http://informahealthcare.com/xen ISSN: 0049-8254 (print), 1366-5928 (electronic) Xenobiotica, Early Online: 1–9 ! 2014 Informa UK Ltd. DOI: 10.3109/00498254.2014.995149
RESEARCH ARTICLE
The mechanism of rapamycin in the intervention of paraquat-induced acute lung injury in rats Da Chen1, Guangyu Jiao2, Tao Ma1, Xiaowei Liu1, Chen Yang1, and Zhi Liu1 Emergency Department, the First Affiliated Hospital of China Medical University, Shenyang, China and 2Respiratory Department and Intensive Care Unit, Shengjing Hospital of China Medical University, Shenyang, China
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Abstract
Keywords
1. Paraquat (PQ) is an organic nitrogen heterocyclic herbicide that is widely used in agriculture throughout the world. Numerous studies have reported PQ intoxication on humans. 2. In this study, we established a rat lung injury model induced by PQ and evaluated the intervention effect of rapamycin on the model, exploring the pathogenesis of PQ on lung injury as well as therapeutic effects of rapamycin on PQ-induced lung injury. 3. A rat lung injury model was established by gavage of PQ, and rapamycin was used to treat the model animals with PQ-induced lung injury. Different physiological indices were measured through Western blot and real-time polymerase chain reaction to evaluate the effect of rapamycin on the PQ-induced lung injury. 4. The analyses showed that application of rapamycin could significantly reduce the lung injury damage caused by PQ, with lung tissue wet–dry weight ratio, pathological features, compositions in serum, protein in bronchoalveolar lavage fluid and other indices being significantly improved after the injection of rapamycin. 5. It was inferred that the use of rapamycin could improve the PQ-induced lung injury through inhibiting the activity of mTOR. And we expected the use of rapamycin to be a potential treatment method for the PQ intoxication in future.
Lung injury, mTOR, paraquat intoxication
Introduction Paraquat (1,1-dimethyl-4-4-bipiridinium dichloride; PQ) is one of the most potent herbicide that is widely used all around the world, especially in developing countries (Wesseling et al., 2007). This compound is highly human and animal toxic (Neves et al., 2010; Parvez & Raisuddin, 2006; Suntres, 2002). Since PQ was introduced into agriculture in 1962, thousands of deaths have been reported every year due to the accidental or intentional ingestion of PQ (Seok et al., 2009). One of the mostly affected organs during PQ poisoning is the lung, because PQ is actively taken up by the alveolar epithelium. In addition, the most death due to PQ is the respiratory disturbance resulting from the lung injury. The lung damages by PQ are characterized by accumulation of extracellular matrix collagen, fibroblast (FB) proliferation and migration and loss of alveolar gas exchange unit (Aparicio et al., 2009). The toxicity of PQ on the lung depends on the process of alternative reduction and re-oxidation known as redox cycling (Mitsopoulos & Suntres, 2010), which damages reactive oxygen species (Han et al., 2006). Since PQ induces its Address for correspondence: Zhi Liu, Emergency Department, the First Affiliated Hospital of China Medical University, No. 155 Nanjing North Road, Heping District, Shenyang, Postal Code 110001, People’s Republic of China. Tel: 18940251105. E-mail: [email protected]
History Received 28 October 2014 Revised 30 November 2014 Accepted 2 December 2014 Published online 19 December 2014
toxicity mainly via oxidative stress-induced mechanism, researchers and clinicians have placed great emphasis on the factors involved in intervening those processes. Among all the molecules, mTOR and its inhibitor rapamycin have drawn a lot attention recent years. mTOR was identified as the kinase target linked to the cellular protein Fkbp12 (FK506-binding protein). It was also therefore known as FKBP-RAP-associated protein, RAP FKBP12 target and RAP target (Aparicio et al., 2009). This molecule plays a central role in cell growth and cellular response to metabolic stress and is required for signaling translational initiation and cell cycle progression from the G0/G1 to S phase, acting as a master switch of cellular catabolism and anabolism (Aparicio et al., 2009; Lorne et al., 2009). Several drugs have been tested as potential treatments for PQ-induced lung toxicity through regulating the pathways activated by mTOR, but a specific antidote has not yet been decided (Ali et al., 1996; Blanco-Ayala et al., 2014; DinisOliveira et al., 2008; Ghazi-Khansari et al., 2005; Suntres, 2002). Rapamycin is a fungicide that forms a complex with the FKBP12, which then binds to and inhibits the activity of mTOR. Rapamycin is clinically used as an immunosuppressant to prevent the rejection of transplanted organs and to inhibit endothelial proliferation as well as vessel restenosis
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after angioplasty (Kahan, 2000; McAlister et al., 2000; Moses et al., 2003; Spaulding et al., 2006). However, the ability of rapamycin to ameliorate the severity of inflammatory processes, such as the lung injury discussed above, has not been described. In this study, we established a rat lung injury model induced by PQ poisoning and confirmed the sensitivity of the models. By using Western blot and real-time polymerase chain reaction (RT-PCR), we assessed the treatment effect of rapamycin on lung injury caused by PQ. This study expected to elucidate the considerable treatment effect of rapamycin on lung injury of PQ and its potential as a potential antidote for PQ intoxication.
Materials and methods
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Animals One-hundred and eight healthy Wistar rats weighing 240– 260 g were purchased from the Experimental Animal Center of China Medical University, including 54 males and females. Rats were housed in cages at room temperature (20–25 C). All animal experiments were conducted in the accordance with the Institutional Animal Ethics Committee and Animal Care Guidelines of China Medical University governing the use of experimental animals. Group of model animals and gavage experiment The animals were randomly grouped into three groups: control group (group A), PQ-exposure group (group B) and PQ + rapamycin group (group C). Furthermore, for each group, animals were further grouped into six subgroups corresponding to six sampling time points (first, third, seventh, fourteenth, twenty-first and twenty-eighth days), six animals for each subgroup. For group A, animals received 1 mL normal saline by a disposable gavage and received an intraperitoneal injection of 1-mL excipients two hours after the first injection; for group B, animals received 1-mL PQ (40 mg/kg) by a disposable gavage, and excipients were injected to the rats by intraperitoneal injection two hours later; and for group C, animals were injected with PQ in the same way as those in group B, but excipients were replaced by rapamycin (2 mg/kg). The excipient consisted of 1 mL ethyl alcohol and diluted with 0.25% polyethylene glycol and 0.25% Tween-20 to a final volume of 20 mL (Madala et al., 2011). Concentration of rapamycin was adjusted by dissolving 20 mg of rapamycin using excipient according to previous studies (Bridle et al., 2009; Chen et al., 2012; Madala et al., 2011). Blood sample collection and detection of treatment effect The animals in each subgroup were anesthetized at different sampling points as mentioned above with 10% chloral hydrate (0.3 mL/100 g) through intraperitoneal injection. Blood samples were collected from the abdominal aortic and preserved in 80 C. For each sample, malondialdehyde (MDA) level, superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) activity in the peripheral blood were spectrophotometrically assayed using assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s recommended instructions.
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The level of interleukin (IL)-6, IL-10 and tumor necrosis factor (TNF)-a were measured via ELISA. The detective processes were conducted according to the instructions of assay kits in Nanjing Jiancheng Bioengineering Institute, Nanjing, China. Lung tissue collection and detection of treatment effect After blood collection, the lung tissue of the animals was cut off. The right upper lung, including the right hilar, was fixed with 4% formaldehyde polymerization and cut into pieces for hematoxylin-eosin (HE) staining according to the regular protocol. The degree of pulmonary fibrosis was evaluated using Ashcroft standard, which classifies the degree of pulmonary fibrosis into nine levels (range 0–8) (Ashcroft et al., 1988). Immunohistochemistry was conducted using streptavidin–biotin complex method to locate and detect the expression of mTOR, p70S6K, transforming growth factor-b1 (TGF-bl), a-smooth muscle actin (a-SMA), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the lung tissue (Hsu et al., 1981). The right lower lung was then excised and wet weight was recorded followed by oven-drying at 80 C for 72 h to obtain dry weight. The ratio of the wet weight to the dry weight was calculated to assess tissue edema. The left lung was lavaged with 3 mL of bronchoalveolar lavage fluid (BALF) for three times, and the collected BALF was merged for detection by Coomassie brilliant blue. The right middle lobe of the lung was removed, flashfrozen in liquid nitrogen and stored in refrigerator at 80 C. Detection of hydroxyproline (HYP) was carried out according to the instructions of Hydroxyproline Assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The protein products of mTOR, p70S6K, TGF-bl, a-SMA, MMP-2 and TIMP-1 were extracted from the lung tissue. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as the reference. The extracts were boiled with loading buffer for five minutes and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% gels. Targeted proteins were transferred onto polyvinylidene difluoride sheets. The membranes were washed with Trisbuffered saline Tween for three times, 20 min for each time. Then the membranes were incubated with antibody overnight at room temperature. After additional three washes, secondary antibody was added and the membrane was incubated for four hours. After three final washes, the blots were developed using Beyo ECL Plus reagent, and the results were detected in the Gel Imaging System. The expression levels were calculated with Bio-Rad Quantity One. The whole RNA of lung tissue of each sample was extracted using Trizol reagent. RT-PCR was used to detect the gene expression levels of mTOR, p70S6K, TGF- l, a-SMA and the reference gene NAPDH. Primers and annealing temperature information is listed in Table S1. The RNA was reversely transcribed to cDNA using RT-PCR kit (Fermentas, Ottawa, Canada), and the final reaction mixture of volume 20 mL contained 4 mL of 5 first-strand buffer, 1 mL RNase inhibitor, 1 mL of 0.1 M dithiothreitol (DTT), 2 mL of 10 mM dNTP, 1 mL of moloney murine leukemia virus (M-MLV)
Intervention of paraquat-induced lung injury
DOI: 10.3109/00498254.2014.995149
reverse transcriptase and 1 mL of the RNA template. Thermal cycling parameters for the amplification were as follows: a denaturation step at 94 C for 3 min, followed by 30 cycles at 94 C for 15 s, annealing temperature in Table S1 for 30 s and 72 C for 45 s. The products of RT-PCR reactions were semiquantified in UVP Gel Imaging System. Statistical analysis
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No significant difference was detected between males and females in each subgroup (Tables S2–S11), so the mean values of different indicators were calculated based on the data of all the samples in each subgroup. All the data were expressed in the form of mean ± SD. Multiple comparisons were conducted by least significant difference method with significant level of 0.05. All the statistical analysis were conducted using SPSS version 16.0 (IBM, Armonk, NY).
