Nitrative inactivation of thioredoxin-1 loses its protective effect in bleomycin-induced pulmonary �brosis

Pulmonary �brosis is in the development of in�ammatory lung diseases with no effective clinical currently. As an essential redox enzyme, thioredoxin (Trx) to be involved in Bleomycin-indued Trx and Trx were examined. p38-MAPK apoptosis pathway was determined in lung C57/BL6 were treated with aminoguanidine (AG, a peroxynitrite scavenger), recombinant human Trx-1 (rhTrx-1), or SIN-1 (a peroxynitrite donor) nitrated Trx-1 (N-Trx-1).


Results
In bleomycin (BLM)-induced pulmonary brosis model in C57/BL6 mice, we observed that nitrated Trx increased, while its activity decreased, with the increase of lung cells apoptosis rate by p38-MAPK pathway. We demonstrated that AG or rhTrx-1, but not N-Trx-1 signi cantly reduced pulmonary brosis.

Conclusion
Blockade of Trx-1 nitration, or supplementation of exogenous rhTrx-1, might represent novel therapies to attenuate pulmonary brosis in idiopathic pulmonary brosis patients.

Background
Pulmonary brosis can be the results of lung injury resulting from radiation, severe infection, chemotherapy, environmental exposure or many unknown reasons, as idiopathic pulmonary brosis (IPF) [1]. As a fetal pulmonary brosis, the annual incidence of IPF is rising and is estimated to be over 150000 patients annually in the USA, and more than 5 million worldwide, resulting in a median survival time less than 5 years [2,3]. However, as its pathogenesis is not fully revealed, effective therapeutic interventions are still needed [4].
Oxidative stress has been revealed to be involved in the pathological process of IPF by regulating the apoptosis rate of alveolar epithelial cells (AECs) through Mitogen-activated protein kinases (MAPK) pathway [5]. However, many fundamental questions cannot be explained by ROS overproduction alone. As another resource of redox stimulations, nitric oxide-derived reactive (RNS) has been reported to be another contributor of cell injury by protein modi cations [6,7].
Trx1 is a 12 k Da redox protein with important antioxidative and cell signaling functions, which has been revealed to take part in the pathogenesis of several diseases, such as ischemia, stroke and lung diseases [8, 9,10]. Some researchers investigated that Trx suppressed BLM-induced pulmonary brosis progression in mice [11,12]. Tao and her colleagues have revealed that in the myocardial ischemia models of the rats, Trx's activity decreased after being nitrated by RNS, resulting in myocardial apoptosis by ASK1-p38MAPK pathway [13]. In our previous research, we have demonstrated the role of Trx's nitration modi cation in BLM-induced pulmonary brosis in rats [14]. But our previous results have not provided direct cause-effect relations among Trx nitration, apoptosis and pulmonary brosis. Therefore, in the BLM-induced pulmonary brosis model, we investigated the direct evidence to support our hypothesis that increased nitrative inactivation of Trx-1 is causatively related to BLM-induced pulmonary brosis in mice by regulating p38-MAPK apoptosis pathway.  ). Mice housed in a room with controlled temperature (22±2℃) and humanity (65±5%) had free access to standard mice chow and water. All mice were acclimated to laboratory conditions for 7 days prior to the experiment. The mice were administered BLMA5 by intracheal injection (5.0 mg/kg body mass in phosphate-buffered saline) as described previously [14]. At 7 or 28 days after administration of BLMA5, the mice were euthanized using an overdose of chloral hydrate (10%). Before BLM administration, mice were randomized to receive either vehicle (0.9%NaCl), aminoguanidine (AG, a peroxynitrite scavenger, 40 mg/kg, continuously for 14 days), recombinant human Trx-1 (40ug/20g, continuously every 2 day for 14 days), or nitrated Trx-1 (please see details, continuously every 2 day for 14 days) by intraperitoneal injection. Pulmonary brosis was assessed from the lung histology. Lung histology was performed as described [15].

