Protective effect of anisodamine on bleomycin-induced acute lung injury in immature rats via modulating oxidative stress, inflammation, and cell apoptosis by inhibiting the JAK2/STAT3 pathway

Abstract

Background: Pediatric acute lung injury (ALI) is one of the most common causes of infant mortality. Although lung-protective strategies have developed in recent years, no ALI treatment is currently available. Anisodamine (Ani) is a common drug used to treat gastrointestinal smooth muscle spasm. The protective effects of Ani against acute kidney injury and myocardial injury have been reported. However, the efficacy of Ani on bleomycin (BLM)-induced ALI has not been examined previously. In the present study, we aimed to examine the effects of Ani on bleomycin (BLM)-induced ALI on immature rats.

Methods: The ALI rat model was established by intratracheally administration of BLM. Ani treatment was performed by an intravenous injection at different concentrations. The lung function of each rat was measured, and then lung tissue structures, apoptosis, and collagen deposition were observed by hematoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, and Masson's staining, respectively. Enzyme-linked immunosorbent assay was used to detect the levels of inflammatory cytokines. The expression of apoptosis-related proteins and fibrosis-related markers was determined by reverse transcription-polymerase chain reaction and/or Western blot analysis. Finally, the expression levels of Janus tyrosine kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) were determined.

Results: Our findings indicated that lung function was remarkably decreased in BLM-induced rats, which could be reversed by Ani. Ani treatment increased the levels of antioxidant enzymes, decreased the apoptotic rate and apoptosis-related proteins, and downregulated the expression of fibrosis-related markers. Additionally, Ani treatment also attenuated inflammatory response and suppressed the activation of the JAK2/STAT3 pathway.

Conclusions: Our results demonstrated that Ani had potent activity against BLM-induced ALI in immature rats through inhibiting the JAK2/STAT3 signaling pathway. Our findings provide supporting evidence to further investigate the therapeutic effect of Ani against ALI in children.

Keywords: Anisodamine; acute lung injury (ALI); bleomycin; inflammation; oxidative stress.

Figure 1

Anisodamine (Ani) increased lung function and reduced oxidative stress induced by bleomycin (BLM). (A) Airway resistance value measured in each group; (B) lung elastance value measured in each group; (C) glutathione (GSH) activity in lung tissue homogenate; (D) superoxide dismutase (SOD) activity in lung tissue homogenate; (E) lactate dehydrogenase (LDH) level in lung tissue homogenate. *, P<0.05 vs. control group; **, P<0.05 vs. BLM group.

Figure 2

Anisodamine (Ani) inhibited cell proliferation and cell apoptosis in rats with acute lung injury. (A) Pathological changes of lung tissue observed after hematoxylin-eosin (HE) staining (magnification 400×, scale bar =25 µm) and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay (magnification 400×, scale bar =25 µm); (B) comparison of apoptotic rate in each group; (C) mRNA levels of Ki-67 and survivin in each group measured by reverse transcription-polymerase chain reaction; (D) expression of apoptosis-related proteins in each group measured by western blot. *, P<0.05 vs. control group; **, P<0.05 vs. BLM group; Bax, Bcl2-associated X protein; Bcl-2, B-cell lymphoma 2.

Figure 3

Anisodamine (Ani) inhibited lung tissue fibrosis in rats with acute lung injury. (A) Representative images of Masson staining in each group (magnification 100×); collagen is stained in blue and cells in red (scale bar =100 µm); (B) quantification of lung fibrosis shown as an Ashcroft score; (C) mRNA levels of transforming growth factor β (TGF-β), fibronectin, and α-smooth muscle actin (α-SMA) measured by reverse transcription-polymerase chain reaction; (D) representative images of western blot analysis of TGF-β, fibronectin, and α-SMA; (E) protein levels of TGF-β measured by western blot; (F) protein levels of fibronectin measured by western blot; (G) protein levels of α-SMA measured by western blot. *, P<0.05 vs. control group; **, P<0.05 vs. bleomycin (BLM) group.

Figure 4

Anisodamine (Ani) attenuated inflammation, cell infiltration, and cytokine expression in rats with acute lung injury. (A) Interleukin (IL)-6 levels in bronchoalveolar lavage fluid (BALF) and serum measured by ELISA; (B) IL-1β levels in BALF and serum measured by enzyme-linked immunosorbent assay (ELISA); (C) tumor necrosis factor-α (TNF-α) levels in BALF and serum measured by ELISA; (D) IL-4 levels in BALF and serum measured by ELISA; (E) IL-10 levels in BALF and serum measured by ELISA; (F) number of total leukocytes measured in BALF; (G) number of neutrophils measured in BALF. *, P<0.05 vs. control group; **, P<0.05 vs. bleomycin (BLM) group.

Figure 5

Anisodamine (Ani) suppressed the Janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in rats with acute lung injury. (A) Downregulated effect of Ani on JAK2/STAT3 pathway; (B) effect of AG490 and dimethyl sulfoxide (DMSO) on the JAK2/STAT3 signaling pathway; (C) comparison of AG490 and AG490+Ani on the JAK2/STAT3 signaling pathway; (D) results of hematoxylin-eosin staining (magnification 400×) and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay (magnification 400×) for lung tissues of rats in each group (scale bar =25 µm); (E) comparison of apoptotic rate in each group; (F) expression of transforming growth factor β (TGF-β), fibronectin, and α-smooth muscle actin (α-SMA) in each group measured by western blot; (G) expression levels of lactate dehydrogenase (LDH); (H) expression levels of superoxide dismutase (SOD); (I) serum interleukin (IL)-6 and IL-10 levels measured by enzyme-linked immunosorbent assay. Note: For C–I, rats were divided into the control, bleomycin (BLM), BLM + AG490, and BLM + AG490 + Ani groups. *, P<0.05 vs. control group; **, P<0.05 vs. BLM group; ***, P<0.01 vs. BLM + AG490 group.