Results Effect of rapamycin on the clinical symptom due to PQ poisoning There was no death due to other factors during the experiment time period. The animals in group B showed signs of PQ intoxication, including deceased activity and body weight, polypnea, cyanosis around mouth, crouch, diarrhea and anorexia. Those signs receded after the seventh day of gavage. However, the animals in group C had few symptom compared with those in group B, indicating the relieving potential of rapamycin in the lung injury induced by PQ poisoning.
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were detected at the third day after the gavage. At the seventh day, alveolar space and stroma was infiltrated with inflammatory cells. The inflammatory response did not relieve until the fourteenth day after the gavage. All the symptoms mentioned above were eased in the group C at the same sampling points (Figure 1). The scores of Ashcroft did not show significant difference between animals in groups A and B at the first three sampling time points; however, the scores of group B increased dramatically at the last three sampling time points. For group C, Ashcroft scores also increased since fourteenth day compared with group A, but the level was significantly lower than that of group B (Table 2). Protein production in BALF was also influenced by PQ and increased significantly during the first seven days but improved by rapamycin (Table 3). Level of HYP in group B increased from the fourteenth day to the end of the experiment but improved by rapamycin in a similar pattern to other indicators (Table 4). Effects of rapamycin on the level of MOD, the activity of SOD and the activity of GSH-Px Compared with group A, the level of MDA in the group B increased significantly in the first seven days and then declined gradually to normal level at fourteenth day. The level of MDA in group C was also higher than that in group A but significantly lower than that in group B at the first sampling time points (Table 5). The activities of SOD and GSH-Px were clearly influenced by PQ but improved after application of rapamycin (Table 5).
Effect of rapamycin on lung W/D ratio in rats with PQ-induced lung injury
Effect of rapamycin on the cytokine production in the rats poisoned by PQ
The lung wet-dry (W/D) ratio of animals in group B increased significantly compared with those in group A in the first seven days of the experiment. The increase decelerated after the seventh day, and the lung W/D ratio of group B got close to that of group A at fourteenth day. For the animals in group C, the W/D ratio also increased significantly compared with the value of group A during the first seven days; however, the value was significantly lower than that of group B, which was indicative of the treatment effect of rapamycin on the increase of lung W/ D ratio caused by PQ poisoning (Table 1).
The results of ELISA showed that the levels of IL-6, IL-10 and TNF-a in group B increased sharply during the first three days of the experiments, and the level decreased slightly after the seventh day, which were still much higher than those in group A. The levels of cytokine production returned to normal level until the 14th day, whereas the application of rapamycin on the PQ-poisoned animals significantly improved the dysexpressions of the cytokines at the first three sampling time points (Table 6).
Effect of rapamycin on the histopathological characteristics in the lung tissue of rats with PQ-induced lung injury The result of HE staining showed that in the group B, clear alveolitis, alveolar edema and vascular endothelial cell injury
Effect of rapamycin on the distribution and expression of mTOR, p70S6K, TGF-bl, a-SMA, MMP-2 and TIMP-1 in the rats with PQ-induced lung injury The distribution of mTOR, p70S6K, TGF-bl, a-SMA, MMP-2 and TIMP-1 is shown in Figures S1–S6. For mTOR and p70S6K, in group A, the positive signals were detected
Table 1. Effect of rapamycin on lung W/D ratio in lung tissue of PQ-induced lung injury rats (mean ± SD). Time points Group A B C a
First day
Third day
Seventh day
Fourteenth day
Twenty-first day
Twenty-eighth day
4.17 ± 0.07 5.21 ± 0.09a 4.57 ± 0.07a,b
4.23 ± 0.09 5.73 ± 0.18a 5.01 ± 0.09a,b
4.23 ± 0.12 5.41 ± 0.15a 4.71 ± 0.11a,b
4.17 ± 0.07 4.71 ± 0.11 4.48 ± 0.07
4.17 ± 0.08 4.51 ± 0.10 4.36 ± 0.03
4.21 ± 0.03 4.42 ± 0.03 4.30 ± 0.07
p50.01, compared with group A. p50.05, compared with group B.
b
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Figure 1. Effect of rapamycin on the histopathological changes in lung tissue illustrated by HE staining. Table 2. Effect of rapamycin on pulmonary fibrosis in lung tissue of PQ-induced lung injury rats illustrated by Ashcroft scores (mean ± SD). Time point Group A B C
First day
Third day
Seventh day
Fourteenth day
Twenty-first day
Twenty-eighth day
0.45 ± 0.12 0.76 ± 0.23 0.56 ± 0.21
0.47 ± 0.15 1.03 ± 0.30 0.86 ± 0.23
0.50 ± 0.13 1.51 ± 0.32 1.06 ± 0.27
0.49 ± 0.14 3.47 ± 0.56a 2.15 ± 0.33a,b
0.50 ± 0.12 4.56 ± 0.76a 2.36 ± 0.46a,b
0.48 ± 0.15 5.43 ± 0.83a 3.02 ± 0.40a,b
a
p50.01, compared with group A. p50.05, compared with group B.
b
Table 3. Effect of rapamycin on proteinproduction in BALF (mg/L) in lung tissue of PQ-induced lung injury rats (mean ± SD). Time point Group A B C
First day
Third day
Seventh day
Fourteenth day
Twenty-first day
Twenty-eighth day
137.45 ± 12.35 254.56 ± 22.49a 178.86 ± 16.7a,b
140.16 ± 12.89 288.56 ± 25.45a 212.53 ± 18.93a,b
138.67 ± 12.56 306.45 ± 30.05a 248.67 ± 20.42a,b
139.43 ± 12.63 155.56 ± 13.78 154.75 ± 13.06
141.32 ± 12.45 143.42 ± 12.89 142.41 ± 12.63
139.24 ± 12.48 141.44 ± 12.67 141.69 ± 12.53
a
p50.01, compared with group A. p50.05, compared with group B.
b
in bronchial epithelial cells and some endothelial cells. In group B, expressions of mTOR and p70S6K were significantly increased three days after PQ administration (Figures S1 and 2) and reached the peak value at the fourteenth day. After treated by rapamycin, the expressions of mTOR and p70S6K were significantly weakened. The results were also illustrated by Western-blot and RT-PCR (Figures 2A and B and 3A and B). For the expressions of TGF-bl and a-SMA, no significant difference between the groups A and B during the first three days was detected. Significant increase in the expression
levels in group B was observed since the seventh day with expanded distribution of the TGF-bl and a-SMA (Figures S3 and 4). The over-expression levels of TGF-bl and a-SMA in group B were highest at the fourteenth day and retained even at twenty-eighth day. Injection of rapamycin in group C alleviated the expressions of TGF-bl and a-SMA in the PQ-induced injured lungs. The results of Western-blot and RT-PCR were consistent with the results of immunohistochemistry (Figures 2C and D, and 3C and D). In the case of MMP-2 and TIMP-1, the distribution in health lung tissue centered around the pulmonary intravascular
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DOI: 10.3109/00498254.2014.995149
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Table 4. Effect of rapamycin on protein production in HYP (mg/mg of lung tissue) in lung tissue of PQ-induced lung injury rats (mean ± SD). Time points Group A B C
First day
Third day
Seventh day
Fourteenth day
Twenty-first day
Twenty-eighth day
0.77 ± 0.07 0.83 ± 0.13 0.78 ± 0.11
0.80 ± 0.10 0.88 ± 0.15 0.81 ± 0.12
0.78 ± 0.11 1.29 ± 0.17 1.03 ± 0.11
0.79 ± 0.12 1.82 ± 0.23a 1.12 ± 0.13a,b
0.81 ± 0.10 2.12 ± 0.28a 1.34 ± 0.21a,b
0.78 ± 0.09 2.87 ± 0.30a 1.56 ± 0.23a,b
a
p50.01, compared with group A. p50.05, compared with group B.
