Ulinastatin enhances autophagy against radiation-induced lung injury in mice

Abstract

Background: To investigate the enhancement of autophagy by ulinastatin for protecting against radiation-induced lung injury (RILI) in mice.

Methods: Forty C57BL/6 mice were equally divided into (I) control (C), (II) irradiation (R), (III) ulinastatin (U), (IV) 3-methyladenine (3-MA) (M), and (V) ulinastatin plus 3-MA (U+M) groups. Three mice in each group were infected with adeno-associated virus (AAV) carrying green fluorescent protein (GFP)-1A/1B-light chain 3 (GFP-LC3) in the lung for the marker of autophagy. All mice in R, U, M and U+M groups were given chest irradiation (1 Gy/min, 12 min), following injection with normal saline in C and U groups, ulinastatin (500,000 IU/kg·d, i.p., 7 d) in U group, 3-MA (10 mg/kg·d, i.p., 7 d) in M group, and ulinastatin plus 3-MA in U+M group. The effects of ulinastatin on lung injury and autophagy were evaluated by electron microscope (EM), immunohistochemistry, mRNA expression levels of collagen alpha-1 (COL1A1), collagen alpha-2 (COL1A2), α-smooth muscle actin (α-SMA) and transforming growth factor β1 (TGF-β1), and protein levels of LC3, α-SMA, COL1A2, TGF-β1, matrix metalloproteinase-2 (MMP-2) and MMP-9.

Results: EM observation revealed that the radiation caused the injury of type I and II alveolar epithelial cells, which was improved by ulinastatin treatment associated with increased the numbers of autophagosomes. GFP-LC3 signals was significantly enhanced by ulinastatin detected by immune histochemical tests. At transcriptional and/or translational levels, ulinastatin significantly enhanced the expression levels of TGF-β1 and LC3 but reduced COL1A1, COL1A2, α-SMA, MMP-2 and MMP-9 after radiation-induced RILI.

Conclusions: Ulinastatin reduces RILI by enhancing autophagy, which might be a potential therapeutic drug in the protection against RILI.

Keywords: Ulinastatin; autophagy; radiation-induced lung injury (RILI).

Figure 1

Ulinastatin improves cell damage induced by RILI in lung tissues detected by electron microscopy. The lung tissue was fixed with 1% glutaraldehyde. The cell structure was observed by scanning EM without any staining. (A) The control group: type I (A1-2) and II (A3-4) alveolar epithelial cells with intact cell membrane and cytoplasmic organelles. (B). The radiation group: type I (B1-2) and II (B3-4) alveolar epithelial cells with rupture, necrosis, mitochondrial cavitation, nuclear bilayer membrane disappears, cytoplasmic cavitation and endoplasmic reticulum expansion, some autophagosomes with the bilayer membrane structure in cells (B5-6); (C) The ulinastatin group: relatively complete type I (C1-2) and II (C3-4) alveolar epithelial cells and more autophagosomes in cells (C5-6); (D) the 3-AM group: the collapsed type I (D1-2) and II (D3-4) alveolar epithelial cells with mitochondria cavitation and endoplasmic reticulum expansion; (E) The ulinastatin plus 3-MA group: type I (E1-2) and II (E3-4) alveolar epithelial cells with mitochondrial cavitation, and some autophagosomes in cells (E5-6). The scale bar =2.5 µm.

Figure 2

Ulinastatin enhances of the GFP-LC3 expression detected by fluorescence confocal microscope. The lung tissues of 3 mice in each group were infected with or without HBAAV2/6-mRFPGFP-LC3. The green fluorescence labelled LC3 (GFP-LC3) in the lung tissue of mice with different treatment were detected under fluorescence microscope without any staining. (A) The control group: no detected GFP-LC3 signals; (B) the radiation group: some GFP-LC3 signals; (C) the ulinastatin group: significantly enhanced GFP-LC3 signals; (D) the 3-MA group: almost invisible GFP-LC3 signals. (E) the ulinastatin +3-MA group: alleviated GFP-LC3 signals. Blue: nuclei; green: GFP-LC3. The scale bar =100 µm.

Figure 3

The relative mRNA expression levels of COL1A1 (A), COL1A2 (B), α-SMA (C) and TGF-β1 (D) in lung tissues of mice in the groups of control (C), radiation (R), ulinastatin (U), 3-MA (M) and ulinastatin plus 3-MA (U+M) (n=3/each group). AVONA and hoc post test, *, P<0.05; **, P<0.01.

Figure 4

Ulinastatin increases LC3 but reduces α-SMA, COL1A2, TGF-β1, MMP-2 and MMP-9 induced by radiation. (A) The lung tissue homogenates of mice in the groups of control (C), radiation (R), ulinastatin (U), 3-MA (M) and ulinastatin plus 3-MA (U+M) (n=3/each group) were subjected to Western blots. (B) The protein signals were normalized to the signal of α-tubulin, and then compared as relative protein expression level by ANOVA and hoc post test. *, P<0.05; **, P<0.01.