Pathogen-free Sprague-Dawley rats were purchased from the Animal Center of Guangxi Medical University (Nanning, China), and approved by the Institutional Animal Care and Use Committee of Tumor Hospital of Guangxi Medical University. Rats were injected intravenously with 500 µg of CpG oligodeoxy nucleotides (ODN2088) 2.0 h before MV [tidal volume (VT) = 40 ml/kg] to inhibit the expression of TLR9 (10). Meanwhile, some rats were intraperitoneally pretreated with an inhibitor of mtDNA called chloroquine (CQ, 30 mg/kg) 2.0 h before MV [VT = 40 ml/kg] (13). EdU (5.0 mg/kg, 2.0 h/time and five times) was intraperitoneally injected to label mtDNA before MV (10). Antimycin A (AmA, 15 mg/kg, bw/day) and cyclosporine A (CsA, 5.0 mg/kg, bw/day) were intraperitoneally injected to inhibit mitochondrial electron transport and membrane permeability transition individually (14–16).

Enzyme linked immunosorbent assay (ELISA) kits were used to test the levels of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and myeloperoxidase (MPO) (CUSABIO, Wuhan, China). DNase I (AMPD1), rat (I4131), and mouse IgG (I8765) were obtained from Sigma-Aldrich for the pretreatment of lung tissues; Percoll (P8370, Pharmacia), RPMI 1640-HEPES medium (22400097, Gibco), RT II-70 monoclonal antibody (gift from Professor Gonzalez, UCSF-Medical Center, San Francisco), goat anti-mouse IgG3 Secondary Antibody Alexa Fluor 488 conjugate (A-21151, Life Technologies) were used to isolate alveolar type II (AT-II) cells. Dulbecco’s modified Eagle’s medium (10567022, Gibco) with rat serum (D110-00-0050, Rockland), recombinant human keratinocyte growth factor (KGF/FGF-7, 251-KG-010, R&D Systems), 8-Bromoadenosine 3′, 5′-cyclic monophosphate (8-bromocyclic AMP, B5386, Sigma-Aldrich) and Engelbreth-Holmes-Swarm Matrix (EHS Matrix, Matrigel, 356234, BD) were employed for culturing AT-II cells. Then CQ and ODN2088 were purchased from Sigma-Aldrich (C6628) and InvivoGen (tlrl-2088), respectively. In addition, TRIzol (15596018, Invitrogen) and real time-quantitative polymerase chain reaction (RT-qPCR) kit (Takara) were used to test the TLR9, cytochrome c oxidase 4 (COX4), MyD88, nuclear factor (NF)-κB (NF-κB), and β-actin’s mRNA levels. LC3B (L7543, Sigma, 1:1,000), PINK1 (BC100-494, Novus biological, 1:1,000), Parkin (SAB4502077, Sigma-Aldrich, 1:800), Mfn1 (M6319, Sigma-Aldrich, 0.7 µg/ml), TLR9 (NBP1-76680, Novus, 1:1,000), MyD88 (4283, Cell Signaling Technology, 1:1,000), COX 4 (NB110-39115, Novus, 1:2,000), NF-κB (4764, Cell Signaling Technology, 1:1,200), and β-actin (4970, Cell Signaling Technology, 1:1,000) were used as primary antibodies and horseradish peroxidase (HRP)-conjugated mouse anti-rabbit antibody (7074, Cell Signaling Technology, 1:1,000) was used as secondary antibody in immunoblot and immunofluorescence assays. Moreover, ATP (MAK190, Sigma-Aldrich), ROS (MAK144, Sigma-Aldrich), and Δψm assay kits (V35116, Invitrogen) were used to evaluate the mitochondrial damage by MV. AmA and CsA were both obtained from Sigma (St. Louis, MO, USA) for regulation of mitophagy. An EdU from Click-iT EdU Alexa Fluor 488 Imaging Kit (C10337, Invitrogen) was applied to detect mtDNA in the section.

The animal model was established successfully based on the previous study (7, 17). Briefly, rats were anesthetized by intraperitoneal injection of 60 mg/kg pentobarbital sodium and 80 mg/kg ketamine, and the rats were given with 15 mg/kg pentobarbital sodium every 30 min and 2 mg/kg/h pancuronium for muscle relaxation. After received 16-gauge tube by tracheotomy, all animals were allowed to breathe spontaneously or ventilated mechanically with room air (FiO₂ = 0.21%) by a small animal ventilator (TOPO, Kent Scientific, Torrington, CT, USA). The ventilation rate was 80 breaths/min and the fraction of inspired oxygen was approximately at 40–50%. The inspiration to expiration ratio was kept at 1:1 throughout the experiment, no positive end expiratory pressure was included, and VT was calculated as previously described. After MV or spontaneous breathing, all rats were sacrificed by carotid artery bleeding, and the bronchoalveolar lavage fluid (BALF), blood serum, and lung tissue were collected and stored at −80°C except the right lung, which was obtained for paraffin embedding, transmission electron microscope (TEM) examining, and wet/dry (W/D) ratio calculating. It should be noted that all animal procedures were performed with great care to minimize activation of an inflammation.