b
Table 5. Effect of rapamycin on the level of MDA, the activity of SOD and activity of GSH-Px in peripheral blood of PQ-induced lung injury rats (mean ± SD). Time points Group
First day
Third day
Seventh day
Fourteenth day
Twenty-first day
Twenty-first day
A B C A B C A B C
0.53 ± 0.13 2.86 ± 0.89a 1.03 ± 0.49a,b 178.53 ± 9.13 122.34 ± 6.78a 145.79 ± 6.89a,b 343.43 ± 17.34 286.15 ± 15.56a 321.85 ± 15.98a,b
0.50 ± 0.12 4.66 ± 1.20a 2.21 ± 0.70a,b 175.04 ± 8.91 100.75 ± 6.20a 132.46 ± 6.76a,b 340.16 ± 17.12 256.56 ± 13.57a 300.98 ± 14.77a,b
0.51 ± 0.11 1.56 ± 0.78a 0.76 ± 0.32a,b 177.14 ± 9.05 112.43 ± 6.65a 148.49 ± 6.55a,b 345.76 ± 18.16 223.41 ± 12.54a 287.76 ± 13.93a,b
0.53 ± 0.12 0.62 ± 0.15 0.52 ± 0.12 175.89 ± 9.03 146.69 ± 7.25 157.19 ± 7.62 339.60 ± 17.85 195.72 ± 10.91 256.98 ± 11.90
0.52 ± 0.13 0.56 ± 0.13 0.51 ± 0.12 176.18 ± 9.09 157.39 ± 7.37 160.65 ± 7.45 342.76 ± 18.12 321.43 ± 15.34 330.67 ± 15.18
0.52 ± 0.11 0.52 ± 0.14 0.50 ± 0.11 177.01 ± 9.10 165.18 ± 8.24 169.67 ± 8.41 343.65 ± 18.42 335.53 ± 16.12 340.76 ± 16.56
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MDA (mmol/mL) SOD (U/mL) GSH-Px (U/0.1 mL)
a
p50.01, compared with group A. p50.05, compared with group B.
b
Table 6. Effect of rapamycin on level IL-6, IL-10, and TNF-a in peripheral blood of PQ-induced lung injury rats (mean ± SD). Time points
IL-6 (pg/mL) TNF-a (pg/mL) IL-10 (pg/mL)
Group
First day
Third day
Seventh day
Fourteenth day
Twenty-first day
Twenty-eighth day
A B C A B C A B C
78.13 ± 8.34 185.54 ± 10.43a 134.42 ± 9.43a,b 48.23 ± 5.18 155.43 ± 8.43a 97.10 ± 7.13a,b 30.76 ± 4.54 45.97 ± 5.87a 37.45 ± 4.67a,b
76.23 ± 7.89 434.67 ± 20.12a 299.67 ± 16.72a,b 47.18 ± 5.03 345.09 ± 15.64a 217.45 ± 13.42a,b 31.05 ± 4.51 69.97 ± 6.15a 55.67 ± 5.76a,b
77.45 ± 8.06 354.79 ± 17.98a 243.18 ± 15.18a,b 49.46 ± 5.21 251.87 ± 13.06a 157.24 ± 9.36a,b 29.89 ± 4.49 58.07 ± 5.96a 45.01 ± 5.56a,b
78.56 ± 8.25 251.41 ± 13.23 167.89 ± 10.98 47.10 ± 5.13 200.43 ± 13.74 109.76 ± 9.74 31.25 ± 4.53 48.76 ± 5.65 37.75 ± 5.33
77.98 ± 8.31 100.32 ± 9.43 90.23 ± 8.43 47.85 ± 5.08 67.12 ± 6.43 55.32 ± 5.98 29.95 ± 4.47 43.86 ± 5.15 34.53 ± 4.76
77.82 ± 8.16 87.47 ± 8.49 81.31 ± 8.41 49.05 ± 5.19 55.65 ± 5.49 51.97 ± 5.43 31.08 ± 4.47 40.34 ± 5.02 32.65 ± 4.63
a
p50.01, compared with group A. p50.05, compared with group B.
b
macrophage, bronchial epithelial cells, alveolar epithelial cell and some endothelial cells (Figures S5 and S6). The expression of MMP-2 in group B increased since the first day of the experiment reached the highest point at the fourteenth day and then decreased to normal level at the twenty-eighth day (Figure 2E). The expression of TIMP-1 in group B kept increasing until the twenty-first day and stayed higher than that in group A until the twenty-eighth day (Figure 2F). Application of rapamycin could reduce the expression of both MMP-2 and TIMP-1 since the first day to the twenty-eighth day end of the experiment and regulated the balance between MMP-2 and TIMP-1. The ratio values of MMP-2/TIMP-1 in group B were 0.46 and 0.39 at the fourteenth and twenty-first day, while those of group C were 0.89 and 0.91.
Discussion The mechanism of PQ-induced lung injury is complicated. It is general view that the process is characterized by the
interaction involving the cytokine, signal transduction, cell damage and inflammatory response (Figure S7) (Ali et al., 1996; Blanco-Ayala et al., 2014; Dinis-Oliveira et al., 2008; Ghazi-Khansari et al., 2005; Suntres, 2002). As the main target organ of PQ poisoning, lung always has the symptoms of alveolar edema, hemorrhage, inflammatory cell infiltration and diffuse alveolar collapse accompanied by wall thickening (Gao et al., 2013). The mTOR, which is identified as pivot of kinds of signal transduction process in cells, has been taken as an important factor involving in the occurrence of PQ-induced lung injury (Ma & Blenis, 2009). In this study, it was found that the treatment with rapamycin, which inhibited the activity of mTOR, was very effective in alleviating PQ-induced lung injury in rat models. Our results demonstrated that the treatment of rapamycin significantly improved the W/D ratios, alveolar edema and other historical characteristics of the lung samples derived from the PQ + rapamycin group (Tables 1–3; Figure 1). Moreover, by binding with FKBP, rapamycin could suppress
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Figure 2. Effects of rapamycin on the expression of mTOR, p70S6K, TGF-bl, a-SMA, MMP-2 and TIMP-1 in lung tissue of PQ-induced acute lung injury rats illustrated by Western blot: (A) mTOR; (B) p70S6K; (C) TGF-bl; (D) a-SMA; (E) MMP-2; and (F) TIMP-1. **p50.01, compared with group A; #p50.05, compared with group B.
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Figure 3. Effects of rapamycin on the expression of mTOR, p70S6K, TGF-bl and a-SMA in lung tissues of PQ-induced acute lung injury rats illustrated by RT-PCR: (A) mTOR; (B) p70S6K; (C) TGF-bl; and (D) a-SMA. **p50.01, compared with group A; #p50.05, compared with group B.
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the expression of mTOR and acts as anti-factor against inflammation (Tables 4–6; Figures 2 and 3) (Sarbassov et al., 2004). As shown in cytokine detection, the dysexpression of IL-6, IL-10, TNF-a and protein in BALF was clearly associated with the increase of mTOR (Tables 3 and 6; Figures S1, 2A and 3A), which was indicative of the involvement of mTOR pathway in the cytokine secretion in PQ-induced lung injury. Previous study also revealed the function of PQ in inducing the secretion of cytokine in inflammatory response (Yost et al., 2004). These cytokines as well as other inflammatory factors, initiate, amplify and perpetuate the inflammatory response. However, the application of rapamycin would inhibit the activation of mTOR in PQ-induced lung injury and further improve the inflammatory response (Lindemann et al., 2004). After injection of rapamycin, the level of MDA, and activities of SOD and GSH-Px, which were known to play critical roles in PQ-induced lung injury (Kim et al., 2010), were significantly improved (Table 5), indicating the antioxidative function of rapamycin in the treatment. Another main characteristic of PQ-induced lung injury was the over-synthesis of collagen, which resulted in the pulmonary fibrosis. mTOR played an important role in the protein synthesis through PI3K/Akt pathway. In this study, it was found that as the final product of collagen metabolism, HYP production was declined in group C after treatment with rapamycin (Table 4). Moreover, the application of rapamycin reduced the level of mTOR as well as p70S6K (Figures S1 and 2, 2A and B and 3A and B). It was reported that p70S6K was the substrate of the phosphorylation of mTOR in the collagen synthesis (Sodhi et al., 2008). Concatenated with our data, it was concluded that rapamycin could influence the synthesis of collagen by reducing the extracellular cell matrix (ECM) phosphorylated by mTOR. The expressions of a-SMA and TGF-bl were also upregulated in the animals poisoned by PQ, and the symptoms were eased after treatment with rapamycin (Figures S3 and 4, 2C and D and 3C and D). a-SMA was an important indicator of cell transforming into myofibroblast (MFB), which resulted in pulmonary fibrosis. Compared with normal FB, MFB has more powerful ability in producing and secreting collagen. This characteristic of MFB would increase the aggregation of ECM (Kelynack et al., 1999). TGF-bl was another key factor involving in the pulmonary fibrosis (Ghaffari et al., 2011), which accelerates the transformation of FB to collagen, fibronectin and other ECMs (Xiong et al., 2005). It was reported that rapamycin could inhibit the pulmonary fibrosis induced by TGF-bl via blocking the mTOR pathway (Korfhagen et al., 2009). Furthermore, this conclusion was confirmed in this study, the expressions of a-SMA and TGF-bl both significantly declined in group C with rapamycin treatment (Figure S3 and 4, 2C and D and 3C and D). The deposition of ECM was not only due to the oversynthesis but also due to the lack of degradation (Tung et al., 2010). The imbalance between the synthesis and degradation of ECM also induces pulmonary fibrosis. In this study, we also detected the expression of MMP and TIMP by immunohistochemistry and Western blot (Figure S5 and S6 and 2E and F), which played important roles in the synthesis/ degradation of ECM. The balance between MMP and TIMP
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would significantly influence the process of fibrosis and was a potential therapy for treatment of pulmonary fibrosis. It was reported that mTOR-binding sites existed in the promoter of MMP genes (Wang et al., 2011). The inhibition of pathways activated by mTOR by rapamycin might adjust the balance between MMP and TIMP. This hypothesis was proved by our experiments, in which animals injected with rapamycin had an improved MMP/TIMP value compared with those injected with PQ only. Although the detail mechanism of rapamycin regulating MMP/TIMP ratio still remained unclear, our results had a reasonable illustration of relieving effect of rapamycin on the balance between MMP and TIMP.
