. Reported values are the mean ± SEM of the transcript fold changes expressed as relative gene expression (RGE) observed in three different experiments. Levels of significance; * p < 0.05; ** p < 0.01. * indicates the comparison between samples treated with TGF−β and all the other conditions: TGF−β versus untreated samples (control); rapamycin and pirfenidone (rapa/pirfe); and samples treated with TGF−β in combination with rapamycin (TGF−β/rapa); pirfenidone (TGF−β/pirfe) or both (TGF−β/rapa/pirfe). n.s statistically not significant