http://informahealthcare.com/iht ISSN: 0895-8378 (print), 1091-7691 (electronic) Inhal Toxicol, 2014; 26(14): 880–884 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/08958378.2014.970784

RESEARCH ARTICLE

The role of p38 MAPK in acute paraquat-induced lung injury in rats Ying-hao Pei1*, Xiao-min Cai2*, Jiao Chen3*, Bao-di Sun4, Zhao-rui Sun4, Xing Wang1, and Xiao-min Qian3 1

Department of Intensive Care Unit, Jiangsu Provincial Hospital of Traditional Chinese Medicine, Nanjing, China, 2Department of Cardiology, Department of Geriatrics, and 4Department of Emergency Medicine, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, China

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Abstract

Keywords

Context: Paraquat (PQ; 1,1’-dimethyl-4,4’-bipyridinium dichloride) is highly toxic and accounts for a large proportion of the herbicide poisonings seen in clinic. The major cause of mortality is respiratory failure. The p38 mitogen-activated protein kinase (MAPK) signal transduction pathway coordinates various cellular stress responses that have been shown to participate in the pathogenesis of PQ-induced lung injury. Objective: To evaluate the effect of the specific p38 MAPK inhibitor SB203580 on PQ-induced lung injury and cytokine secretion. Methods: In groups of 24, rats were treated with PQ, PQ and SB203580 (SB + PQ), SB203580 alone (SB) or normal saline (control group). Six rats from each group were euthanized at 1, 3, 5 or 7 d. Pathology of lung specimens was scored through hematoxylin and eosin staining. Edema in the lung was quantified from wet-to-dry weight ratios. p38 and p-p38MAPK proteins were measured via electrochemiluminescent Western blots. tumor necrosis factor (TNF)-alpha and interleukin-1 beta (IL-1b) concentrations in lung specimens and bronchoalveolar lavage fluid (BALF) were quantified via enzyme-linked immunosorbent assay. Results: The mortality rate of the SB + PQ group (16.7%) was significantly lower than that of the PQ group (33.3%; p50.05). The PQ group had significantly higher pulmonary histology scores, wet-to-dry weight ratios and phosphorylated p-p38 MAPK levels, as well as higher IL-1b and TNF-alpha levels in BALF and lung tissues, that did the SB + PQ and control groups (p50.05, all). Conclusion: The data suggest that the p38 MAPK signaling pathway has an important role in regulating the production of IL-1b and TNF-alpha in PQ-induced lung injury in rats.

Interleukin 1 beta, lung injury, mitogen-activated protein kinase, paraquat, tumor necrosis factor-alpha

Introduction 0

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Paraquat (PQ; 1,1 -dimethyl-4,4 -bipyridinium dichloride) has been used worldwide as a highly efficient vegetative herbicide since it was first introduced for agricultural purposes in 1962. It is also highly toxic in humans, which is an important concern in clinical toxicology. Incidents of suicidal or accidental ingestion of PQ constitute a large proportion of the herbicide poisonings seen in clinic (Jeyaratnam, 1990). The major cause of mortality in PQ poisoning is respiratory failure. This is likely due to damage in the lungs, where PQ selectively accumulates, leading to edema, hemorrhage, inflammation and cell proliferation, and finally lung fibrosis and respiratory failure (Nerlich et al., 1984). Survivors of PQ poisoning were found to have reduced vital capacity and reduced total lung capacity that progressively worsened over a long-term follow-up of 3.4 ± 1.4 years, indicating that *These authors contributed equally to this work. Address for correspondence: Xiao-min Qian, Department of Geriatrics, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, China. Tel: +86-02580860133. Fax: +86-02580860133. E-mail: [email protected] Xing Wang, Department of Intensive Care Unit, Jiangsu Provincial Hospital of Traditional Chinese Medicine, Nanjing 210029, China. Tel: +86-02586618472. Fax: +86-02580860133. Email: [email protected] sina.com