Conclusions In conclusion, this study showed that rapamycin had a considerable treatment effect on PQ-induced lung injury in animal models. The drug functioned via inhibiting the pathways activated by mTOR. After treatment by rapamycin, the PQ-poisoned rats had a lower level of MDA, higher activities of SOD and GSH-Px and lower levels of cytokine such as IL-6, IL-10 and TNF-a in the peripheral blood. Moreover, other productions involved in the pulmonary fibrosis also declined through the blocking of mTOR signaling pathway. The treatment of rapamycin on PQ-induced lung injury without significant side-effects was validated in this study, which offered theoretical support for the treatment potential of rapamycin on PQ intoxication. Further study will be conducted to facilitate the application of rapamycin in practice.
Acknowledgements The authors appreciate the language editing by Edanz English Editing.
Declaration of interest The authors disclose no conflict of interest. This work was supported by the National Natural Science Foundation of China (81170068) and the Natural Science Foundation of Liaoning Province (2013021059).
References Ali S, Jain SK, Abdulla M, Athar M. (1996). Paraquat induced DNA damage by reactive oxygen species. Biochem Mol Biol Int 39:63–7. Aparicio G, Calvo MB, Medina V, et al. (2009). Comprehensive lung injury pathology induced by mTOR inhibitors. Clin Transl Oncol 11: 499–510. Ashcroft T, Simpson JM, Timbrell V. (1988). Simple method of estimating severity of pulmonay fibrosis on a numerical scale. J Clin Pathol 41:467–70. Blanco-Ayala T, Anderica-Romero AC, Pedraza-Chaverri J. (2014). New insights into antioxidant strategies against paraquat toxicity. Free Radical Res 48:623–40. Bridle KR, Popa C, Morgan ML, et al. (2009). Rapamycin inhibits hepatic fibrosis in rats by attenuating multiple profibrogenic pathways. Liver Transpl 15:1315–24. Chen G, Chen H, Wang C, et al. (2012). Rapamycin ameliorates kidney fibrosis by inhibiting the activation of mTOR signaling in interstitial macrophages and myofibroblasts. PLOS One 7:e33626. Dinis-Oliveira RJ, Duarte JA, Sa´nchez-Navarro A, et al. (2008). Paraquat poisonings: mechanisms of lung toxicity, clinical features, and treatment. Crit Rev Toxicol 38:13–71.
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Gao Y, Xu X, Ding K, et al. (2013). Rapamycin inhibits transforming growth factor b1-induced fibrogenesis in primary human lung fibroblasts. Yonsei Med J 54:437–44. Ghaffari AR, Noshad H, Ostadi A, Hasanzadeh N. (2011). Effect of pulse therapy with glucocorticoid and cyclophosphamide in lung fibrosis due to paraquat poisoning in rats. Saudi Med J 32:249–53. Ghazi-Khansari M, Nasiri G, Honarjoo M. (2005). Decreasing the oxidant stress from paraquat in isolated perfused rat lung using captopril and niacin. Arch Toxicol 79:341–5. Han JF, Wang SL, He XY, et al. (2006). Effect of genetic variation on human cytochrome p450 reductase-mediated paraquatcytotoxicity. Toxicol Sci 91:42–8. Hsu RM, Raine L, Fanger H. (1981). The use of antiavidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase techniques. Am J Clin Pathol 75:816–21. Kahan BD. (2000). Efficacy of sirolimus compared with azathioprine for reduction of acute renal allograft rejection: a randomised multicenter study. The Rapamune US Study Group. Lancet 356: 194–202. Kelynack KJ, Hewistson TD, Pedagogos E, et al. (1999). Renal myofibroblast contract collagen I matrix lattices in vitro. Am J Nephrol 19:694–701. Kim JH, Gil HW, Yang JO, et al. (2010). Effect of glutathione administration on serum levels of reactive oxygen metabolites in patients with paraquat intoxication: a pilot study. Korean J Intern Med 2010;25:282–7. Korfhagen TR, Le Cras TD, Davidson CR, et al. (2009). Rapamycin prevents transforming growth factor-alpha-induced pulmonary fibrosis. Am J Respir Cell Mol Biol 41:562–72. Lindemann SW, Yost CC, Denis MM, et al. (2004). Neutrophils alter the inflammatory milieu by signal-dependent translation of constitutive messenger RNAs. Proc Nat l Acad Sci USA 101:7076–81. Lorne E, Zhao X, Zmijewski JW, et al. (2009). Participation of mammalian target of rapamycin complex 1 in toll-like receptor 2- and 4-induced neutrophil activation and acute lung injury. Am J Respir Cell Mol Biol 41:237–45. Ma XM, Blenis J. (2009). Molecular mehanisms of mTOR-mediated translational control. Nat Rev Mol Cell Biol 10:307–18. Madala SK, Maxfield MD, Davidson CR, et al. (2011). Rapamycin regulates bleomycin-induced lung damage in SP-C-deficient mice. Pulm Med 2011:653524. McAlister VC, Gao Z, Peltekian K, et al. (2000). Sirolimus-tacrolimus combination immunosuppression. Lancet 355:376–7.
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Mitsopoulos P, Suntres ZE. (2010). Cytotoxicity and gene array analysis of alveolar epithelial A549 cells exposed to paraquat. Chem Biol Interact 188:427–36. Moses JW, Leon MB, Popma JJ, et al. (2003). Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med 349:1315–23. Neves FF, Sousa RB, Pazin-Filho A, et al. (2010). Severe paraquat poisoning: clinical and radiological findings in a survivor. J Bras Pneumol 36:513–16. Parvez S, Raisuddin S. (2006). Effects of paraquat on the freshwater fish Channa punctata: non-enzymatic antioxidants as biomarkers of exposure. Arch Environ Contam Toxicol 50:392–87. Sarbassov DD, Ali SM, Kim DH, et al. (2004). Rictor, a novel binding partner of mTOR, defines a rapamycin insensitive and raptorindependent pathway that regulates the cytoskeleton. Curr Biol 14: 1296–302. Seok SJ, Gil HW, Jeong DS, et al. (2009). Paraquat intoxication in subjects who attempt suicide: why they chose paraquat. Korean J Intern Med 24:247–51. Sodhi A, Chaisuparat R, Hu J, et al. (2008). The TSC2/mTOR pathway drives endothelial cell transformation induced by the Kaposi’s sarcoma-associated herpesvirus G protein-coupled receptor. Cancer Cell 10:133–43. Spaulding C, Henry P, Teiger E, et al. (2006). Sirolimus-eluting versus uncoated stents in acute myocardial infarction. N Engl J Med 355: 1093–104. Suntres ZE. (2002). Role of antioxidants in paraquat toxicity. Toxicology 180:65–77. Tung JN, Lang YD, Wang LF, Chen CM. (2010). Paraquat increases connective tissue growth factor and collagen expression via angiotensin signaling pathway in human lung fibroblasts. Toxicol In Vitro 24:803–8. Wang BL, Tu YY, Fu JF. (2011). Unbalanced MMP/TIMP-1 expression during the development of experimental pulmonary fibrosis with acute paraquat poisoning. Mol Med Report 4:243–8. Wesseling C, van Wendel de Joode B, Ruepert C, et al. (2007). Paraquat in developing countries. Int J Occup Environ Health 7:275–86. Xiong XM, Jiang HL, Liu BF, et al. (2005). Influence of interleukin-10 on the cytokine in acute lung injury in rats induced by endotoxin. Chin J Emerg Med 14:380–3. Yost CC, Denis MM, Lindermann S, et al. (2004). Activated polymorphonuclear leukocytes rapidly synthesize retinoic acid receptora1pha: a mechanism for translational control of transcriptional events. J Exp Med 200:671–80.
Supplementary material available online Supplementary Tables S1–11 and Figures S1–7.
