Korean J Physiol Pharmacol.  2015 Sep;19(5):401-411. 10.4196/kjpp.2015.19.5.401.

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

• Li X
• Shen Y
• Lu Y
• Yang J

• KMID: 2285584
• DOI: http://doi.org/10.4196/kjpp.2015.19.5.401

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-beta1, alpha-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-beta1, AR, alpha-SMA and collagen I (both mRNA and protein). In vitro, epalrestat remarkably attenuated proliferation of pulmonary fibroblasts and expression of alpha-SMA and collagen I induced by TGF-beta1, and this inhibitory effect of epalrestat was accompanied by inhibiting AR expression. Knockdown of AR gene expression reversed TGF-beta1-induced proliferation of fibroblasts, up-regulation of alpha-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.

Keyword

Aldose reductase; Epalrestat; Pulmonary fibroblasts; Pulmonary fibrosis

MeSH Terms

Airway Remodeling
Aldehyde Reductase*
Animals
Asthma
Bleomycin
Blotting, Western
Bromodeoxyuridine
Collagen
Diabetic Neuropathies
Fibroblasts
Fibrosis
Flow Cytometry
Gene Expression
Negotiating
Pulmonary Fibrosis*
Rats*
Real-Time Polymerase Chain Reaction
RNA, Messenger
Transforming Growth Factor beta1
Up-Regulation
Aldehyde Reductase
Bleomycin
Bromodeoxyuridine
Collagen
RNA, Messenger
Transforming Growth Factor beta1

Figure

• 

  Fig. 1 Lung histology and α-SMA expression in bleomycin-induced pulmonary fibrosis of rats. (A) Hematoxylin-eosin staining of lung tissue. (B and C) The expression of α-SMA mRNA and protein were determined by real-time PCR and Western blot. Data are means±S.E.M. n=8. **p<0.01 vs. Control; #p<0.05, ##p<0.01 vs. Bleomycin. EPS, Epalrestat; α-SMA, α-smooth muscle actin.

• 

  Fig. 2 Collagen expression of lung tissue in bleomycin-induced pulmonary fibrosis rats. (A) Masson's trichrome staining of lung tissue. (B) The expression of collagen I mRNA was determined by real-time PCR. (C) The expression of collagen I protein was determined by Western blot. Data are means±S.E.M. n=8. **p<0.01 vs. Control; #p<0.05, ##p<0.01 vs. Bleomycin. EPS, Epalrestat; α-SMA, α-smooth muscle actin.

• 

  Fig. 3 Effect of Epalrestat on the expression of TGF-β1 and AR in bleomycin-induced pulmonary fibrosis rats. (A and B) The expression of TGF-β1 mRNA and protein in lung tissue were determined by real-time PCR and Western blot. (C and D) The expression of AR mRNA and protein in lung tissue were determined by real-time PCR and Western blot. (E) The expression of AR in lung tissue was determined with immunohistochemisty staining (arrows indicate AR positive staining). Data are means±S.E.M. n=8. **p<0.01 vs. Control; #p<0.05, ##p<0.01 vs. Bleomycin. EPS, Epalrestat; AR, aldose reductase.

• 

  Fig. 4 Effect of epalrestat on TGF-β1-induced expression of AR and proliferation of pulmonary fibroblasts. (A) The expression of AR mRNA was determined by real-time PCR. (B) The expression of AR protein was determined by 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. EPS, Epalrestat; AR, aldose reductase.

• 

  Fig. 5 Effect of Epalrestat on TGF-β1-induced expression of α-SMA and collagen I in cultured pulmonary fibroblasts. (A and B) The expression of α-SMA mRNA and protein were determined by real-time PCR and Western blot. (C and D) The expression of collagen I mRNA and protein were determined by real-time PCR and Western blot. 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. EPS, Epalrestat; α-SMA, α-smooth muscle actin.

• 

  Fig. 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.

• 

  Fig. 7 Effect of AR knockdown on TGF-β1-induced expression of α-SMA and collagen I in cultured pulmonary fibroblasts. (A, B and E, F) The expression of α-SMA mRNA and protein were determined by real-time PCR and Western blot. (C, D and G, H) The expression of collagen I mRNA and protein were determined by real-time PCR and Western blot. The values are means±S.E.M. from three independent experiments in vitro. (A, B and C, D) **p<0.01 vs. Control; #p<0.05, ##p<0.01 vs. TGF-β1; (E, F and G, H) **p<0.01 vs. Control; ##p<0.01 vs. EPS. EPS, Epalrestat; α-SMA, α-smooth muscle actin; siRNA, small interfering RNA.

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