Histopathological examination of lung
Part of the upper lungs were in ated with 1 ml of 10% paraformaldehyde / PBS solution and embedded in para n. Sections (5µm thick) were treated for sample preparation. The lung tissues were stained with hematoxylin-eosin (H&E) and Masson's trichrome, then evaluated under a light microscopy conducted by experienced pathologists. The lung brosis score, based on the severity and extent of lung brosis present in the peribronchial and interstitial tissues was assessed by a pulmonary pathologist blinded to the experimental protocol. Lungs were assigned a severity score from 0 to 4 (0=absence of brosis, 1=1% to 25%, 2= 26% to 50%, 3=51% to75%, 4=76% to 100% brosis) [16].

Determination of pulmonary cells apoptosis
Pulmonary cells apoptosis was determined by terminal deoxynucleotidyltransferase-mediated dUTP nick and labeling (TUNEL) staining and caspase-3 activity assay. Sections were stained as the manufacturer's protocol. Brie y, the sections were depara nized and treated by ethylenediaminetetraacetic acid solution while being heated in a microwave. The sections were then reacted in a solution of terminal deoxynucleotidyl transferase enzyme mixed with biotinylated nucleotide. Horseradish peroxidase-labeled streptavidin was bound to these biotinylated nucleotides, which were detected using the peroxidase substrate, hydrogen peroxide, and diaminobenzidine (DAB). The index of apoptosis (number of TUNEL positive nuclei / total number of nuclei×100) was automatically calculated for further analysis. The caspase-3 activity was determined as the instruction provided by the manufacturer.

Quanti cation of tissue nitrotyrosine content
Paraformaldehyde-xed lung tissues were cut into semi-thin sections 4 to 5 µm thick and stained with a primary antibody against nitrotyrosine. Immunostaining was determined as described in our previous study [14]. Quanti cation of lung tissue nitrotyrosine content was performed by ELISA assay. Results are presented as micrograms per milligram(µg/mg) protein.

Western blotting analysis
Lung tissues were cut into small pieces and homogenized in lysis buffer on ice. The protein concentration was determined according to a protein assay kit, where 40 µg protein was loaded and separated by electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and transferred to 0.22 µm polyvinylidene di uoride (PVDF) membranes. Membranes were blocked using 5% nonfat milk in TRISbuffered saline with Tween-20 (TBST) for 1 h and then were incubated with Trx antibody or iNOS antibody overnight. After washing with TBST, protein bands were detected with secondary antibody conjugated with horseradish peroxidase. The membranes were washed 3 times in TBST for 10 min each.
The blot was developed with a super-signal chemiluminescent detection kit (Pierce) and visualized with a Kodak Image Station 400. The blot density was analyzed with Vision Works LS Acquisition and Analysis software.

Determination of Trx enzyme activity
Trx activity was determined by the insulin disul de reduction assay [17]. In brief, 40 mg of tissue homogenates were pre-incubated at 37℃ for 15 min with 2 mL activation buffer (100 mmol/L HEPES, 2 mmol/L EDTA, 1 mg/mL bovine serum albumin, and 2 mmol/L dithiothreitol) for Trx reduction. Samples were then mixed with 20 uL reaction buffer (100 mmol/L HEPES, 2.0 mmol/L EDTA, 0.2 mmol/L NADPH, and 140 mmol/L insulin), the reaction was initiated by the addition of mammalian Trx reductase (1 mL, 15 mU, Sigma) or water to the controls, and the samples were incubated for 30 min at 37℃. Finally, the reaction was stopped by adding 125uL of stopping solution (0.2 mmol/L Tris-HCL, 10 mmol/L guanidine-HCl, and 1.7 mmol/L3-carboxy-4-nitrophenyl disul de; DTNB) followed by absorption measurement at 412 nm.