RESEARCH ARTICLE
The mechanism of rapamycin in the intervention of paraquat-induced acute lung injury in rats Da Chen1, Guangyu Jiao2, Tao Ma1, Xiaowei Liu1, Chen Yang1, and Zhi Liu1 Emergency Department, the First Affiliated Hospital of China Medical University, Shenyang, China and 2Respiratory Department and Intensive Care Unit, Shengjing Hospital of China Medical University, Shenyang, China
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Abstract
Keywords
1. Paraquat (PQ) is an organic nitrogen heterocyclic herbicide that is widely used in agriculture throughout the world. Numerous studies have reported PQ intoxication on humans. 2. In this study, we established a rat lung injury model induced by PQ and evaluated the intervention effect of rapamycin on the model, exploring the pathogenesis of PQ on lung injury as well as therapeutic effects of rapamycin on PQ-induced lung injury. 3. A rat lung injury model was established by gavage of PQ, and rapamycin was used to treat the model animals with PQ-induced lung injury. Different physiological indices were measured through Western blot and real-time polymerase chain reaction to evaluate the effect of rapamycin on the PQ-induced lung injury. 4. The analyses showed that application of rapamycin could significantly reduce the lung injury damage caused by PQ, with lung tissue wet–dry weight ratio, pathological features, compositions in serum, protein in bronchoalveolar lavage fluid and other indices being significantly improved after the injection of rapamycin. 5. It was inferred that the use of rapamycin could improve the PQ-induced lung injury through inhibiting the activity of mTOR. And we expected the use of rapamycin to be a potential treatment method for the PQ intoxication in future.
Lung injury, mTOR, paraquat intoxication
Introduction Paraquat (1,1-dimethyl-4-4-bipiridinium dichloride; PQ) is one of the most potent herbicide that is widely used all around the world, especially in developing countries (Wesseling et al., 2007). This compound is highly human and animal toxic (Neves et al., 2010; Parvez & Raisuddin, 2006; Suntres, 2002). Since PQ was introduced into agriculture in 1962, thousands of deaths have been reported every year due to the accidental or intentional ingestion of PQ (Seok et al., 2009). One of the mostly affected organs during PQ poisoning is the lung, because PQ is actively taken up by the alveolar epithelium. In addition, the most death due to PQ is the respiratory disturbance resulting from the lung injury. The lung damages by PQ are characterized by accumulation of extracellular matrix collagen, fibroblast (FB) proliferation and migration and loss of alveolar gas exchange unit (Aparicio et al., 2009). The toxicity of PQ on the lung depends on the process of alternative reduction and re-oxidation known as redox cycling (Mitsopoulos & Suntres, 2010), which damages reactive oxygen species (Han et al., 2006). Since PQ induces its Address for correspondence: Zhi Liu, Emergency Department, the First Affiliated Hospital of China Medical University, No. 155 Nanjing North Road, Heping District, Shenyang, Postal Code 110001, People’s Republic of China. Tel: 18940251105. E-mail: [email protected]
History Received 28 October 2014 Revised 30 November 2014 Accepted 2 December 2014 Published online 19 December 2014
toxicity mainly via oxidative stress-induced mechanism, researchers and clinicians have placed great emphasis on the factors involved in intervening those processes. Among all the molecules, mTOR and its inhibitor rapamycin have drawn a lot attention recent years. mTOR was identified as the kinase target linked to the cellular protein Fkbp12 (FK506-binding protein). It was also therefore known as FKBP-RAP-associated protein, RAP FKBP12 target and RAP target (Aparicio et al., 2009). This molecule plays a central role in cell growth and cellular response to metabolic stress and is required for signaling translational initiation and cell cycle progression from the G0/G1 to S phase, acting as a master switch of cellular catabolism and anabolism (Aparicio et al., 2009; Lorne et al., 2009). Several drugs have been tested as potential treatments for PQ-induced lung toxicity through regulating the pathways activated by mTOR, but a specific antidote has not yet been decided (Ali et al., 1996; Blanco-Ayala et al., 2014; DinisOliveira et al., 2008; Ghazi-Khansari et al., 2005; Suntres, 2002). Rapamycin is a fungicide that forms a complex with the FKBP12, which then binds to and inhibits the activity of mTOR. Rapamycin is clinically used as an immunosuppressant to prevent the rejection of transplanted organs and to inhibit endothelial proliferation as well as vessel restenosis
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after angioplasty (Kahan, 2000; McAlister et al., 2000; Moses et al., 2003; Spaulding et al., 2006). However, the ability of rapamycin to ameliorate the severity of inflammatory processes, such as the lung injury discussed above, has not been described. In this study, we established a rat lung injury model induced by PQ poisoning and confirmed the sensitivity of the models. By using Western blot and real-time polymerase chain reaction (RT-PCR), we assessed the treatment effect of rapamycin on lung injury caused by PQ. This study expected to elucidate the considerable treatment effect of rapamycin on lung injury of PQ and its potential as a potential antidote for PQ intoxication.
Materials and methods
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Animals One-hundred and eight healthy Wistar rats weighing 240– 260 g were purchased from the Experimental Animal Center of China Medical University, including 54 males and females. Rats were housed in cages at room temperature (20–25 C). All animal experiments were conducted in the accordance with the Institutional Animal Ethics Committee and Animal Care Guidelines of China Medical University governing the use of experimental animals. Group of model animals and gavage experiment The animals were randomly grouped into three groups: control group (group A), PQ-exposure group (group B) and PQ + rapamycin group (group C). Furthermore, for each group, animals were further grouped into six subgroups corresponding to six sampling time points (first, third, seventh, fourteenth, twenty-first and twenty-eighth days), six animals for each subgroup. For group A, animals received 1 mL normal saline by a disposable gavage and received an intraperitoneal injection of 1-mL excipients two hours after the first injection; for group B, animals received 1-mL PQ (40 mg/kg) by a disposable gavage, and excipients were injected to the rats by intraperitoneal injection two hours later; and for group C, animals were injected with PQ in the same way as those in group B, but excipients were replaced by rapamycin (2 mg/kg). The excipient consisted of 1 mL ethyl alcohol and diluted with 0.25% polyethylene glycol and 0.25% Tween-20 to a final volume of 20 mL (Madala et al., 2011). Concentration of rapamycin was adjusted by dissolving 20 mg of rapamycin using excipient according to previous studies (Bridle et al., 2009; Chen et al., 2012; Madala et al., 2011). Blood sample collection and detection of treatment effect The animals in each subgroup were anesthetized at different sampling points as mentioned above with 10% chloral hydrate (0.3 mL/100 g) through intraperitoneal injection. Blood samples were collected from the abdominal aortic and preserved in 80 C. For each sample, malondialdehyde (MDA) level, superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) activity in the peripheral blood were spectrophotometrically assayed using assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s recommended instructions.
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The level of interleukin (IL)-6, IL-10 and tumor necrosis factor (TNF)-a were measured via ELISA. The detective processes were conducted according to the instructions of assay kits in Nanjing Jiancheng Bioengineering Institute, Nanjing, China. Lung tissue collection and detection of treatment effect After blood collection, the lung tissue of the animals was cut off. The right upper lung, including the right hilar, was fixed with 4% formaldehyde polymerization and cut into pieces for hematoxylin-eosin (HE) staining according to the regular protocol. The degree of pulmonary fibrosis was evaluated using Ashcroft standard, which classifies the degree of pulmonary fibrosis into nine levels (range 0–8) (Ashcroft et al., 1988). Immunohistochemistry was conducted using streptavidin–biotin complex method to locate and detect the expression of mTOR, p70S6K, transforming growth factor-b1 (TGF-bl), a-smooth muscle actin (a-SMA), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the lung tissue (Hsu et al., 1981). The right lower lung was then excised and wet weight was recorded followed by oven-drying at 80 C for 72 h to obtain dry weight. The ratio of the wet weight to the dry weight was calculated to assess tissue edema. The left lung was lavaged with 3 mL of bronchoalveolar lavage fluid (BALF) for three times, and the collected BALF was merged for detection by Coomassie brilliant blue. The right middle lobe of the lung was removed, flashfrozen in liquid nitrogen and stored in refrigerator at 80 C. Detection of hydroxyproline (HYP) was carried out according to the instructions of Hydroxyproline Assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The protein products of mTOR, p70S6K, TGF-bl, a-SMA, MMP-2 and TIMP-1 were extracted from the lung tissue. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as the reference. The extracts were boiled with loading buffer for five minutes and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% gels. Targeted proteins were transferred onto polyvinylidene difluoride sheets. The membranes were washed with Trisbuffered saline Tween for three times, 20 min for each time. Then the membranes were incubated with antibody overnight at room temperature. After additional three washes, secondary antibody was added and the membrane was incubated for four hours. After three final washes, the blots were developed using Beyo ECL Plus reagent, and the results were detected in the Gel Imaging System. The expression levels were calculated with Bio-Rad Quantity One. The whole RNA of lung tissue of each sample was extracted using Trizol reagent. RT-PCR was used to detect the gene expression levels of mTOR, p70S6K, TGF- l, a-SMA and the reference gene NAPDH. Primers and annealing temperature information is listed in Table S1. The RNA was reversely transcribed to cDNA using RT-PCR kit (Fermentas, Ottawa, Canada), and the final reaction mixture of volume 20 mL contained 4 mL of 5 first-strand buffer, 1 mL RNase inhibitor, 1 mL of 0.1 M dithiothreitol (DTT), 2 mL of 10 mM dNTP, 1 mL of moloney murine leukemia virus (M-MLV)
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reverse transcriptase and 1 mL of the RNA template. Thermal cycling parameters for the amplification were as follows: a denaturation step at 94 C for 3 min, followed by 30 cycles at 94 C for 15 s, annealing temperature in Table S1 for 30 s and 72 C for 45 s. The products of RT-PCR reactions were semiquantified in UVP Gel Imaging System. Statistical analysis
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No significant difference was detected between males and females in each subgroup (Tables S2–S11), so the mean values of different indicators were calculated based on the data of all the samples in each subgroup. All the data were expressed in the form of mean ± SD. Multiple comparisons were conducted by least significant difference method with significant level of 0.05. All the statistical analysis were conducted using SPSS version 16.0 (IBM, Armonk, NY).