History Received 26 May 2014 Revised 3 September 2014 Accepted 24 September 2014 Published online 30 October 2014

exposure results in restrictive pulmonary dysfunction over time (Yamashita et al., 2000). Clinical treatments focus on alleviating early inflammation as much as possible to delay lung fibrosis. Unfortunately, there are still no known pharmacological antidotes for PQ poisoning and mortality due to exposure has remained high (Suntres, 2002; Yoon, 2009). The mechanism of PQ toxicity is mainly associated with a sustained redox-cycling effect, resulting in elevated levels of reactive oxygen species. This suggests that superoxide scavengers may be helpful in treating PQ poisoning, but therapies based on antioxidants such vitamin C, N-acetylcysteine and melatonin did not significantly reduce mortality (Awadalla, 2012; Suntres, 2002; Yeh et al., 2006). Many studies have shown that mitogen-activated protein kinase (MAPK) has an important role in intracellular signal pathways of inflammation and oxidative stress responses (Davis, 1993; Hommes et al., 2003; Runchel et al., 2011). Among members of the MAPK family, p38 MAPK may be induced by several types of stress to mediate the production of the cytokines tumor necrosis factor (TNF)-alpha and interleukin-1 beta (IL-1b) (Foey et al., 1998), which participate in the pathogenesis of PQ-induced lung injury (Erroi et al., 1992). In rodent models of trauma and endotoxemia, inhibition of p38 MAPK has been reported to decrease the release

p38 MAPK regulates IL-1 and TNF-alpha

DOI: 10.3109/08958378.2014.970784

of TNF-alpha and IL-1b and reduce lung injury (Chen et al., 2003; Kim et al., 2006). In this study, we investigated whether inhibition of p38 MAPK could protect against PQ-induced lung injury and reduce generation of PQ-induced cytokines in rats.

Materials and methods Animals

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Adult male Sprague-Dawley rats weighing 250–300 g were purchased from the Animal Center of Jinling Hospital (Nanjing, China) and maintained at least one week in a specific pathogen-free animal facility to allow adjustment to the environment. All procedures were performed in accordance with the guidelines of the National Research Council’s Guide for the Humane Care and Use of Laboratory Animals. Reagents SB203580, PQ, TNF-alpha and IL-1b enzyme-linked immunosorbent assay (ELISA) kits were purchased from Sigma (St. Louis, MO). Both anti-phospho-p38 MAPK and anti-p38 MAPK were from Cell Signaling Technologies (Beverly, MA). Study protocol Rats were divided randomly into four groups of 24 and treated with the following via intraperitoneal injection: rats of the PQ group were injected with PQ (18 mg/kg); the SB + PQ group received SB203580 (10 mg/kg) 30 min before PQ treatment (18 mg/kg); the SB group were given only SB203580 (10 mg/kg); and the control group received only normal saline (NS; volume equal to PQ). The doses of these drugs were chosen on the basis of previous studies (Liu et al., 2008; Zhi et al., 2011) and our preliminary experiments. In each group, six rats were euthanized on days 1, 3, 5 or 7 after the above treatments for collecting bronchoalveolar lavage fluid (BALF) and lung samples. BALF samples were evaluated for TNF-alpha and IL-1b protein levels, which were also determined from frozen (liquid nitrogen) specimens of the superior lobe of the right lung. The superior lobe of the left lung was kept in 10% formaldehyde for histology. The middle lobe of the right lung was used for calculating the wet-to-dry weight ratios. The remaining lung tissues were used for p38 MAPK and p-p38MAPK protein quantification via Western blot. Effect of treatments on lung edema To evaluate the effect of the treatments on lung edema, the water content of the lungs was determined by calculating the wet-to-dry weight ratio of lung tissues at 1, 3, 5 and 7 d. In each of the four groups, lung samples were taken at the end timepoints, rinsed free of blood, weighed, incubated at 80  C for 72 h, and the wet-to-dry ratio was calculated by dividing the wet weight by the dry weight. TNF-alpha and IL-1b protein levels in lung tissue and BALF Concentrations of TNF-alpha and IL-1b in lung tissue and BALF were quantified using commercially available ELISA kits conducted in accordance with the manufacturer’s instructions.

