Melatonin prevents lung injury by regulating apelin 13 to improve mitochondrial dysfunction

Abstract

Pulmonary fibrosis is a progressive disease characterized by epithelial cell damage, fibroblast proliferation, excessive extracellular matrix (ECM) deposition, and lung tissue scarring. Melatonin, a hormone produced by the pineal gland, plays an important role in multiple physiological and pathological responses in organisms. However, the function of melatonin in the development of bleomycin-induced pulmonary injury is poorly understood. In the present study, we found that melatonin significantly decreased mortality and restored the function of the alveolar epithelium in bleomycin-treated mice. However, pulmonary function mainly depends on type II alveolar epithelial cells (AECIIs) and is linked to mitochondrial integrity. We also found that melatonin reduced the production of reactive oxygen species (ROS) and prevented apoptosis and senescence in AECIIs. Luzindole, a nonselective melatonin receptor antagonist, blocked the protective action of melatonin. Interestingly, we found that the expression of apelin 13 was significantly downregulated in vitro and in vivo and that this downregulation was reversed by melatonin. Furthermore, ML221, an apelin inhibitor, disrupted the beneficial effects of melatonin on alveolar epithelial cells. Taken together, these results suggest that melatonin alleviates lung injury through regulating apelin 13 to improve mitochondrial dysfunction in the process of bleomycin-induced pulmonary injury.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1. Melatonin increased survival and improved pulmonary epithelial function in response to bleomycin.

Mice were intratracheally treated with PBS or bleomycin (BLM) with or without melatonin (MLN), (PBS group: n = 25, BLM group: n = 26, BLM + MLN group: n = 25). a Survival plots of mice in the PBS, BLM, and BLM + MLN groups. *P < 0.05. b Mice were intraperitoneally injected with Evans blue solution (1 ml/kg 3% Evans blue solution in PBS) for 30 min before euthanization. The amount of Evans blue was measured in the whole lungs of mice in the PBS, BLM, and BLM + MLN groups and normalized to the Evans blue concentration in methanamide to generate an Evans blue index. n = 3; *P < 0.05. c H&E staining of representative lung sections from mice in the PBS, BLM, and BLM + MLN groups. Scale bars, 50 μm. d The mRNA expression of inflammatory factors (IL1β, IL6, and TNFα). n = 4; *P < 0.05, **P < 0.01. e The Immunohistochemical assay of E-cadherin in lung samples from mice in the PBS, BLM, and BLM + MLN groups. Scale bar, 50 μm. f–g Quantitative RT-PCR was performed to analyze the mRNA expression of E-cadherin (Cdh1) and Sftpc. n = 5; *P < 0.05, **P < 0.01. h E-cadherin, a marker of alveolar epithelial cells, was identified by immunoblot analysis. n = 6; *P < 0.05, **P < 0.01

Fig. 2. Melatonin attenuated apoptosis during lung injury.

a Immunoblot analysis showed the expression of apoptosis-related proteins (Bcl2, Bax, and Cyt-c) in the PBS, BLM, and BLM + MLN groups. TC-1 cells were treated with H2O2 for 12 h with or without 10 nM, 1 μM, or 100 μM melatonin for 24 h. *P < 0.05. b An MTT assay was performed to assess TC-1 cells viability. n = 5; *P < 0.05, **P < 0.01. c Left, a TUNEL assay was performed to detect apoptotic cells, and TUNEL-positive cells were counted in TC-1 cells. Green, TUNEL-positive TC-1 cells; blue, DAPI. Scale bars, 50 μm. **P < 0.01. d Immunoblot analysis of the protein expression of Bcl2, Bax, and Cyt-c. n = 5; *P < 0.05. e Luzindole inhibited the effect of melatonin on cell viability in TC-1 cells, as indicated by an MTT assay. n = 6; **P < 0.01. f The number of TUNEL-positive cells was increased in the MLN + luzindole + H2O2 group compared with that in the MLN + H2O2 group. Green, TUNEL-positive TC-1 cells; blue, DAPI. Scale bars, 50 μm. Right, relative percentage of TUNEL-positive TC-1 cells. *P < 0.05, **P < 0.01

Fig. 3. Melatonin inhibited senescence during lung injury.

a Western blot analysis of the expression of p53 and p21 in whole lungs from mice in the PBS, BLM, and BLM + MLN groups. *P < 0.05. b Western blot analysis of p53 and p21 expression in TC-1 cells. *P < 0.05, **P < 0.01. c β-Gal staining showed increased senescence-associated β-gal activity in TC-1 cells 12 h after H2O2 treatment, and treatment with 100 μM melatonin reversed senescence in these cells. Scale bar, 50 μm. **P < 0.01. d Luzindole significantly blocked melatonin-induced senescence, as evidenced by staining for senescence-associated β-gal activity. Cells with green staining are senescent cells. Scale bar, 50 μm. **P < 0.01