Results Effect of rapamycin on the clinical symptom due to PQ poisoning There was no death due to other factors during the experiment time period. The animals in group B showed signs of PQ intoxication, including deceased activity and body weight, polypnea, cyanosis around mouth, crouch, diarrhea and anorexia. Those signs receded after the seventh day of gavage. However, the animals in group C had few symptom compared with those in group B, indicating the relieving potential of rapamycin in the lung injury induced by PQ poisoning.
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were detected at the third day after the gavage. At the seventh day, alveolar space and stroma was infiltrated with inflammatory cells. The inflammatory response did not relieve until the fourteenth day after the gavage. All the symptoms mentioned above were eased in the group C at the same sampling points (Figure 1). The scores of Ashcroft did not show significant difference between animals in groups A and B at the first three sampling time points; however, the scores of group B increased dramatically at the last three sampling time points. For group C, Ashcroft scores also increased since fourteenth day compared with group A, but the level was significantly lower than that of group B (Table 2). Protein production in BALF was also influenced by PQ and increased significantly during the first seven days but improved by rapamycin (Table 3). Level of HYP in group B increased from the fourteenth day to the end of the experiment but improved by rapamycin in a similar pattern to other indicators (Table 4). Effects of rapamycin on the level of MOD, the activity of SOD and the activity of GSH-Px Compared with group A, the level of MDA in the group B increased significantly in the first seven days and then declined gradually to normal level at fourteenth day. The level of MDA in group C was also higher than that in group A but significantly lower than that in group B at the first sampling time points (Table 5). The activities of SOD and GSH-Px were clearly influenced by PQ but improved after application of rapamycin (Table 5).
Effect of rapamycin on lung W/D ratio in rats with PQ-induced lung injury
Effect of rapamycin on the cytokine production in the rats poisoned by PQ
The lung wet-dry (W/D) ratio of animals in group B increased significantly compared with those in group A in the first seven days of the experiment. The increase decelerated after the seventh day, and the lung W/D ratio of group B got close to that of group A at fourteenth day. For the animals in group C, the W/D ratio also increased significantly compared with the value of group A during the first seven days; however, the value was significantly lower than that of group B, which was indicative of the treatment effect of rapamycin on the increase of lung W/ D ratio caused by PQ poisoning (Table 1).
The results of ELISA showed that the levels of IL-6, IL-10 and TNF-a in group B increased sharply during the first three days of the experiments, and the level decreased slightly after the seventh day, which were still much higher than those in group A. The levels of cytokine production returned to normal level until the 14th day, whereas the application of rapamycin on the PQ-poisoned animals significantly improved the dysexpressions of the cytokines at the first three sampling time points (Table 6).
Effect of rapamycin on the histopathological characteristics in the lung tissue of rats with PQ-induced lung injury The result of HE staining showed that in the group B, clear alveolitis, alveolar edema and vascular endothelial cell injury
Effect of rapamycin on the distribution and expression of mTOR, p70S6K, TGF-bl, a-SMA, MMP-2 and TIMP-1 in the rats with PQ-induced lung injury The distribution of mTOR, p70S6K, TGF-bl, a-SMA, MMP-2 and TIMP-1 is shown in Figures S1–S6. For mTOR and p70S6K, in group A, the positive signals were detected
Table 1. Effect of rapamycin on lung W/D ratio in lung tissue of PQ-induced lung injury rats (mean ± SD). Time points Group A B C a
First day
Third day
Seventh day
Fourteenth day
Twenty-first day
Twenty-eighth day
4.17 ± 0.07 5.21 ± 0.09a 4.57 ± 0.07a,b
4.23 ± 0.09 5.73 ± 0.18a 5.01 ± 0.09a,b
4.23 ± 0.12 5.41 ± 0.15a 4.71 ± 0.11a,b
4.17 ± 0.07 4.71 ± 0.11 4.48 ± 0.07
4.17 ± 0.08 4.51 ± 0.10 4.36 ± 0.03
4.21 ± 0.03 4.42 ± 0.03 4.30 ± 0.07
p50.01, compared with group A. p50.05, compared with group B.
b
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Figure 1. Effect of rapamycin on the histopathological changes in lung tissue illustrated by HE staining. Table 2. Effect of rapamycin on pulmonary fibrosis in lung tissue of PQ-induced lung injury rats illustrated by Ashcroft scores (mean ± SD). Time point Group A B C
First day
Third day
Seventh day
Fourteenth day
Twenty-first day
Twenty-eighth day
0.45 ± 0.12 0.76 ± 0.23 0.56 ± 0.21
0.47 ± 0.15 1.03 ± 0.30 0.86 ± 0.23
0.50 ± 0.13 1.51 ± 0.32 1.06 ± 0.27
0.49 ± 0.14 3.47 ± 0.56a 2.15 ± 0.33a,b
0.50 ± 0.12 4.56 ± 0.76a 2.36 ± 0.46a,b
0.48 ± 0.15 5.43 ± 0.83a 3.02 ± 0.40a,b
a
p50.01, compared with group A. p50.05, compared with group B.
b
Table 3. Effect of rapamycin on proteinproduction in BALF (mg/L) in lung tissue of PQ-induced lung injury rats (mean ± SD). Time point Group A B C
First day
Third day
Seventh day
Fourteenth day
Twenty-first day
Twenty-eighth day
137.45 ± 12.35 254.56 ± 22.49a 178.86 ± 16.7a,b
140.16 ± 12.89 288.56 ± 25.45a 212.53 ± 18.93a,b
138.67 ± 12.56 306.45 ± 30.05a 248.67 ± 20.42a,b
139.43 ± 12.63 155.56 ± 13.78 154.75 ± 13.06
141.32 ± 12.45 143.42 ± 12.89 142.41 ± 12.63
139.24 ± 12.48 141.44 ± 12.67 141.69 ± 12.53
a
p50.01, compared with group A. p50.05, compared with group B.
b
in bronchial epithelial cells and some endothelial cells. In group B, expressions of mTOR and p70S6K were significantly increased three days after PQ administration (Figures S1 and 2) and reached the peak value at the fourteenth day. After treated by rapamycin, the expressions of mTOR and p70S6K were significantly weakened. The results were also illustrated by Western-blot and RT-PCR (Figures 2A and B and 3A and B). For the expressions of TGF-bl and a-SMA, no significant difference between the groups A and B during the first three days was detected. Significant increase in the expression
levels in group B was observed since the seventh day with expanded distribution of the TGF-bl and a-SMA (Figures S3 and 4). The over-expression levels of TGF-bl and a-SMA in group B were highest at the fourteenth day and retained even at twenty-eighth day. Injection of rapamycin in group C alleviated the expressions of TGF-bl and a-SMA in the PQ-induced injured lungs. The results of Western-blot and RT-PCR were consistent with the results of immunohistochemistry (Figures 2C and D, and 3C and D). In the case of MMP-2 and TIMP-1, the distribution in health lung tissue centered around the pulmonary intravascular
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Table 4. Effect of rapamycin on protein production in HYP (mg/mg of lung tissue) in lung tissue of PQ-induced lung injury rats (mean ± SD). Time points Group A B C
First day
Third day
Seventh day
Fourteenth day
Twenty-first day
Twenty-eighth day
0.77 ± 0.07 0.83 ± 0.13 0.78 ± 0.11
0.80 ± 0.10 0.88 ± 0.15 0.81 ± 0.12
0.78 ± 0.11 1.29 ± 0.17 1.03 ± 0.11
0.79 ± 0.12 1.82 ± 0.23a 1.12 ± 0.13a,b
0.81 ± 0.10 2.12 ± 0.28a 1.34 ± 0.21a,b
0.78 ± 0.09 2.87 ± 0.30a 1.56 ± 0.23a,b
a
p50.01, compared with group A. p50.05, compared with group B.
