Research ArticleISCHEMIA/REPERFUSION INJURY

Irisin protects mitochondria function during pulmonary ischemia/reperfusion injury

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Science Translational Medicine  29 Nov 2017:
Vol. 9, Issue 418, eaao6298
DOI: 10.1126/scitranslmed.aao6298
  • Fig. 1. Limb RIPC caused an increase in circulating irisin and translocation of irisin to injured lung alveoli.

    (A) Western blot of serum samples derived from mice subjected to remote ischemic preconditioning (RIPC) (20 μl of serum per lane). Colloidal staining served as loading control. Time course of RIPC-induced elevation of irisin in serum (*P < 0.05 versus control; n = 4). (B) Effect of ischemia/reperfusion (IR) on irisin concentration in mouse serum (*P < 0.05 versus sham and #P < 0.05 versus IR group; n = 6). (C) Time-dependent changes in irisin concentration in serum from mice after RIPC, with or without IR injury, determined by liquid chromatography–mass spectrometry (LC-MS) (*P < 0.05 versus non-IR sham mice and #P < 0.05 versus IR mice 0 min after RIPC; n = 6). (D) Immunohistochemical staining showed irisin expression in the IR-injured lung tissue from mice subjected to RIPC and lung IR injury (*P < 0.05 versus non-IR sham group and #P < 0.05 versus IR only group; n = 5; scale bars, 50 μm). OD, optical density.

  • Fig. 2. Infants with NRDS had reduced serum concentrations of irisin.

    (A) Heat map of irisin expression in serum or bronchoalveolar lavage fluid (BALF) from each newborn patient or control infant was performed by protein liquid-chip assay (n = 15 in control, n = 7 in NRDS group for serum samples; six BALF samples were used in the study). Serum and BALF concentrations of irisin in NRDS infants were detected by an irisin protein liquid-chip assay and are provided as irisin concentration (B) or peak area (C) [*P < 0.05 versus control; n = 15 in control and n = 7 in NRDS group (B); *P < 0.05 versus the irisin concentration in serum; n = 7 for serum samples and n = 6 for BALF samples (C)]. Serum and BALF concentrations of irisin in NRDS infants were detected by LC-MS and are provided as irisin concentration (D) or peak area (E) [*P < 0.05 versus control; n = 6 in each group (D);*P < 0.05 versus the irisin concentration in serum; n = 6 in each group (E)].

  • Fig. 3. RIPC and exogenous irisin protected against IR injury in the mouse lung.

    (A) The animal survival rate after lung IR injury with RIPC treatment or irisin administration (1 μg/kg) (*P < 0.05 versus others; n = 16 in sham group, n = 15 in IR group, and n = 8 in other groups). (B) Lung edema was evaluated as the wet/dry weight ratio of the excised lung from mice (*P < 0.05 versus others; n = 9 in sham group, n = 8 in IR group, and n = 4 in other groups). The plasma PaO2 (C) and PaCO2 (D) were measured (*P < 0.05 versus others; n = 8 in sham and IR group and n = 4 in other groups). A.U., arbitrary units. Serum concentrations of interleukin-1β (IL-1β) (E) and IL-6 (F) were measured using enzyme-linked immunosorbent assay (*P < 0.05 versus others; n = 4).

  • Fig. 4. Irisin protected against oxidative stress and apoptosis in IR-injured lung tissue.

    (A) Immunostaining showed that irisin was detected in the mouse lung tissue after IR injury with irisin treatment but not in sham-operated animals (scale bars, 50 μm). (B) Lung tissue sections were immunostained for irisin (green) and AT1a (red, alveolar type I epithelial cell marker) (scale bars, 20 μm). (C) The extent of reactive oxygen species (ROS) production in mouse lung tissue was determined by dihydroethidium staining (*P < 0.05 versus sham group and #P < 0.05 versus IR group; n = 5; scale bars, 100 μm). (D) Assessment of cytochrome c (cyc c) in mitochondria and cytosol derived from lung tissue subjected to IR (*P < 0.05 versus sham and #P < 0.05 versus IR; n = 4). COX IV, cyclooxygenase IV; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (E) Caspase 9 activation in lung tissue subjected to IR (*P < 0.05 versus sham and #P < 0.05 versus IR; n = 4).

  • Fig. 5. Irisin protected mitochondria from apoptosis in cultured lung epithelial cells subjected to AR injury.