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Histological examinations For pathology assessments, lung tissue slices were fixed in 4% formaldehyde for 48 h. These were then embedded in paraffin and cut into sections (4 mm in thickness), which were placed on glass microscope slides and stained with hematoxylin and eosin for histopathological analysis. All histology procedures were performed and graded by two observers who were blinded to the treatment group. Lung tissues were graded (Gloor et al., 2000) for intraalveolar edema, intra-alveolar hemorrhage and neutrophil infiltration (absent, 0; mild, 1; moderate, 2; severe, 3; and overwhelming, 4), and the scores summed to a maximum of 12. Western blot analysis The p38 MAPK activity in lung tissue was assessed by measuring phosphorylation via Western blot as previously described (Chen et al., 2003). In brief, tissue proteins were obtained from the lung at each timepoint in each group. Tissues were lysed in ice-cold extraction buffer containing protease inhibitor cocktail for 30 min. The whole lysates were then centrifuged at 12 000  g for 30 min, and the protein concentration in the supernatant was determined using bicinchoninic acid assays. Thirty micrograms of total lung protein per lane were resolved through 12% denaturingpolyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk and then incubated overnight serially with affinitypurified rabbit polyclonal antibodies against phospho-p38 MAPK (Cell Signaling Technology, catalogue # 9216) or non-phosphorylated p38 MAPK (Cell Signaling Technology, catalogue # 9212). A horseradish peroxidase-conjugated antirabbit secondary antibody was used (diluted to 1:2000 from Cell Signaling Technology). The protein bands were visualized using electrochemiluminescence Plus Western blotting detection reagents (Amersham Biosciences, Castle Hill, Australia) and exposure to X-ray film (Fujifilm, Stafford, Australia). Statistical analyses Data are presented as mean ± standard deviation. Statistical significance in multiple comparison was evaluated by oneway analysis of variance using SPSS version 19.0 software (SPSS, Chicago, IL). Values were considered statistically significant at probability (p)50.05.

Results The effect of SB203580 on PQ-induced lung injury To evaluate the effect of p38 MAPK in PQ-induced lung injury, in a preliminary experiment, we compared the mortality rate of rats treated with PQ alone (the PQ group) with that of rats administered the specific p38 MAPK inhibitor SB203580 prior to PQ administration (the SB + PQ group; Figure 1). In the PQ group, the mortality rate associated with PQ treatment was 33.3%. However, in the SB + PQ group, the mortality rate was 16.7% (p50.05; Figure 1). The mortality of the NS group and SB groups were

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and IL-1b levels were highest on day 1 in both lung tissues and BALF and then gradually declined over time (all p50.05, compared with NS). Relative to the these levels in the PQ group, those of the SB + PQ group were significantly lower at each time point (p50.05) except for the TNF-alpha levels in BALF on days 5 and 7. There were no significant differences between the control and SB groups at any time point for either cytokine.

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Pulmonary histology

Figure 1. SB203580 pretreatment associated with prolonged survival in PQ rats.

The pulmonary histologic scores were significantly higher in the PQ group compared with those of the NS group after the administration of PQ (Figure 4). SB203580 treatment prior to PQ administration was associated with significantly lower histology scores compared with those of the PQ group (p50.05). Expressions of p38 MAPK and phosphorylated p38 MAPK in lung tissue The total p38 MAPK density of the Western blot bands of each group appeared to be similar (Figure 5). Phosphorylated p38 MAPK was significantly higher in the PQ group compared with the NS control group (p50.05), whereas phosphorylated p38 MAPK levels were markedly lower in the SB203580 treatment group (p50.05). The mean density ratios of the bands for phosphorylated p38 MAPK, normalized to the NS control group, were 0.8, 5.3 and 3.4 for the SB, PQ and SB + PQ groups, respectively.

Discussion Figure 2. Effect of SB203580 pretreatment on pulmonary edema in paraquat-treated rats. Pulmonary edema was measured as the increase in lung wet-to-dry weight ratio. Paraquat treatment was associated with a higher wet-to-dry weight ratio at days 1, 3, 5 and 7, while SB203580 treatment appeared to have a protective effect. #p50.05 compared with NS (control) group; *p50.05 compared with PQ group.

0% and 4%, respectively. This indicated that inhibition of p38 MAPK might reduce PQ-induced death in the rat. Pulmonary edema mitigated in PQ-treated rats by pretreatment with SB203580 The lung wet-to-dry ratios were considered to reflect relative changes in pulmonary edema (Figure 2). In the PQ group, the lung wet-to-dry ratios were significantly higher at days 1, 3, 5 and 7 compared with those of the control group at the corresponding time points (p50.05). At each timepoint, pretreatment with SB203580 (the SB + PQ group) before PQ administration was associated with significantly less lung edema compared with that of the PQ group. At day 1, the lung wet-to-dry weight ratios of the control and SB groups were similar. Measurement of TNF-alpha and IL-1b in lung and BALF TNF-alpha and IL-1b levels in rat lung tissue and BALF were measured by ELISA (Figure 3). In the PQ group, TNF-alpha