The W/D ratio was calculated to estimate the condition of lung edema during VILI. The middle lobe of right lung was weighed and then dried to a constant weight at 60°C for 48 h. Total protein of BALF was assessed for pulmonary permeability by bicinchonininc acid (BCA) assay, and cells were counted for inflammatory infiltration by hemocytometer. Moreover, IL-1β, IL-6, TNF-α, and MPO in plasma and BALF were detected by ELISA kits according to the manufacturer’s instructions.

The right lower lung lobe was dissected and fixed with 10% formaldehyde. The lung tissues were embedded with paraffin and stained with hematoxylin and eosin. The degree of lung injury was estimated by scores according to four criteria: (1) alveolar congestion; (2) hemorrhages; (3) neutrophils’ infiltration; and (4) incrassation of the alveolar wall. Criterion were scaled as five-point scores: 0, minimal injury; 1, mild injury; 2, moderate injury; 3, serious injury; and 4, maximal injury. The cumulative histology score for all of the parameters was calculated, then the overall score of VILI was obtained. A mean ± SD was generated from the cohort of spontaneous breathing or ventilated lungs to generate a cumulative histological VILI (7, 18). Simultaneously, lung samples were cut for TEM analysis to observe lung cell epithelial cells and other cell injuries.

Total RNA was isolated from lung tissue using TRIzol reagent. After RNA quality and quantity were determined by 260/280 nm absorbance, single-stranded cDNA was synthesized using the Takara RNA PCR kit. Total RNA was determined, and 1 µg of total RNA was reverse-transcribed into cDNA and amplified using SYBR Premix Ex Taq II and specific primers for TLR9, COX4, MyD88, NF-κΒ, and β-actin. The level of each target gene was normalized relative to that of β-actin in each sample using the ΔCt method. Relative differences in gene expression among groups of the lung tissues were determined using the comparative Ct (ΔΔCt) method and fold expression was calculated by the formula 2^−ΔΔCt, where ΔΔCt represents ΔCt values normalized relative to the mean ΔCt of healthy control samples. The final data were shown as the ratio of the mean value of triplicate detection of the mtDNA samples (COX 4) and the average value of the nuclear gene (β-actin), namely mtDNA/β-actin (19).

Total protein was extracted, and the concentrations were assessed by BCA assay. Then, the molecular weight marker and each sample were added to the lanes of sodium dodecyl sulfate (SDS)-polyacrylamide gel. The proteins were transferred onto a nitrocellulose membrane and then blocked. Subsequently, the membranes were incubated by LC3B, PINK1, Parkin, Mfn1, COX 4, TLR9, MyD88, NF-κB, and β-actin primary antibodies and HRP-conjugated mouse anti-rabbit antibody as a secondary antibody. The bands of each protein from different samples were scanned and detected via a West Pico enhanced chemiluminescence kit (Thermo Fisher Scientific).

The slides from the paraffin-embedded lung tissues were dewaxed, hydrated, used for antigen retrieval, blocked, and cleared of endogenous peroxidases. Then, anti-LC3B was used as the primary antibody and anti-rabbit IgG (H + L), F(ab′)2 Fragment (Alexa Fluor 594 Conjugate) served as secondary antibody, along with DAPI for nuclear staining. Meanwhile, cells from the EdU-labeled rats were detected via Click-iT EdU Alexa Fluor 488 Imaging Kit according to the manufacturer’s directions to observe mtDNA accumulation in lung tissues, as well as for analyzing the source of escaped mtDNA. The slides were coated with Prolong Gold quenching resistant reagent and preserved at 4°C for imaging with fluorescence microscopy. The whole process was kept out of the sun.

The MV-induced mitochondrial damage with mitophagy was evaluated via measuring ATP levels, ROS production, and Δψm. The ATP level, ROS production, and Δψm were assayed using corresponding assay kits from Sigma-Aldrich according to the manufacturer’s instruction. Briefly, the ATP levels were determined via firefly luciferase-associated chemiluminescence and ROS production was detected by 2′7′-dichlorofluorescin diacetate using flow cytometry analysis. The Δψm was observed using a fluorescent probe JC-1 that assembles as J-aggregates with red fluorescence in the mitochondrial matrix during higher Δψm, but is depolymerized as monomer and results in green fluorescence.