Immunoprecipitation and immunoblotting
Trx-1 nitration and Trx-1/ASK1 interaction detection were performed as described [18]. In brief, Trx-1 in homogenized lung tissues was immunoprecipitated with a monoclonal antibody against Trx-1. Trx-1 nitration and Trx-1/ASK1 interaction were examined, respectively, by immunoblotting using a primary antibody against nitrotyrosine or against ASK1 after sample separation. After incubation with horseradish peroxidase conjugated secondary antibody, the blot was developed with an ECL-Plus chemiluminescence reagent kit and visualized with UVP Bio-Imaging Systems. Blot densities were analyzed with Vision Works LS Acquisition and Analysis software.
p38 MAPK activity assay p38 MAPK activity assay was performed by p38 MAPK assay kit according to the manufacturer's instructions.
In vitro nitration of Trx-1 Human Trx-1 was subjected to in vitro nitration with a provided procedure described by Guo et al [19]. In brief, puri ed human Trx-1(dissolved in 0.1 µM phosphate buffer, pH 7.4, nal concentration of 50 µM) was incubated at 37℃for 30 min with SIN-1 ( nal concentration of 100 µM). Unreacted SIN-1 was removed by ultra ltration over membranes with a 3-kDa cutoff.

Statistical analysis
All values in the text and gures were presented as mean±SD of independent experiments. All data (except immunoblotting density) were subjected to two-way ANOVA followed by Bonferroni correction for post hoc t test. Immunoblotting densities were analyzed with the Kruskal-Wallis test followed by Dunn post hoc test. Probabilities of 0.05 or less were considered to be statistically signi cant.

BLM-induced pulmonary brosis in mice
As C57/BL6 mice are more susceptible to BLM induced brosis [20], C57/BL6 mice were selected in the present study. After BLM exposure, the mice weight dropped signi cantly data not shown). In the lungs of control group, both the HE staining and Masson staining results showed that intact and clear alveoli, normal interstitium, few in ammatory cells were observed in the lungs at 7 d or 28 d. However, BLM administration caused progressive lung damage, demonstrating as destruction of lung alveoli, in ammatory cells in ltration and thickening of lung interstitium (Fig. 1).
The apoptosis rate of pulmonary cells increased in the BLM-induced pulmonary brosis in mice Apoptosis of AECs has been revealed to play a critical role in IPF [21,22]. To investigate whether BLMinduced pulmonary brosis was associated with increased pulmonary cells apoptosis rate, the TUNEL straining and caspase-3 activity were examined. Compared with the control group, the rate of TUNELpositive nuclei or caspase-3 activity in BLM group increased signi cantly, which revealed that apoptosis might be involved in BLM-induced pulmonary brosis (Fig.2).

BLM-induced protein nitration in mice
Nitrotyrosine has been revealed as a footprint of protein nitration [23,24]. The enzyme-linked immunosorbent assay (ELISA) kit was used to examine nitrotyrosine content. BLM group manifested nitration formation, while absent in control groups. As iNOS is a key enzyme to induce NO production, its expression was determined in the research. The expression of iNOS was induced in the lungs of BLM group, which means that the increase of nitrotyrosine content might be the result of iNOS overexpression (Fig.3).

The activity and nitrated Trx in BLM-induced pulmonary brosis in mice
Trx is a key regulator of oxidative stress [25], therefore, we examined Trx's activity. Compared with the control group, in the BLM-induced pulmonary brosis group, Trx's activity decreased while its expression increased. As Trx is susceptible to nitrative modi cation, resulting in irreversible inhibition, the expression of nitrated Trx was determined. Compared with control group, the expression of nitrated Trx increased dramatically in BLM group (Fig.4). The results are in accordance with our previous results [14].
The inhibition of Trx-1/ASK1 interaction and increased p38MAPK activity was involved in BLM-induced pulmonary brosis in C57/BL6 mice Previous researches have revealed that Trx exerts its antiapoptotic effect by binding to ASK1 (causing inhibition of the downstream proapoptotic kinases) [26]. As the brosis rate of pulmonary cells is more obvious at day 28 after BLM administration, therefore, 28 d was chosen in the following study. We investigated the signaling pathway by which nitration of Trx increased the BLM-induced pulmonary brosis. Trx was physically associated with ASK1 in control group, and p38MAPK activity was inhibited.
In BLM group, the association between Trx and ASK1 was decreased, with the increase of p38 MAPK activity. The results suggested that increased activation of the p38-MAPK signaling pathway may contribute to the increased ACE apoptosis in BLM-induced pulmonary brosis in mice (Fig.5).