Fig. 4. Melatonin reversed bleomycin-induced mitochondrial abnormalities in alveolar epithelial cells.

a Representative transmission electron microscopy (TEM) images of lung sections from mice treated with PBS, BLM, or BLM + MLN. The red arrows indicate normal mitochondria or damaged and swollen mitochondria. b Cellular ATP content was measured in TC-1 cells incubated with the indicated concentrations of H2O2 and melatonin. Cells were lysed, and the ATP content was determined using an ATP assay and normalized to the protein concentration. n = 5; *P < 0.05, **P < 0.01. c Comparison of ROS production in TC-1 cells exposed to H2O2 with or without melatonin (10 nM, 1 μM, or 100 μM). Green, ROS; blue, DAPI. Scale bars, 50 μm. *P < 0.05, **P < 0.01. d Measurement of ATP content in TC-1 cells after exposure to H2O2, melatonin, and luzindole; the ATP content was normalized to the corresponding protein concentration. n = 5; **P < 0.01. e Effect of luzindole on ROS production in TC-1 cells. Green, ROS; blue, DAPI. Scale bars, 50 μm. *P < 0.05, **P < 0.01

Fig. 5. Apelin 13 participated in the process of by which melatonin prevents lung injury.

a Western blot assays were performed to determine the protein levels of apelin 13 in lung tissues from mice in the PBS, BLM, and BLM + MLN groups. b The protein expression of apelin 13 in TC-1 cells after treatment with different concentrations of melatonin. c The MTT assay showed the effect of apelin 13 on the viability of H2O2-impaired TC-1 cells. n = 7; **P < 0.01. d Comparison of ROS production in TC-1 cells exposed to apelin 13. Green, ROS; blue, DAPI. Scale bars, 50 μm. **P < 0.01. e Apelin 13 impacted the mitochondrial ATP content in cells treated with H2O2. n = 5; **P < 0.01. f Representative images of the TUNEL assay in TC-1 cells are shown. Apelin 13 decreased the H2O2-induced increase in apoptosis. Green, TUNEL-positive TC-1 cells; blue, DAPI. Scale bars, 50 μm. **P < 0.01. g Apelin 13 significantly decreased cell senescence, as evidenced by staining for senescence-associated β-gal activity. Right, relative percentage of senescence-associated β-gal activity. **P < 0.01

Fig. 6. Blocking Apelin 13 by ML221 inhibited the effect of melatonin on alveolar epithelial cells.

a Survival curves of mice after the injection of PBS, BLM, BLM + MLN, or BLM + MLN + ML221. n = 21; *P < 0.05. b E-cadherin, a marker of alveolar epithelial cells, was assessed by immunoblot analysis. n = 4; *P < 0.05, **P < 0.01. c The Immunohistochemical assay of E-cadherin in lung samples from mice in the PBS, BLM, BLM + MLN, and BLM + MLN + ML221 groups. Scale bar, 50 μm. d The effect of ML221 on cell viability was determined using an MTT assay. n = 7; **P < 0.01. e Comparison of ROS production in TC-1 cells exposed to ML221. Green, ROS; blue, DAPI. Scale bars, 50 μm. *P < 0.05. f ATP content in TC-1 cells treated with H2O2, melatonin, or ML221. The ATP content was normalized to the corresponding protein concentration. n = 5; **P < 0.01. g Treatment with ML221 inhibited the antiapoptotic effect of apelin 13. Green, TUNEL-positive TC-1 cells; blue, DAPI. Scale bars, 50 μm. *P < 0.05, **P < 0.01. h Left, TC-1 cells were treated with ML221 and were then fixed and stained for senescence-associated β-gal activity. **P < 0.01. i Representative profiles and summary data for oxygen consumption in TC-1 cells. The blue arrow indicates the oxygen concentration in the detection chamber. The blue line indicates the oxygen concentration in the detection chamber. j Western blot assays were performed to determine the protein levels of p-ERK1/2 and apelin 13 in TC-1 cells. n = 4; *P < 0.05

Fig. 7. Schematic model describing the protective effect of melatonin on the restoration of mitochondrial homeostasis and function in AECIIs.

Injury of AECIIs led to mitochondrial dysfunction, ROS release, and decreased ATP content. Melatonin supplementation modified injury through an epithelial protective effect via apelin 13 and its receptors, which resulted in the restoration of normal mitochondrial function and, eventually, rescued from mitochondria-regulated apoptosis and senescence