PGC1-Alpha/Sirt3 Signaling Pathway Mediates the Anti-Pulmonary Fibrosis Effect of Hirudin by Inhibiting Fibroblast Senescence

Abstract

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic lung disease for which there is a lack of effective pharmacological treatments. Hirudin, a natural peptide extracted from leeches, has been used for broad pharmacological purposes. In this study, we investigated the therapeutic effects of hirudin on IPF and its related mechanism of action. By constructing a mouse model of pulmonary fibrosis and treating it with hirudin in vivo, we found that hirudin exerted anti-fibrotic, anti-oxidative, and anti-fibroblast senescence effects. Moreover, using an in vitro model of stress-induced premature senescence in primary mouse lung fibroblasts and treating with hirudin, we observed inhibition of fibroblast senescence and upregulation of PGC1-alpha and Sirt3 expression. However, specific silencing of PGC1-alpha or Sirt3 suppressed the anti-fibroblast senescence effect of hirudin. Thus, the PGC1-alpha/Sirt3 pathway mediates the anti-fibroblast senescence effect of hirudin, potentially serving as a molecular mechanism underlying its anti-fibrosis and anti-oxidative stress effects exerted on the lungs.

Keywords: PGC1-alpha/Sirt3 pathway; fibroblast senescence; hirudin; idiopathic pulmonary fibrosis.

Figure 1

Hirudin has a therapeutic effect in bleomycin-induced lung fibrosis in mice. Bleomycin tracheal injection was used to create a pulmonary fibrosis model. From days 15 to 28, intraperitoneal injections of hirudin at high, moderate, and low doses were then administered. Histopathological structure and ECM deposition changes in mouse lung tissue (A,B). The overall fibrosis of lung tissue in groups of mice (C). The level of hydroxyproline in mouse lung tissue (D). The mRNA expression levels of Col1a1 and ACTA2 (E,F). The amount of type I collagen and α-SMA in mouse lung tissues (G). BLM + H, 10 mg/kg group; BLM + M, 3 mg/kg group; BLM + L, 1 mg/kg group; BLM, pulmonary fibrosis model group; and Control, control group. All data are expressed in the mean ± standard deviation; n = 8, * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001.

Figure 2

Hirudin inhibits oxidative stress in bleomycin-induced lung fibrosis. The levels of ROS in lung tissues (A).The MDA levels, GSH levels, and total SOD levels in lung tissues (B–D). BLM + H, 10 mg/kg group; BLM + M, 3 mg/kg group; BLM + L, 1 mg/kg group; BLM, pulmonary fibrosis model group; and Control, control group. All data are expressed in the mean ± standard deviation; n = 8; ** p < 0.01; and **** p < 0.0001.

Figure 3

Hirudin inhibits fibroblast senescence in bleomycin-induced lung fibrosis. The levels of fibrosis and fibroblast senescence in mouse lung tissues (A). The mRNA expression levels of IL1b, IL6, IL8, and TNF-α in mouse lung tissues (B–E). The protein expression levels of p21, p16, PGC1-alpha, and Sirt3 in mouse lung tissues (F,I). The mRNA expression levels of p21 and p16 in mouse lung tissues (G,H). The mRNA expression levels of PGC1-alpha and Sirt3 in mouse lung tissues (J,K). BLM + H, 10 mg/kg group; BLM + M, 3 mg/kg group; BLM + L, 1 mg/kg group; BLM, pulmonary fibrosis model group; and Control, control group. All data are expressed in the mean ± standard deviation; n = 8; **** p < 0.0001; *** p < 0.001; ** p < 0.01; and * p < 0.05.

Figure 4

Hirudin has an inhibitory effect on hydrogen peroxide-induced senescence in fibroblasts. The effect of hirudin on the viability of fibroblasts (A). The level of ROS in fibroblasts (B). The β-galactosidase levels in fibroblasts (C). The mRNA expression levels of IL1b, IL6, IL8, and TNF-α in fibroblasts (D–G). The mRNA expression levels of p21 and p16 in fibroblasts (I,J). The expression of p21 in fibroblasts (H). The protein expression levels of p21 and p16 in fibroblasts (K). All data are presented as mean ± standard deviation, and all experiments were conducted independently and repeated a minimum of three times. * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001.

Figure 5

Hirudin activated the PGC1-alpha/Sirt3 pathway to inhibit fibroblast senescence. The protein expression levels of PGC1-alpha and Sirt3 in fibroblasts (A). The mRNA expression levels of PGC1-alpha and Sirt3 in fibroblasts (B,C). The levels of ROS in fibroblasts (D). β-galactosidase levels in fibroblasts (E). The mRNA expression levels of IL1b, TNF-α, IL8, and IL6 in fibroblasts (F–I). The expression of p21 in fibroblasts (J). The protein expression levels of p21 and p16 in fibroblasts (K). The mRNA expression levels of p21 and p16 in fibroblasts (L,M). All data are expressed in the mean ± standard deviation, and all experiments were conducted independently and repeated a minimum of three times. ** p < 0.01; **** p < 0.0001.

Figure 6

Schematic of a model of the anti-pulmonary fibrosis effect of hirudin.