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Amelioration of Bleomycin-induced Pulmonary Fibrosis of Rats by an Aldose Reductase Inhibitor, Epalrestat.

Li X, Shen Y, Lu Y, Yang J - Korean J. Physiol. Pharmacol. (2015)

Bottom Line: Aldose reductase (AR) is known to play a crucial role in the mediation of diabetic and cardiovascular complications.Epalrestat is an aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy.Knockdown of AR gene expression reversed TGF-β1-induced proliferation of fibroblasts, up-regulation of α-SMA and collagen I expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Wannan Medical College, Wuhu 241002, China.

ABSTRACT
Aldose reductase (AR) is known to play a crucial role in the mediation of diabetic and cardiovascular complications. Recently, several studies have demonstrated that allergen-induced airway remodeling and ovalbumin-induced asthma is mediated by AR. Epalrestat is an aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Whether AR is involved in pathogenesis of pulmonary fibrosis and whether epalrestat attenuates pulmonary fibrosis remains unknown. Pulmonary fibrosis was induced by intratracheal instillation of bleomycin (5 mg/kg) in rats. Primary pulmonary fibroblasts were cultured to investigate the proliferation by BrdU incorporation method and flow cytometry. The expression of AR, TGF-β1, α-SMA and collagen I was analyzed by immunohistochemisty, real-time PCR or western blot. In vivo, epalrestat treatment significantly ameliorated the bleomycin-mediated histological fibrosis alterations and blocked collagen deposition concomitantly with reversing bleomycin-induced expression up-regulation of TGF-β1, AR, α-SMA and collagen I (both mRNA and protein). In vitro, epalrestat remarkably attenuated proliferation of pulmonary fibroblasts and expression of α-SMA and collagen I induced by TGF-β1, and this inhibitory effect of epalrestat was accompanied by inhibiting AR expression. Knockdown of AR gene expression reversed TGF-β1-induced proliferation of fibroblasts, up-regulation of α-SMA and collagen I expression. These findings suggest that AR plays an important role in bleomycin-induced pulmonary fibrosis, and epalrestat inhibited the progression of bleomycin-induced pulmonary fibrosis is mediated via inhibiting of AR expression.

No MeSH data available.


Related in: MedlinePlus

Effect of AR knockdown on TGF-β1-induced cell proliferation in cultured pulmonary fibroblasts. (A and B) The expression of AR mRNA and protein were determined by real-time PCR and Western blot. (C) Cell proliferation was measured by BrdU incorporation assay. (D) The percentage of cells in S+G2 phase. (E) Cell cycle distribution was monitored by flow cytometry using a propidium iodide staining assay. The values are means±S.E.M. from three independent experiments in vitro. **p<0.01 vs. Control; #p<0.05, ##p<0.01 vs. TGF-β1. AR, aldose reductase; siRNA, small interfering RNA.
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Figure 6: Effect of AR knockdown on TGF-β1-induced cell proliferation in cultured pulmonary fibroblasts. (A and B) The expression of AR mRNA and protein were determined by real-time PCR and Western blot. (C) Cell proliferation was measured by BrdU incorporation assay. (D) The percentage of cells in S+G2 phase. (E) Cell cycle distribution was monitored by flow cytometry using a propidium iodide staining assay. The values are means±S.E.M. from three independent experiments in vitro. **p<0.01 vs. Control; #p<0.05, ##p<0.01 vs. TGF-β1. AR, aldose reductase; siRNA, small interfering RNA.

Mentions: To confirm the role of AR in mediating TGF-β1-induced the expression of AR in pulmonary fibroblasts, we developed AR specific siRNA. In our pilot study, we used three siRNA TargetSeq against AR to establish the AR siRNA pulmonary fibroblasts, and compared the effects of the three siRNA TargetSeq. The result showed that transfection with the first sequence for 24 h had the best efficiency to inhibit AR expression (data not shown). We therefore used the first siRNA TargetSeq for the sequent experiments. As shown in Fig. 6A and B, AR siRNA inhibited TGF-β1-induced up-regulation of AR expression. Importantly, we found that AR siRNA reversed the effect of TGF-β1-induced proliferation of pulmonary fibroblasts as shown by an increase in BrdU incorporation and the percentage of cells in S+G2 phase (Fig. 6C~E). Accordingly, AR siRNA also reversed the effect of TGF-β1-induced up-regulation of α-SMA and collagen I (both mRNA and protein) (p<0.05) (Fig. 7A~D). But compared with TGF-β1+AR siRNA, epalrestat (100 µM) had no effect on α-SMA and collagen I expression in TGF-β1+AR siRNA cells (Fig. 7E~H). These results strongly suggested that epalrestat reduced pulmonary fibrosis via inhibition of AR.


