Anti‐fibrotic effects of pirfenidone by interference with the hedgehog signalling pathway in patients with systemic sclerosis‐associated interstitial lung disease

Abstract Aim To determine whether pirfenidone attenuates lung fibrosis by interfering with the hedgehog (Hh) signalling pathway in patients with systemic sclerosis‐associated interstitial lung disease ( SS c‐ ILD ). Methods Twenty‐five SS c‐ ILD patients (20 first visit, five who underwent pirfenidone treatment for 6 months) and 10 healthy controls were recruited. Lung tissues were obtained by open‐chest surgery, and primary lung fibroblasts were isolated, cultured and stimulated with pirfenidone. The levels of the proteins glioma‐associated oncogene 1 ( GLI 1), suppressor of fused (Sufu), α‐smooth muscle actin, and fibronectin in lung tissues or fibroblasts were determined by Western blotting. The messenger RNA levels of GLI 1, glioma‐associated oncogene 2, protein patched homolog 1, and Sufu in lung tissues or fibroblasts were determined by quantitative reverse‐transcription polymerase chain reaction. Meanwhile, the levels of phosphorylation glycogen synthase kinasep‐3β ( pGSK ‐3β), phosphorylation SMAD 2 ( pSMAD 2), and phosphorylation c‐Jun N‐terminal kinase (p JNK ) in fibroblasts were determined by Western blotting. Results Hh pathway activation was increased in the lung tissue of SS c‐ ILD patients and was decreased by pirfenidone, Sufu was upregulated in lung fibroblasts isolated from SS c‐ ILD patients after pirfenidone challenge, and pirfenidone inhibited the phosphorylation of GSK ‐3β signalling. Conclusion Pirfenidone has anti‐fibrotic effects in SS c‐ ILD patients by interfering with both the Hh signalling pathway and the GSK ‐3β signalling pathway via the regulation of Sufu expression. These results might promote its use in other Hh driven lung diseases such as idiopathic pulmonary fibrosis and especially the interstitial lung disease associated with connective tissue diseases.