b
Table 5. Effect of rapamycin on the level of MDA, the activity of SOD and activity of GSH-Px in peripheral blood of PQ-induced lung injury rats (mean ± SD). Time points Group
First day
Third day
Seventh day
Fourteenth day
Twenty-first day
Twenty-first day
A B C A B C A B C
0.53 ± 0.13 2.86 ± 0.89a 1.03 ± 0.49a,b 178.53 ± 9.13 122.34 ± 6.78a 145.79 ± 6.89a,b 343.43 ± 17.34 286.15 ± 15.56a 321.85 ± 15.98a,b
0.50 ± 0.12 4.66 ± 1.20a 2.21 ± 0.70a,b 175.04 ± 8.91 100.75 ± 6.20a 132.46 ± 6.76a,b 340.16 ± 17.12 256.56 ± 13.57a 300.98 ± 14.77a,b
0.51 ± 0.11 1.56 ± 0.78a 0.76 ± 0.32a,b 177.14 ± 9.05 112.43 ± 6.65a 148.49 ± 6.55a,b 345.76 ± 18.16 223.41 ± 12.54a 287.76 ± 13.93a,b
0.53 ± 0.12 0.62 ± 0.15 0.52 ± 0.12 175.89 ± 9.03 146.69 ± 7.25 157.19 ± 7.62 339.60 ± 17.85 195.72 ± 10.91 256.98 ± 11.90
0.52 ± 0.13 0.56 ± 0.13 0.51 ± 0.12 176.18 ± 9.09 157.39 ± 7.37 160.65 ± 7.45 342.76 ± 18.12 321.43 ± 15.34 330.67 ± 15.18
0.52 ± 0.11 0.52 ± 0.14 0.50 ± 0.11 177.01 ± 9.10 165.18 ± 8.24 169.67 ± 8.41 343.65 ± 18.42 335.53 ± 16.12 340.76 ± 16.56
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MDA (mmol/mL) SOD (U/mL) GSH-Px (U/0.1 mL)
a
p50.01, compared with group A. p50.05, compared with group B.
b
Table 6. Effect of rapamycin on level IL-6, IL-10, and TNF-a in peripheral blood of PQ-induced lung injury rats (mean ± SD). Time points
IL-6 (pg/mL) TNF-a (pg/mL) IL-10 (pg/mL)
Group
First day
Third day
Seventh day
Fourteenth day
Twenty-first day
Twenty-eighth day
A B C A B C A B C
78.13 ± 8.34 185.54 ± 10.43a 134.42 ± 9.43a,b 48.23 ± 5.18 155.43 ± 8.43a 97.10 ± 7.13a,b 30.76 ± 4.54 45.97 ± 5.87a 37.45 ± 4.67a,b
76.23 ± 7.89 434.67 ± 20.12a 299.67 ± 16.72a,b 47.18 ± 5.03 345.09 ± 15.64a 217.45 ± 13.42a,b 31.05 ± 4.51 69.97 ± 6.15a 55.67 ± 5.76a,b
77.45 ± 8.06 354.79 ± 17.98a 243.18 ± 15.18a,b 49.46 ± 5.21 251.87 ± 13.06a 157.24 ± 9.36a,b 29.89 ± 4.49 58.07 ± 5.96a 45.01 ± 5.56a,b
78.56 ± 8.25 251.41 ± 13.23 167.89 ± 10.98 47.10 ± 5.13 200.43 ± 13.74 109.76 ± 9.74 31.25 ± 4.53 48.76 ± 5.65 37.75 ± 5.33
77.98 ± 8.31 100.32 ± 9.43 90.23 ± 8.43 47.85 ± 5.08 67.12 ± 6.43 55.32 ± 5.98 29.95 ± 4.47 43.86 ± 5.15 34.53 ± 4.76
77.82 ± 8.16 87.47 ± 8.49 81.31 ± 8.41 49.05 ± 5.19 55.65 ± 5.49 51.97 ± 5.43 31.08 ± 4.47 40.34 ± 5.02 32.65 ± 4.63
a
p50.01, compared with group A. p50.05, compared with group B.
b
macrophage, bronchial epithelial cells, alveolar epithelial cell and some endothelial cells (Figures S5 and S6). The expression of MMP-2 in group B increased since the first day of the experiment reached the highest point at the fourteenth day and then decreased to normal level at the twenty-eighth day (Figure 2E). The expression of TIMP-1 in group B kept increasing until the twenty-first day and stayed higher than that in group A until the twenty-eighth day (Figure 2F). Application of rapamycin could reduce the expression of both MMP-2 and TIMP-1 since the first day to the twenty-eighth day end of the experiment and regulated the balance between MMP-2 and TIMP-1. The ratio values of MMP-2/TIMP-1 in group B were 0.46 and 0.39 at the fourteenth and twenty-first day, while those of group C were 0.89 and 0.91.
Discussion The mechanism of PQ-induced lung injury is complicated. It is general view that the process is characterized by the
interaction involving the cytokine, signal transduction, cell damage and inflammatory response (Figure S7) (Ali et al., 1996; Blanco-Ayala et al., 2014; Dinis-Oliveira et al., 2008; Ghazi-Khansari et al., 2005; Suntres, 2002). As the main target organ of PQ poisoning, lung always has the symptoms of alveolar edema, hemorrhage, inflammatory cell infiltration and diffuse alveolar collapse accompanied by wall thickening (Gao et al., 2013). The mTOR, which is identified as pivot of kinds of signal transduction process in cells, has been taken as an important factor involving in the occurrence of PQ-induced lung injury (Ma & Blenis, 2009). In this study, it was found that the treatment with rapamycin, which inhibited the activity of mTOR, was very effective in alleviating PQ-induced lung injury in rat models. Our results demonstrated that the treatment of rapamycin significantly improved the W/D ratios, alveolar edema and other historical characteristics of the lung samples derived from the PQ + rapamycin group (Tables 1–3; Figure 1). Moreover, by binding with FKBP, rapamycin could suppress
D. Chen et al.
Figure 2. Effects of rapamycin on the expression of mTOR, p70S6K, TGF-bl, a-SMA, MMP-2 and TIMP-1 in lung tissue of PQ-induced acute lung injury rats illustrated by Western blot: (A) mTOR; (B) p70S6K; (C) TGF-bl; (D) a-SMA; (E) MMP-2; and (F) TIMP-1. **p50.01, compared with group A; #p50.05, compared with group B.
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DOI: 10.3109/00498254.2014.995149
Figure 3. Effects of rapamycin on the expression of mTOR, p70S6K, TGF-bl and a-SMA in lung tissues of PQ-induced acute lung injury rats illustrated by RT-PCR: (A) mTOR; (B) p70S6K; (C) TGF-bl; and (D) a-SMA. **p50.01, compared with group A; #p50.05, compared with group B.
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D. Chen et al.
the expression of mTOR and acts as anti-factor against inflammation (Tables 4–6; Figures 2 and 3) (Sarbassov et al., 2004). As shown in cytokine detection, the dysexpression of IL-6, IL-10, TNF-a and protein in BALF was clearly associated with the increase of mTOR (Tables 3 and 6; Figures S1, 2A and 3A), which was indicative of the involvement of mTOR pathway in the cytokine secretion in PQ-induced lung injury. Previous study also revealed the function of PQ in inducing the secretion of cytokine in inflammatory response (Yost et al., 2004). These cytokines as well as other inflammatory factors, initiate, amplify and perpetuate the inflammatory response. However, the application of rapamycin would inhibit the activation of mTOR in PQ-induced lung injury and further improve the inflammatory response (Lindemann et al., 2004). After injection of rapamycin, the level of MDA, and activities of SOD and GSH-Px, which were known to play critical roles in PQ-induced lung injury (Kim et al., 2010), were significantly improved (Table 5), indicating the antioxidative function of rapamycin in the treatment. Another main characteristic of PQ-induced lung injury was the over-synthesis of collagen, which resulted in the pulmonary fibrosis. mTOR played an important role in the protein synthesis through PI3K/Akt pathway. In this study, it was found that as the final product of collagen metabolism, HYP production was declined in group C after treatment with rapamycin (Table 4). Moreover, the application of rapamycin reduced the level of mTOR as well as p70S6K (Figures S1 and 2, 2A and B and 3A and B). It was reported that p70S6K was the substrate of the phosphorylation of mTOR in the collagen synthesis (Sodhi et al., 2008). Concatenated with our data, it was concluded that rapamycin could influence the synthesis of collagen by reducing the extracellular cell matrix (ECM) phosphorylated by mTOR. The expressions of a-SMA and TGF-bl were also upregulated in the animals poisoned by PQ, and the symptoms were eased after treatment with rapamycin (Figures S3 and 4, 2C and D and 3C and D). a-SMA was an important indicator of cell transforming into myofibroblast (MFB), which resulted in pulmonary fibrosis. Compared with normal FB, MFB has more powerful ability in producing and secreting collagen. This characteristic of MFB would increase the aggregation of ECM (Kelynack et al., 1999). TGF-bl was another key factor involving in the pulmonary fibrosis (Ghaffari et al., 2011), which accelerates the transformation of FB to collagen, fibronectin and other ECMs (Xiong et al., 2005). It was reported that rapamycin could inhibit the pulmonary fibrosis induced by TGF-bl via blocking the mTOR pathway (Korfhagen et al., 2009). Furthermore, this conclusion was confirmed in this study, the expressions of a-SMA and TGF-bl both significantly declined in group C with rapamycin treatment (Figure S3 and 4, 2C and D and 3C and D). The deposition of ECM was not only due to the oversynthesis but also due to the lack of degradation (Tung et al., 2010). The imbalance between the synthesis and degradation of ECM also induces pulmonary fibrosis. In this study, we also detected the expression of MMP and TIMP by immunohistochemistry and Western blot (Figure S5 and S6 and 2E and F), which played important roles in the synthesis/ degradation of ECM. The balance between MMP and TIMP
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would significantly influence the process of fibrosis and was a potential therapy for treatment of pulmonary fibrosis. It was reported that mTOR-binding sites existed in the promoter of MMP genes (Wang et al., 2011). The inhibition of pathways activated by mTOR by rapamycin might adjust the balance between MMP and TIMP. This hypothesis was proved by our experiments, in which animals injected with rapamycin had an improved MMP/TIMP value compared with those injected with PQ only. Although the detail mechanism of rapamycin regulating MMP/TIMP ratio still remained unclear, our results had a reasonable illustration of relieving effect of rapamycin on the balance between MMP and TIMP.