Amelioration of Bleomycin-induced Pulmonary Fibrosis of Rats by an Aldose Reductase Inhibitor, Epalrestat.

Li X, Shen Y, Lu Y, Yang J - Korean J. Physiol. Pharmacol. (2015)

Effect of AR knockdown on TGF-β1-induced cell proliferation in cultured pulmonary fibroblasts. (A and B) The expression of AR mRNA and protein were determined by real-time PCR and Western blot. (C) Cell proliferation was measured by BrdU incorporation assay. (D) The percentage of cells in S+G2 phase. (E) Cell cycle distribution was monitored by flow cytometry using a propidium iodide staining assay. The values are means±S.E.M. from three independent experiments in vitro. **p<0.01 vs. Control; #p<0.05, ##p<0.01 vs. TGF-β1. AR, aldose reductase; siRNA, small interfering RNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4553399&req=5

Figure 6: Effect of AR knockdown on TGF-β1-induced cell proliferation in cultured pulmonary fibroblasts. (A and B) The expression of AR mRNA and protein were determined by real-time PCR and Western blot. (C) Cell proliferation was measured by BrdU incorporation assay. (D) The percentage of cells in S+G2 phase. (E) Cell cycle distribution was monitored by flow cytometry using a propidium iodide staining assay. The values are means±S.E.M. from three independent experiments in vitro. **p<0.01 vs. Control; #p<0.05, ##p<0.01 vs. TGF-β1. AR, aldose reductase; siRNA, small interfering RNA.
Mentions: To confirm the role of AR in mediating TGF-β1-induced the expression of AR in pulmonary fibroblasts, we developed AR specific siRNA. In our pilot study, we used three siRNA TargetSeq against AR to establish the AR siRNA pulmonary fibroblasts, and compared the effects of the three siRNA TargetSeq. The result showed that transfection with the first sequence for 24 h had the best efficiency to inhibit AR expression (data not shown). We therefore used the first siRNA TargetSeq for the sequent experiments. As shown in Fig. 6A and B, AR siRNA inhibited TGF-β1-induced up-regulation of AR expression. Importantly, we found that AR siRNA reversed the effect of TGF-β1-induced proliferation of pulmonary fibroblasts as shown by an increase in BrdU incorporation and the percentage of cells in S+G2 phase (Fig. 6C~E). Accordingly, AR siRNA also reversed the effect of TGF-β1-induced up-regulation of α-SMA and collagen I (both mRNA and protein) (p<0.05) (Fig. 7A~D). But compared with TGF-β1+AR siRNA, epalrestat (100 µM) had no effect on α-SMA and collagen I expression in TGF-β1+AR siRNA cells (Fig. 7E~H). These results strongly suggested that epalrestat reduced pulmonary fibrosis via inhibition of AR.

Bottom Line: Aldose reductase (AR) is known to play a crucial role in the mediation of diabetic and cardiovascular complications.Epalrestat is an aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy.Knockdown of AR gene expression reversed TGF-β1-induced proliferation of fibroblasts, up-regulation of α-SMA and collagen I expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Wannan Medical College, Wuhu 241002, China.

ABSTRACT
Aldose reductase (AR) is known to play a crucial role in the mediation of diabetic and cardiovascular complications. Recently, several studies have demonstrated that allergen-induced airway remodeling and ovalbumin-induced asthma is mediated by AR. Epalrestat is an aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Whether AR is involved in pathogenesis of pulmonary fibrosis and whether epalrestat attenuates pulmonary fibrosis remains unknown. Pulmonary fibrosis was induced by intratracheal instillation of bleomycin (5 mg/kg) in rats. Primary pulmonary fibroblasts were cultured to investigate the proliferation by BrdU incorporation method and flow cytometry. The expression of AR, TGF-β1, α-SMA and collagen I was analyzed by immunohistochemisty, real-time PCR or western blot. In vivo, epalrestat treatment significantly ameliorated the bleomycin-mediated histological fibrosis alterations and blocked collagen deposition concomitantly with reversing bleomycin-induced expression up-regulation of TGF-β1, AR, α-SMA and collagen I (both mRNA and protein). In vitro, epalrestat remarkably attenuated proliferation of pulmonary fibroblasts and expression of α-SMA and collagen I induced by TGF-β1, and this inhibitory effect of epalrestat was accompanied by inhibiting AR expression. Knockdown of AR gene expression reversed TGF-β1-induced proliferation of fibroblasts, up-regulation of α-SMA and collagen I expression. These findings suggest that AR plays an important role in bleomycin-induced pulmonary fibrosis, and epalrestat inhibited the progression of bleomycin-induced pulmonary fibrosis is mediated via inhibiting of AR expression.

No MeSH data available.


Related in: MedlinePlus