Blockade of Trx-1 nitrative inactivation, or Trx-1 supplement, is protective of BLM-induced pulmonary brosis
To further obtain more direct evidence to support the hypothesis that increased nitrative Trx is causatively related to increased BLM-induced pulmonary brosis in mice, aminoguanidine (AG), or rhTrx was administered before BLM administration. As shown in Figs.6 and 7, treatment with AG reduced Trx nitration, preserved Trx activity, restored TRX1/ASK1 interaction, and inhibited p38 MAPK activity in BLMinduced pulmonary brosis model, suggesting that AG could prevent the nitration of Trx, resulting in inhibiting p38MAPK pathway in BLM-induced pulmonary brosis model. In addition, treatment with AG or Trx signi cantly decreased the brosis rate, attenuated lung cells apoptosis rate, evidenced by decreasing TUNNEL staining and caspase3 activity. Finally, in vitro incubation of Trx with a peroxynitrite donor SIN-1(100µM) for 30 min inhibited Trx activity (135±15 μmol/min/mg Trx-1 vs. 762±53 μmol/min/mg Trx-1 invehicle-incubated Trx-1, P 0.001). Administration with N-Trx lost Trx's protecting effect in BLM-induced pulmonary brosis (Fig.8). All the results above fully provided the direct proof that Trx nitration modi cation plays an essential role in BLM-induced pulmonary brosis in mice.

Discussion
IPF is a chronic in ammatory interstitial lung disease with poor prognosis [2]. Despite signi cant progress in the understanding of pathological mechanisms of IPF, effective therapeutic interventions are to be revealed. In IPF, apoptosis of AECs has been reported as the main initiator event, though the brotic cascade is still unknown. Martinez et al reviewed a connection between ROS overload and the apoptosis rate of AECs in IPF patients [27]. ROS/RNS may induce apoptosis by stimulating caspase 3, inducing the release of cytochrome c and DNA fragmentation, resulting in activation of ASK1-mitogen-activated protein kinase (MAPK) pathway [5]. We attempted to reveal a key protein target susceptible and relevant for nitration. Trx is a small ubiquitous protein which exerts plenty of biological functions [28]. Except for its redox regulating effect, Trx exerts its apoptosis regulating effect by Trx-1/ASK1 interaction mechanism. In resting situations, ASK1 is inhibited by Trx-1 binding. When Trx loses its activity, Trx separates from ASK1, ASK1/p38MAPK apoptosis pathway is stimulated [26].
Trx family includes thioredoxin, thioredoxin reductase, NADPH. Trx contains ve cystines, which is essential in maintaining its function. Cysteines 32 and 35 are the main domains in regulating its redox state. When the functional groups of cysteines32 and cysteines35 are oxidized, Trx cannot bind to its target protein, resulting in Trx inactivation. When oxidized, Trx itself is reduced by thioredoxin reductase to keep its redox activity [29]. Moreover, Trx activity decreased when cysteine-73 was glutathionylazed under oxidative conditions [30]. Tao et al revealed that the S-nitrosylation of Trx at cysteine-73 contributed to the cardioprotective and anti-apoptotic functions of Trx in myocardial-ischemia model of mice [31].