The single-phase enzyme diffusion (21) method was used to compare DNase-II expression in the serum. In brief, “Solution A” [1 ml of 6 g/l calf thymus DNA; 50 µl of 10 g/l ethidium bromide; pH 7.2, 0.05 M Tris–HCl (0.05 M MgCl₂) 8~10 μl; 40 µl of 1 M CaCl₂] and “Solution B” (10 ml of 2% agarose in diethyl pyrocarbonate water) were mixed to make agar board which was then bored with a 1.5 mm-diameter puncher. 4 µl of either serum samples or standard DNase-II were added to the agar board holes, followed by 36 h of incubation at 37°C. The agar board was then soaked with 0.1 M elhylene diamine tetraacetic acid (EDTA) to stop the reaction and the diameter of DNA hydrolyzed loop was measured using ultraviolet radiation.

The extraction and purification of mtDNA were modified from the previous study to improve the yield and quality of mtDNA (22). Briefly, a pellet composed of 10⁷ cells were resuspended with 5.5 ml “Solution I” of (10 M Tris–HCl; 10 M NaCl; 5 M MgCl₂; pH = 7.5) and centrifuged at 2,000 rpm for 10 min. The pellet was resuspended in 5.5 ml of “Solution II” (10 mM Tris–HCl; 0.4 M NaCl; 2 mM EDTA; pH = 7.5) and centrifuged at 3,500 rpm for 10 min. Then the pellet was resuspended in 0.95 ml of “Solution II” and 50 µl of 20 mg/ml protease K and 10% SDS were added, followed by incubation at 4°C overnight. 0.3 ml of saturated sodium acetate was added to the suspension liquid and lightly shaken, followed by centrifugation at 15,000 rpm for 15 min at 4°C. The supernatant was obtained, and the last step was repeated. A phenol:chloroform:isopropanol (25:24:1) solution was added to the supernatant, agitated by light shaking and centrifuged at 10,000 rpm for 10 min at 4°C. The upper water phase was transferred to a new tube, and the previous step was repeated. Two volumes of ice-cold ethanol were added to the upper water phase and incubated on ice for 30 min, followed by centrifugation at 15,000 rpm for 10 min at 4°C. 70% ice-cold ethanol was next used to wash the pellet, centrifuging the sample at 15,000 rpm for 2 min at 4°C. The pellet was dried for 25 min at room temperature and finally dissolved by recombinational trypsin/EDTA solution at storage of 4°C.

The analyses of data were conducted by SPSS 13.0 software. All quantitative data were demonstrated as mean ± SD. One-way ANOVA was operated in order to analyze multiple comparisons of each groups, followed by the LSD-t test and the SNK test for pair-wise comparisons. Chi-square test was used for analysis of categorical variables. The statistical difference was defined by P value less than 0.05.

Anesthetized animals were randomly allocated into three groups (n = 12) using table of random number: non-ventilated (CON), ventilated with normal VT (10 ml/kg, NTV), and ventilated with high VT (40 ml/kg, HTV). All animals survived the 4-h period of spontaneous breathing or MV at normal or high VT. The rats treated with high VT exhibited significantly severe pulmonary edema and higher BALF total protein levels than the rats treated with spontaneous breathing and normal VT by determining the lung W/D ratios (Figures 1A,C). The lung histopathology score was higher in high VT rats as compared with the CON and NTV group. However, no differences were noted between the CON and NTV group (Figure 1B). In the HTV group, significantly more cells were infiltrated than in the NTV group and non-ventilated animals (Figure 1D). In addition, the levels of cytokine profiles, including IL-1β, IL-6, TNF-α, and MPO, in BALF and plasma were significantly higher in the HTV group than that in the CON and NTV groups. And these cytokine profiles were similar in animals ventilated with normal VT and control animals (Figures 1E–H).

Lungs from animals ventilated with high VT showed acute inflammatory infiltration, perivascular edema, and more alveolar septal thickening, whereas no major histological differences were observed between animals ventilated with normal VT and spontaneous breathing control animals (Figure 1I). Using TEM to examine alveolar histopathology in greater detail, we found that, as expected, alveolar cells in the tissue of the rats ventilated with high VT exhibited a disrupted cytoplasmic and nuclear structure, as well as cell membrane discontinuities. However, tissue from rats ventilated with normal VT and spontaneous breathing control animals showed a normal cytoplasmic and nuclear structure and continuous cell membrane for types I and II alveolar epithelial cells. The osmilphilic multilamellar body is the unique structure of AT-IIs. In addition, the osmilphilic multilamellar bodies show characteristic vacuolation in the AT-IIs of HTV group (Figure 1J).