    (A) Accumulation of exogenous irisin in A594 cells after anoxia recorded by a time-lapse live cell system (see also movies S1 to S3). (B) Pharmacological agents that disrupt endocytic pathways were used to test their effect on uptake of irisin into A594 cells. Representative images and quantification data are presented. CPZ (chloropyrazine) is an inhibitor of clathrin-mediated endocytosis; DMA (dimethylacetamide) is an inhibitor of large endocytic uptake pathway; and nystatin is an inhibitor of lipid raft–mediated endocytosis. The staining was repeated five times, and 15 cells in each slide were randomly chosen for fluorescence intensity analysis [*P < 0.05 versus other anoxia/reoxygenation (AR) groups; scale bars, 50 μm]. (C) A549 cells were incubated with MitoTracker and fluorescein isothiocyanate (FITC)–conjugated irisin for 60 min. Cells did not take up FITC-irisin at the normal condition (top), with measurable uptake of irisin after AR treatment for 2 hours (middle). FITC-boiled irisin was used as a negative control (bottom). Note that FITC-irisin shows overlap with MitoTracker in AR-treated A549 cells. Scale bars, 10 μm. (D) The amount of ROS in the A549 cells was determined by MitoSOX Red (green) (*P < 0.05 versus others; n = 4; scale bar, 5 μm). (E) Assessment of cyc c in mitochondria and cytosol derived from AR-treated A549 cells (*P < 0.05 versus control and #P < 0.05 versus AR; n = 4). (F) Caspase 9 activation in AR-treated A549 cells (*P < 0.05 versus control and #P < 0.05 versus AR; n = 4).

  • Fig. 6. The protective effect of irisin on mitochondrial function is associated with UCP2.

    (A) Uncoupling protein 2 (UCP2) protein expression in mouse lung tissue after IR, with or without irisin treatment, was determined by immunoblotting. IR injury reduced the expression of UCP2, whereas irisin (1 μg/kg) treatment partially rescued UCP2 expression (*P < 0.05 versus sham control mice and #P < 0.05 versus lung IR injury mice; n = 5). (B) UCP2 protein expression in A549 cells after AR with or without irisin treatment was determined by immunoblotting (*P < 0.05 versus control group and #P < 0.05 versus AR group; n = 4). (C) Colocalization of irisin and UCP2 in A549 cells. A549 cell was first treated with FITC-irisin (0.1 μg/ml) or FITC-boiled irisin and then immunostained with anti-UCP2 antibody. Colocalization of irisin and UCP2 was observed after AR injury (scale bars, 10 μm). (D) Coimmunoprecipitation of irisin and UCP2 in A549 cells. Cell lysates were immunoprecipitated with anti-UCP2 antibody and then immunoblotted with anti-irisin antibody. For negative control, rabbit immunoglobulin G was used for immunoprecipitation. Anti-UCP2 antibody was used for both immunoprecipitation and immunoblot as a positive control. (E) Time course of UCP2 degradation in A549 cells during AR treatment. The cells were incubated with cycloheximide (10−5 M) to inhibit protein synthesis (*P < 0.05 versus without irisin treatment; n = 8).

  • Fig. 7. Genetic ablation of UCP2 compromised the protective effect of exogenous irisin on lung IR injury.

    (A) Histopathological changes in IR-injured lung in WT or Ucp2−/− mice, with or without irisin administration (scale bars, 50 μm). (B) Histopathological assessment (lung injury score) of pulmonary tissue for each group is summarized. The staining for each group was repeated at least five times, and 15 fields of vision in each slide were chosen for histopathological assessment (*P < 0.05 versus WT mice under same conditions and #P < 0.05 versus IR group in WT mice). (C) Immunohistochemical staining shows targeting of irisin to the injured alveolar cells in WT and Ucp2−/− mice (scale bars, 50 μm). (D) Lung edema, determined by wet/dry ratio in WT and Ucp2−/− mice after IR injury (*P < 0.05 versus WT mice under same conditions and #P < 0.05 versus IR group in WT mice; n = 5). Measurement of PaO2 (E) and PaCO2 (F) in WT and Ucp2−/− mice after IR injury (*P < 0.05 versus WT mice under same conditions and #P < 0.05 versus IR group in WT mice; n = 5).

  • Fig. 8. Irisin inhibitor eliminated the protective effect of irisin on IR-injured lung.

    (A) Effect of irisin and genipin administration on lung edema in IR mice. Arterial blood samples were drawn from individual mice, and plasma PaO2 (B) and PaCO2 (C) were measured; n = 5 for control, 4 for genipin alone, 4 for IR, 4 for (IR + irisin), and 4 for (IR + irisin + genipin) (*P < 0.05 versus control and #P < 0.05 versus irisin-treated lung IR mice). (D) Effect of genipin on lactate dehydrogenase (LDH) concentration in supernatant from A549 cells after AR with or without irisin administration (*P < 0.05 versus control cells and #P < 0.05 versus irisin-treated AR cells; n = 4). (E and F) Effect of genipin on mitochondria-mediated apoptosis in A549 cells after AR, with or without irisin administration. Cyc c release (E) was expressed as the ratio of cyc c in cytosol and mitochondria, and caspase 9 activity (F) was expressed as the ratio of active caspase 9 and GAPDH (*P < 0.05 versus control cells and #P < 0.05 versus irisin-treated AR cells; n = 4).