Nitration has been revealed as Trx's fourth post-transcriptional modi cation. Tao and Zhang reported that
Trx nitration at the tyrosine residue caused its inactivation in an ischemia-reperfusion rat model [13]. Yin revealed that changes in Trx nitration might contribute to exaggerated ischemia/reperfusion injury by regulating the apoptosis rate in cardiac cells [32]. These results demonstrated that the inhibition of Trx nitration may attenuate cardiac injury after myocardial ischemia. In our previous study, we observed that in BLM-induced pulmonary brosis in rats, the activity of thioredoxin decreased while the level of nitrated thioredoxin increased, we proposed that nitration of thioredoxin resulting in the increasing of AECs apoptosis by p38MAPK-ASK1 pathway, but we did not provide the direct proof [14].
In this research, we revealed that in BLM-induced pulmonary brosis in mice, decreased activity of Trx caused the activation of ASK1-p38 MAPK pathway, resulting in lung cells apoptosis. To provide further cause-effect evidence, we examined whether blockade of Trx-1 nitrative inactivation or supplementation with exogenous Trx-1 could affect TRX-ASK1 activity and p38MAPK activity, reduce ACEs apoptosis rate, protect mice against BLM-induced pulmonary brosis. We demonstrated that either treatment reduced pulmonary brosis to some extent by decreasing AEC apoptosis rate, while N-Trx lost the effect. As administration of oxidized Trx-1 will not lose antiapoptotic effect unless thioredoxin reductase is inhibited concomitantly [33], suggesting that oxidative modi cation of Trx1 is reversible, which indicates that redox modi cation of Trx1 may not play essential role in regulating apoptosis. However, nitrative modi cation of Trx is irreversible [34]. These results provide the rst evidence that Trx-1 nitrative inactivation plays a causative role in pulmonary brosis. But the BLM-induced pulmonary brosis is different from the IPF patients, so the conclusion needed to be in clinical practice.

Conclusions
Our research's novel results strongly suggest that in BLM-induced pulmonary brosis in mice, overload of ONOO − formation promote of RNS production, enhancing nitrative inactivation of Trx-1, and thereby sensitizing lung injury by elevated ASK1-p38 MAPK signaling pathway-mediated AEC apoptosis. Therefore, inhibition of thioredoxin nitration might be one of the potential therapeutic targets for the treatment of idiopathic pulmonary brosis.

Declarations
Ethics approval and consent to participate All animal experiments were approved by the Animal Research Committee of Zheng Zhou University, Zheng Zhou, China.

Consent for publication
All the authors have consented to the publication of the research.

Availability of data and materials
Data could be obtained upon request to the corresponding author.

Competing interests
The authors declare that there is no con ict of interests. Semiquanti ed histological analysis of pulmonary brosis on day 7 and 28 after BLM administration.
Data are means ± SD, n = 6-8 in each group. ** P < 0.01 vs. control group.  The iNOS expression in lungs was determined by Western-blotting method(C). * P 0.05 vs. control group, ** P 0.01 vs. control group. n=6 to 8 in each group.

Figure 4
Effect of BLM on the activity, expression of Trx and the nitrated Trx in mice. BLM (5.0 mg/kg body mass in phosphate buffered saline, intratracheal) was given once to the mice. Sections of pulmonary tissues were prepared on day 7 and 28 after BLM administration, the activity (4A) and expression of thioredoxin (4B) and nitrated Trx (4C) were examined. Data are the mean ± SD, n = 6-8 C57 /BL6 mice in each group; * P < 0.05 compared with the control group, ** P 0.01 vs. control group.  groups. Data are the mean ± SD, n = 6-8 C57 mice in each group; * P < 0.05 compared with the control group, ** P < 0.01 compared with the control group; # P < 0.05 compared with BLM group, ## P < 0.01 compared with BLM group.

Figure 7
Effect of AG on p38-MAPK pathway in BLM-induced lung brosis in C57/BL6 mice. BLM (5.0 mg/kg body mass in phosphate buffered saline, intratracheal) was given once to the mice. Before BLM administration, mice were randomized to receive either vehicle (0.9%NaCl), aminoguanidine (AG, a peroxynitrite scavenger, 40 mg/kg) by intraperitoneal injection. Sections of pulmonary tissues were prepared on day 28 after BLM administration. (A) BLM-induced dissociation of Trx from ASK1 in lung sections. (B)p38MAPK activation. Data are the mean ± SD, n = 6-8 C57 mice in each group; ** P < 0.01 compared with the control group; # P < 0.05 compared with BLM group, ## P < 0.01 compared with BLM group. Samples were electrophoretically size fractionated on SDS-PAGE and transferred onto a polyvinylidene di uoride (PVDF)-plus membrane, and nitrated Trx-1 was detected with antinitrotyrosine antibody. Data are the mean ± SD, n = 6-8 C57/BL6 mice in each group; ** P < 0.01 compared with the control group; # P < 0.05 compared with BLM group, ## P < 0.01 compared with BLM group.

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