The protein levels of TLR9, MyD88, and NF-κB were significantly higher in the HTV group as compared with the other groups. Moreover, pretreated with ODN2088 could significantly ameliorated the HTV-induced these proteins in the lungs. It might suggest that HTV induces inflammatory response in the lungs via TLR9/MyD88/NF-κB signaling pathway (Figure 2).

In order to explore whether MV with HTV can favor the damage of mitochondrial and the release of mtDNA, ATP levels, ROS production, Δψm variation, and expression level of mtDNA were measured. The level of ATP in the HTV was decreased in comparison to the rats treated with spontaneous breathing and pretreated CQ could alleviate the result (Figure 3A). Then, the production of ROS in HTV group was significantly increased in comparison to the rats treated with spontaneous breathing, but pretreated with CQ could reduce these ROS induced by MV with HTV, especially in AT-IIs (Figures 3B,G). Moreover, the Δψm in the HTV group was decreased in comparison to the rats treated with spontaneous breathing, while compared with the animals over-ventilated with CQ (Figure 3C).

The level of serum DNase-II (a main enzyme for degradation of mtDNA) in the HTV was decreased in comparison to the rats treated with spontaneous breathing or normal VT (Figure 3D). The outcome of RT-qPCR in isolated mtDNA in the HTV group was significantly higher than that in CON group, but level of mtDNA was significantly lower in the CQ pretreatment group compare with the HTV group (Figures 3E,F).

The mRNA and protein levels of COX4, TLR9, MyD88, and NF-κB were significantly higher for animals ventilated with high VT than animals in the CON and NTV groups. Expectedly, after pretreated with mtDNA inhibitor CQ, the mRNA and protein expression of COX4, TLR9, MyD88, and NF-κB in animals treated with high VT were significantly inhibited (Figures 4A–C).

The expression of LC3B conjugated with a fluorescence moiety was recommended in order to observe the autophagy state and the EdU was used for mtDNA distribution. The fluorescence of mtDNA and LC3B, which specifically marked mitophagy, turned up in same places, indicating that mtDNA could be released due to autophagic lysosomes. The fluorescence intensity showed that the expression of LC3B and mtDNA in the HTV was increased compared to CON group. More importantly, the expression of LC3B and mtDNA in the HTV + CQ group was lower than that in the HTV group (Figure 5).

The role of mitophagy in VILI was shown by presence of antimycin A (inhibitor of mitochondrial electron transport) and cyclosporine A (inhibitor of mitochondrial membrane permeability transition). AmA pretreatment aggravated lung edema (Figure 6A) and morphological injuries (Figures 6B,I,J), but CsA pretreatment attenuated the lung edema and injury. Total protein level for pulmonary permeability in BALF was significantly increased in the AmA group compared to the HTV group and CON group (Figure 6C), while infiltrated cells counted in BALF was consistent with the variation of total proteins (Figure 6D). The concentrations of inflammatory factors, including IL-1β, IL-6, TNF-α, and MPO, in the AmA group (plasma and BALF) were higher than those in the HTV and control groups, and CsA pretreatment could attenuate these inflammatory factors (Figures 6E–H).

The expression levels of mitophagy related genes (LC3B-II/LC3B-I, PINK1, Parkin, and Mfn1) in animals ventilated with high VT were significantly upregulated (Figures 7A–D). AmA favored the higher ratio of LC3B-II/LC3B-I, expression of PINK1, Parkin, and Mfn1, while CsA inhibited the expression of these mitophagy related proteins (Figures 7A–D). What is more, AmA pretreatment upregulated the mRNA and protein level for COX4, TLR9, MyD88, and NF-κB, but CsA pretreatment attenuated the expression of these genes (Figures 7E–I).

Although mtDNA is clearly involved in inflammation through the TLR9/MyD88/NF-κB pathway in regulating inflammatory cytokines during MV, there are several limitations that warrant further discussion. First of all, there are some other factors can affect injury severity, such as positive end expiratory pressure and variations in pressure support (48). For minimizing these impacts, we continuously monitored hemodynamic stability and oxygen saturation in the anesthetized rats. Second, according to the literature, the type and dose of anesthetic (e.g., sevoflurane, ketamine, or protofol) use can also affect animal studies on acute lung injury (49, 50). In addition, our study, however, were done only in living animals. We cannot say whether the results would be the same in vitro experiments.