This study investigated whether inhibition of p38 MAPK through pretreatment with the specific p38 MAPK inhibitor SB203580 could reduce generation of PQ-induced cytokines and protect against subsequent lung injury in a rat model. We found that, compared with the rats administered only PQ, the SB203580 pretreatment was associated with a significantly lower mortality rate, less pathology and lung edema, lower levels of the inflammatory cytokines IL-1b and TNF-alpha in lung and BALF, and inhibition of p38 MAPK activation reflected by significantly lower phosphorylated p-p38 MAPK levels. These results indicate that the p38 MAPK signaling pathway has an important role in promoting PQ-induced lung injury in rats. Inflammation in acute lung injury induced by PQ involves multiple signal transduction processes and is not completely clear. Some signaling pathways, such as NF-kB, have been shown to participate in the inflammatory process (Everhart et al., 2006). An increasing number of studies suggest that MAPKs closely regulate inflammatory reactions by integrating and coordinating cellular stress responses, including the secretion of cytokines, cell migration and apoptosis. Given the central role of p38 MAPK in the regulation of cellular stress response mechanisms, manipulation of p38 kinase activity is an attractive therapeutic approach in the treatment of several diseases. Some studies have shown that lipopolysaccharide and burn trauma enhance the activation of p38 MAPK to regulate signal transduction pathways leading to cytokine synthesis (Chen et al., 2003; Yoshinari et al.,

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Figure 3. Paraquat-induced alterations of IL-1b and TNF-alpha levels in BALF (A and B) and lung tissue. (C and D) #p50.05 compared with NS group, *p50.05 compared with PQ group.

Figure 4. Pulmonary histology. After the administration of paraquat, a significant increase in histologic scores was shown in the PQ group compared with those of the NS control group. In the SB + PQ group, the pulmonary histologic scores were significantly lower than those of the PQ group. #p50.05 compared with NS, *p50.05 compared with PQ.

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the p38 MAPK signaling pathway may have a crucial role in the generation of the inflammatory cytokines TNF-alpha and IL-1b. These findings do not necessarily exclude the potential role of other MAPK subfamilies, such as ERKs and JNKs, in sepsis-induced lung injury. Inhibition of p38 MAPK activity by administration of SB203580 was associated with a reduction in not only TNF-alpha and IL-1b levels but also edema formation in the lung. These novel findings suggest that the p38 MAPK signaling pathway may be a useful target to restrain pathologic inflammation in PQ-induced lung injury.

Declaration of interest The authors report no declarations of interest.

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References

Figure 5. p38 MAPK and phosphorylated p-p38 MAPK levels. (A) Bands of p38 MAPK were identified in the four groups by Western blot, and the mean density levels in each group were almost identical. (B) Phosphorylated p38 MAPK was significantly higher in the PQ group compared with the NS control group, whereas phosphorylated p38 MAPK levels were markedly lower in the SB + PQ group compared with the PQ group. #p50.05 compared with NS; *p50.05 compared with PQ.

2001). It is reported that PQ can prolong neutrophil lifespan, induce the production of reactive oxygen species and increase TNF-alpha concentrations by activating the p38 MAPK signaling pathway (Wang et al., 2014). However, it is surprising to note that little is known of changes in p38 MAPK activity associated with acute lung injury induced by PQ. In this study, after in vivo injection of PQ, increases in cytokines and inflammatory cell infiltration were observed in pulmonary tissue, concomitant with elevated levels of phosphorylated p38 MAPK. Overexpressed cytokines can positively feed back to the activation of p38 MAPK, promoting a vicious cycle that amplifies inflammatory injury. With an in vivo treatment of SB203580 prior to PQ intoxication, we found an associated decrease in TNF-alpha and IL-1b, in both BALF and lung tissue, and improved pulmonary histology. These results indicate that p38 MAPK contributes to PQ-induced acute lung inflammation and mediates the amplification of TNF-alpha and IL-1b in the lung. The results also imply that SB305080 treatment may limit the lethality of PQ-induced acute lung injury via the p38 MAPK signaling pathway. Of note, we were able to demonstrate the important role of p38 MAPK in PQ-induced lung injury, but not that of other MAPK signaling pathways like extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). Novel treatments such as bosentan or cyclophosphamide for PQinduced lung injuries may elucidate the crucial role of p38MPAK as a therapeutic target. These will be addressed in a future study.

Conclusion In summary, this study shows that in PQ-induced acute lung injury phosphorylation of p38 MAPK increased, and therefore

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The role of p38 MAPK in acute paraquat-induced lung injury in rats.

Paraquat (PQ; 1,1'-dimethyl-4,4'-bipyridinium dichloride) is highly toxic and accounts for a large proportion of the herbicide poisonings seen in clin...
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