  • Table 1. The characteristics of patients included in the study.
    GroupsnGender (male/female)Gestational age (weeks)Birth weight (kg)5-min Apgar scoreSpO2 (%)
    Control157/833.5 ± 3.33.12 ± 0.129.3 ± 0.296.1 ± 1.8
    NRDS73/431.6 ± 4.82.65 ± 0.29*6.9 ± 0.3*Acute illness82.7 ± 5.1*
    Recovery92.9 ± 2.7

    *P < 0.05, versus control.

    P < 0.05, versus neonatal respiratory distress syndrome (NRDS) infants after recovery.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/9/418/eaao6298/DC1

      Fig. S1. Specificity of irisin antibody and absence of endogenous irisin expression in lung epithelial cells.

      Fig. S2. Mass spectrogram profile of irisin peptide.

      Fig. S3. Irisin expression in lung tissues from sham-treated mice after RIPC.

      Fig. S4. Irisin expression in mouse lung tissues at different time points after RIPC, with or without IR injury.

      Fig. S5. OCR of lung tissue from IR-injured mice.

      Fig. S6. Time-dependent accumulation of exogenous irisin in RLE-6TN and BEAS-2B cells.

      Fig. S7. Localization of irisin in mitochondria of cultured lung epithelial cells.

      Fig. S8. Measurement of cell viability and LDH concentration in culture medium derived from lung epithelial cells after AR.

      Fig. S9. Colocalization of irisin and UCP2 in lung epithelial cells.

      Fig. S10. Basal UCP2 expression, inflammation markers, and amount of ROS in WT and Ucp2−/− mice.

      Table S1. The characteristics of control and NRDS newborn patients in Fig. 2B.

      Table S2. The characteristics of control and NRDS newborn patients in Fig. 2C.

      Table S3. Original data and P values (Excel file).

      Movie S1. Uptake of exogenous irisin into BEAS-2B cells.

      Movie S2. Uptake of exogenous irisin into RLE-6TN cells.

      Movie S3. Uptake of exogenous irisin into A549 cells.

    • Supplementary Material for:

      Irisin protects mitochondria function during pulmonary ischemia/reperfusion injury

      Ken Chen, Zaicheng Xu, Yukai Liu, Zhen Wang, Yu Li, Xuefei Xu, Caiyu Chen, Tianyang Xia, Qiao Liao, Yonggang Yao, Cindy Zeng, Duofen He, Yongjian Yang, Tao Tan, Jianxun Yi, Jingsong Zhou, Hua Zhu, Jianjie Ma,* Chunyu Zeng*

      *Corresponding author. Email: chunyuzeng01{at}163.com (C.Z.); jianjie.ma{at}osumc.edu (J.M.)

      Published 29 November 2017, Sci. Transl. Med. 9, eaao6298 (2017)
      DOI: 10.1126/scitranslmed.aao6298

      This PDF file includes:

      • Fig. S1. Specificity of irisin antibody and absence of endogenous irisin expression in lung epithelial cells.
      • Fig. S2. Mass spectrogram profile of irisin peptide.
      • Fig. S3. Irisin expression in lung tissues from sham-treated mice after RIPC.
      • Fig. S4. Irisin expression in mouse lung tissues at different time points after RIPC, with or without IR injury.
      • Fig. S5. OCR of lung tissue from IR-injured mice.
      • Fig. S6. Time-dependent accumulation of exogenous irisin in RLE-6TN and BEAS-2B cells.
      • Fig. S7. Localization of irisin in mitochondria of cultured lung epithelial cells.
      • Fig. S8. Measurement of cell viability and LDH concentration in culture medium derived from lung epithelial cells after AR.
      • Fig. S9. Colocalization of irisin and UCP2 in lung epithelial cells.
      • Fig. S10. Basal UCP2 expression, inflammation markers, and amount of ROS in WT and Ucp2−/− mice.
      • Table S1. The characteristics of control and NRDS newborn patients in Fig. 2B.
      • Table S2. The characteristics of control and NRDS newborn patients in Fig. 2C.
      • Legends for movies S1 to S3

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Table S3. Original data and P values (Excel file).
      • Movie S1 (.wmv format). Uptake of exogenous irisin into BEAS-2B cells.
      • Movie S2 (.wmv format). Uptake of exogenous irisin into RLE-6TN cells.
      • Movie S3 (.wmv format). Uptake of exogenous irisin into A549 cells.

      [Download Table S3]

      [Download Movies S1 to S3]

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