Conclusions In conclusion, this study showed that rapamycin had a considerable treatment effect on PQ-induced lung injury in animal models. The drug functioned via inhibiting the pathways activated by mTOR. After treatment by rapamycin, the PQ-poisoned rats had a lower level of MDA, higher activities of SOD and GSH-Px and lower levels of cytokine such as IL-6, IL-10 and TNF-a in the peripheral blood. Moreover, other productions involved in the pulmonary fibrosis also declined through the blocking of mTOR signaling pathway. The treatment of rapamycin on PQ-induced lung injury without significant side-effects was validated in this study, which offered theoretical support for the treatment potential of rapamycin on PQ intoxication. Further study will be conducted to facilitate the application of rapamycin in practice.
Acknowledgements The authors appreciate the language editing by Edanz English Editing.
Declaration of interest The authors disclose no conflict of interest. This work was supported by the National Natural Science Foundation of China (81170068) and the Natural Science Foundation of Liaoning Province (2013021059).
References Ali S, Jain SK, Abdulla M, Athar M. (1996). Paraquat induced DNA damage by reactive oxygen species. Biochem Mol Biol Int 39:63–7. Aparicio G, Calvo MB, Medina V, et al. (2009). Comprehensive lung injury pathology induced by mTOR inhibitors. Clin Transl Oncol 11: 499–510. Ashcroft T, Simpson JM, Timbrell V. (1988). Simple method of estimating severity of pulmonay fibrosis on a numerical scale. J Clin Pathol 41:467–70. Blanco-Ayala T, Anderica-Romero AC, Pedraza-Chaverri J. (2014). New insights into antioxidant strategies against paraquat toxicity. Free Radical Res 48:623–40. Bridle KR, Popa C, Morgan ML, et al. (2009). Rapamycin inhibits hepatic fibrosis in rats by attenuating multiple profibrogenic pathways. Liver Transpl 15:1315–24. Chen G, Chen H, Wang C, et al. (2012). Rapamycin ameliorates kidney fibrosis by inhibiting the activation of mTOR signaling in interstitial macrophages and myofibroblasts. PLOS One 7:e33626. Dinis-Oliveira RJ, Duarte JA, Sa´nchez-Navarro A, et al. (2008). Paraquat poisonings: mechanisms of lung toxicity, clinical features, and treatment. Crit Rev Toxicol 38:13–71.
Intervention of paraquat-induced lung injury
Xenobiotica Downloaded from informahealthcare.com by Selcuk Universitesi on 12/23/14 For personal use only.
DOI: 10.3109/00498254.2014.995149
Gao Y, Xu X, Ding K, et al. (2013). Rapamycin inhibits transforming growth factor b1-induced fibrogenesis in primary human lung fibroblasts. Yonsei Med J 54:437–44. Ghaffari AR, Noshad H, Ostadi A, Hasanzadeh N. (2011). Effect of pulse therapy with glucocorticoid and cyclophosphamide in lung fibrosis due to paraquat poisoning in rats. Saudi Med J 32:249–53. Ghazi-Khansari M, Nasiri G, Honarjoo M. (2005). Decreasing the oxidant stress from paraquat in isolated perfused rat lung using captopril and niacin. Arch Toxicol 79:341–5. Han JF, Wang SL, He XY, et al. (2006). Effect of genetic variation on human cytochrome p450 reductase-mediated paraquatcytotoxicity. Toxicol Sci 91:42–8. Hsu RM, Raine L, Fanger H. (1981). The use of antiavidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase techniques. Am J Clin Pathol 75:816–21. Kahan BD. (2000). Efficacy of sirolimus compared with azathioprine for reduction of acute renal allograft rejection: a randomised multicenter study. The Rapamune US Study Group. Lancet 356: 194–202. Kelynack KJ, Hewistson TD, Pedagogos E, et al. (1999). Renal myofibroblast contract collagen I matrix lattices in vitro. Am J Nephrol 19:694–701. Kim JH, Gil HW, Yang JO, et al. (2010). Effect of glutathione administration on serum levels of reactive oxygen metabolites in patients with paraquat intoxication: a pilot study. Korean J Intern Med 2010;25:282–7. Korfhagen TR, Le Cras TD, Davidson CR, et al. (2009). Rapamycin prevents transforming growth factor-alpha-induced pulmonary fibrosis. Am J Respir Cell Mol Biol 41:562–72. Lindemann SW, Yost CC, Denis MM, et al. (2004). Neutrophils alter the inflammatory milieu by signal-dependent translation of constitutive messenger RNAs. Proc Nat l Acad Sci USA 101:7076–81. Lorne E, Zhao X, Zmijewski JW, et al. (2009). Participation of mammalian target of rapamycin complex 1 in toll-like receptor 2- and 4-induced neutrophil activation and acute lung injury. Am J Respir Cell Mol Biol 41:237–45. Ma XM, Blenis J. (2009). Molecular mehanisms of mTOR-mediated translational control. Nat Rev Mol Cell Biol 10:307–18. Madala SK, Maxfield MD, Davidson CR, et al. (2011). Rapamycin regulates bleomycin-induced lung damage in SP-C-deficient mice. Pulm Med 2011:653524. McAlister VC, Gao Z, Peltekian K, et al. (2000). Sirolimus-tacrolimus combination immunosuppression. Lancet 355:376–7.
9
Mitsopoulos P, Suntres ZE. (2010). Cytotoxicity and gene array analysis of alveolar epithelial A549 cells exposed to paraquat. Chem Biol Interact 188:427–36. Moses JW, Leon MB, Popma JJ, et al. (2003). Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med 349:1315–23. Neves FF, Sousa RB, Pazin-Filho A, et al. (2010). Severe paraquat poisoning: clinical and radiological findings in a survivor. J Bras Pneumol 36:513–16. Parvez S, Raisuddin S. (2006). Effects of paraquat on the freshwater fish Channa punctata: non-enzymatic antioxidants as biomarkers of exposure. Arch Environ Contam Toxicol 50:392–87. Sarbassov DD, Ali SM, Kim DH, et al. (2004). Rictor, a novel binding partner of mTOR, defines a rapamycin insensitive and raptorindependent pathway that regulates the cytoskeleton. Curr Biol 14: 1296–302. Seok SJ, Gil HW, Jeong DS, et al. (2009). Paraquat intoxication in subjects who attempt suicide: why they chose paraquat. Korean J Intern Med 24:247–51. Sodhi A, Chaisuparat R, Hu J, et al. (2008). The TSC2/mTOR pathway drives endothelial cell transformation induced by the Kaposi’s sarcoma-associated herpesvirus G protein-coupled receptor. Cancer Cell 10:133–43. Spaulding C, Henry P, Teiger E, et al. (2006). Sirolimus-eluting versus uncoated stents in acute myocardial infarction. N Engl J Med 355: 1093–104. Suntres ZE. (2002). Role of antioxidants in paraquat toxicity. Toxicology 180:65–77. Tung JN, Lang YD, Wang LF, Chen CM. (2010). Paraquat increases connective tissue growth factor and collagen expression via angiotensin signaling pathway in human lung fibroblasts. Toxicol In Vitro 24:803–8. Wang BL, Tu YY, Fu JF. (2011). Unbalanced MMP/TIMP-1 expression during the development of experimental pulmonary fibrosis with acute paraquat poisoning. Mol Med Report 4:243–8. Wesseling C, van Wendel de Joode B, Ruepert C, et al. (2007). Paraquat in developing countries. Int J Occup Environ Health 7:275–86. Xiong XM, Jiang HL, Liu BF, et al. (2005). Influence of interleukin-10 on the cytokine in acute lung injury in rats induced by endotoxin. Chin J Emerg Med 14:380–3. Yost CC, Denis MM, Lindermann S, et al. (2004). Activated polymorphonuclear leukocytes rapidly synthesize retinoic acid receptora1pha: a mechanism for translational control of transcriptional events. J Exp Med 200:671–80.
Supplementary material available online Supplementary Tables S1–11 and Figures S1–7.
