Necrostatin-1 Alleviates Bleomycin-Induced Pulmonary Fibrosis and Extracellular Matrix Expression in Interstitial Pulmonary Fibrosis

Abstract

BACKGROUND Interstitial pulmonary fibrosis (IPF) is harmful for patients' life and health. The effective treatment of IPF is lacking because of unclear pathogenesis. Necrostatin-1 has protective effects on lung injury and can suppress the fibrosis development. I this study we investigated whether necrostatin-1 could decrease the proliferation of pulmonary fibroblasts, pulmonary fibrosis and expression of extracellular matrix (ECM) in IPF. MATERIAL AND METHODS The IPF mice model was conducted by intra-tracheal injection of bleomycin (BLM) (2 mg/kg) for C57BL/6N mice. Necrostatin-1 treatment was performed with 1 mg/kg necrostatin-1 by an intravenous injection for C57BL/6N mice. Lung tissue structures and collagen deposition were observed by hematoxylin and eosin staining and Masson staining. IPF in vitro model was constructed by MRC-5 cells induced by transforming growth factor beta 1 (TGF-ß1). And, 20 μM necrostatin-1 was used to treat the TGF-ß1 induced MRC-5 cells. Cell Counting Kit-8 (CCK-8) assay detected the viability of MRC-5 cells. The expression of receptor-interacting protein kinase-1 and -3 (RIPK1 and RIPK3), alpha smooth muscle actin (alpha-SMA), collagen IV, collagen I, fibronectin (FN), and transforming growth factor-ß (TGF-ß) in lung tissues and MRC-5 cells was measured by western blot analysis. The alpha-SMA expression in lung tissues was also analyzed by immunohistochemistry. RESULTS The expression of RIPK1 and RIPK3 in lung tissues of BLM induced mice was increased. The degree of pulmonary fibrosis and expression of alpha-SMA, collagen IV, collagen I, FN, and TGF-ß in lung tissues of BLM induced mice was enhanced. The proliferation of MRC-5 cells was increased when MRC-5 cells were induced by TGF-ß. The expression of RIPK1, RIPK3, alpha-SMA, collagen IV, collagen I, and FN was increased in TGF-ß induced MRC-5 cells. And, necrostatin-1 could effectively reverse the changes of pulmonary fibrosis, RIPK1, RIPK3, and ECM in vivo and in vitro experiments. CONCLUSIONS Necrostatin-1 attenuated pulmonary fibrosis in lung tissues of BLM induced mice and inhibited the fibroblast proliferation. And, necrostatin-1 also decreased the expression of RIPK1, RIPK3, and ECM in lung tissues of BLM induced mice and TGF-ß induced fibroblasts. Necrostatin-1 could be a new effective drug for the treatment of IPF.

Figure 1

Necrostatin-1 reduces the expression of RIPK1 and RIPK3 in BLM-induced lung tissues. (A) The protein expression of RIPK1 and RIPK3 in lung tissues detected by western blot analysis. ** P<0.01 and *** P<0.001 versus control group. ^# P<0.05 versus BML-induced group. ^@ P<0.05 versus BML-induced+DMSO group. (B) The mRNA expression of RIPK1 and RIPK3 in lung tissues detected by RT-qPCR analysis. * P<0.05, ** P<0.01 and *** P<0.001 versus control group. ^### P<0.001 versus BML-induced group. ^@@@ P<0.001 versus BML-induced+DMSO group. RIPK1 – receptor-interacting protein kinase-1; RIPK3 – receptor-interacting protein kinase-3; BLM – bleomycin; DMSO – dimethylsulfoxide; RT-qPCR – real-time quantitative polymerase chain reaction.

Figure 2

Necrostatin-1 alleviates pathological changes and collagen deposition in lung tissues induced by BLM. (A) The pathological changes of lung tissue observed after H&E staining. (B) The lung organization fibrosis degree observed after Masson staining. BLM – bleomycin; H&E – hematoxylin and eosin.

Figure 3

Necrostatin-1 decreases the ECM expression in lung tissues induced by BLM. (A) The expression of α-SMA in lung tissues detected by immunohistochemistry. (B) The expression of α-SMA, collagen IV, collagen I, FN, and TGF-β in lung tissues detected by western blot analysis. ** P<0.01 and *** P<0.001 versus control group. ^# P<0.05 and ^## P<0.01 versus BML-induced group. ^@ P<0.05 and ^@@ P<0.01 versus BML-induced+DMSO group. ECM – extracellular matrix; BLM – bleomycin; α-SMA – smooth muscle actin; FN – fibronectin; TGF-β1 – transforming growth factor beta 1.

Figure 4

Necrostatin-1 decreases the ECM expression in pulmonary fibroblasts induced by TGF-β1. The expression of α-SMA, collagen IV, collagen I, and FN in lung tissues detected by western blot analysis. * P<0.05, ** P<0.01 and *** P<0.001 versus control group. ^### P<0.001 versus necrostatin-1 group. ^@ P<0.05 versus TGF-β1 group. ECM – extracellular matrix; TGF-β1 – transforming growth factor beta 1; α-SMA – smooth muscle actin; FN – fibronectin.

Figure 5

Necrostatin-1 inhibits the proliferation of pulmonary fibroblasts induced by TGF-β1. The cell viability detected by CCK-8 assay. * P<0.05 versus control group. ^### P<0.001 versus necrostatin-1 group. ^@@@ P<0.001 versus TGF-β1 group. TGF-β1 – transforming growth factor beta 1; CCK-8 – Cell Counting Kit-8.

Figure 6

Necrostatin-1 decreases the expression of RIPK1 and RIPK3 in pulmonary fibroblasts induced by TGF-β1. The expression of RIPK1 and RIPK3 in lung tissues detected by western blot analysis. ** P<0.01 and *** P<0.001 versus control group. ^### P<0.001 versus necrostatin-1 group. ^@ P<0.05 and ^@@ P<0.01 versus TGF-β1 group. RIPK1 – receptor-interacting protein kinase-1; RIPK3 – receptor-interacting protein kinase-3; TGF-β1 – transforming growth factor beta 1.