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MyD88 gene knockout attenuates paraquat-induced acute lung injury
Accepted Manuscript Title: MyD88 gene knockout attenuates paraquat-induced acute lung injury Authors: Haitao Shen, Na Wu, Yu Wang, Feng Guo, Lianghong Chen, Zhigang Zhang, Dong Jia, Min Zhao PII: DOI: Reference:
S0378-4274(17)30025-5 http://dx.doi.org/doi:10.1016/j.toxlet.2017.01.015 TOXLET 9686
To appear in:
Toxicology Letters
Received date: Revised date: Accepted date:
23-11-2016 17-1-2017 21-1-2017
Please cite this article as: Shen, Haitao, Wu, Na, Wang, Yu, Guo, Feng, Chen, Lianghong, Zhang, Zhigang, Jia, Dong, Zhao, Min, MyD88 gene knockout attenuates paraquat-induced acute lung injury.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2017.01.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
MyD88 gene knockout attenuates paraquat-induced acute lung injury
Haitao Shena, Na Wub, Yu Wanga, Feng Guoa, Lianghong Chena, Zhigang Zhang, Dong Jiaa, Min Zhaoa, *
a
Department of Emergency Medicine, Shengjing Hospital of China Medical University,
Shenyang 110004, People’s Republic of China b
Department of Endocrinology, Shengjing Hospital of China Medical University, Shenyang
110004, People’s Republic of China
*
Corresponding author: Dr. Min Zhao, E-mail: [email protected]. Department of
Emergency Medicine, Shengjing Hospital of China Medical University, 36 Sanhao Street, Shenyang 110004, People’s Republic of China Tel: +86-24-96615-64131
1
HIGHLIGHT
1 Paraquat poisoning activates the MyD88-dependent pathway causing ALI
2 MyD88 gene knockout attenuates paraquat-induced ALI
3 MyD88 gene knockout reduces the levels of serum inflammatory cytokines in paraquat poisoning
Abstract Objective: This study investigated the role of myeloid differentiation factor 88 (MyD88) in paraquat-induced acute lung injury (ALI). Methods: C57BL mice were divided into the control group, paraquat group, MyD88 knockout (KO) group, and MyD88 KO plus paraquat group. At 48 h after paraquat poisoning, serum and lung tissues were collected. ELISA was employed to detect tumor necrosis factor-α (TNF-α) and interleukine-1β (IL-1β) contents in serum. Lung tissues were processed for hematoxylin-eosin staining, followed by histological scoring. PCR was performed to detect the mRNA expression of MyD88, TNF-α, and IL-1β in the lungs. Immunofluorescence staining was done to evaluate the expression and distribution of MyD88 and nuclear factor κB (NF-κB) in the lungs. Western blotting was conducted to detect the protein level of toll-like receptor (TLR) 4, TLR9, MyD88, and NF-κB in the lungs.
2
Results: Paraquat poisoning significantly increased serum inflammatory cytokines, as well as MyD88, TLR4, TLR9, and NF-κB, and resulted in ALI. After MyD88 KO, the levels of inflammatory cytokines and NF-κB decreased markedly, and ALI was also attenuated although TLR4 and TLR9 expression continued at an elevated level. Conclusion: MyD88 mediates paraquat-induced ALI, and MyD88 gene knockout may attenuate paraquat-induced ALI and reduce the production of proinflammatory cytokines.
Keywords: paraquat; acute lung injury; MyD88; proinflammatory cytokines
3
Introduction Paraquat is a quick-acting organic heterocyclic herbicide that can kill green plant tissue on contact and has been widely used worldwide. Although lots of measures have been taken to treat paraquat poisoning, the mortality is as high as 70%–80%. Death is usually caused by multiple organ failure based on acute lung injury (ALI) for the lungs are target organs of paraquat. Alveolar epithelial cells may actively transport paraquat into cells, which elevates the paraquat concentration in lung tissues 10–90 times than in plasma. Thus, paraquat cause more severe lung injury than other organs, so ALI become the major cause of death in cases of paraquat poisoning [1]. Although a large number of studies have investigated paraquat poisoning, the molecular mechanism underlying the pathogenesis of paraquat-induced ALI is still poorly understood. Myeloid differentiation factor 88 (MyD88) is a key adaptor in the Toll-like receptor/interleukin-1 (TLR/IL-1) receptor (TIR) pathway, and TLRs are important components in the innate immune system that are responsible for the recognition of pathogen-associated molecular patterns and the subsequent activation of the innate immune system. IL-1 is a representative inflammatory cytokine in the acquired immune response and plays a key role in the specific immune response and inflammatory reaction [2]. In recent years, evidence has shown that TIR is able to facilitate the progression of ALI [3–5]. At least two TIR pathways have been identified: the MyD88-dependent and the MyD88-independent 4
pathways. All TIR-related signal transduction depends on MyD88 except for the TLR3-mediated one [6]. MyD88-dependent TIR activation may induce the activation of the downstream molecule nuclear factor κB (NF-κB) via MyD88, leading to the release of inflammatory cytokines that promote lung inflammation and cause ALI [7,8]. Inhibition of TLR activation attenuates ALI and suppresses the production of inflammatory cytokines [9,10]. The severity of paraquat-induced ALI is positively related to the IL-1 level [11], indicating that interventions targeting the TIR/MyD88 pathway may relieve ALI after paraquat poisoning, and the key adaptor in the MyD88-dependent TIR pathway may become a new target in the therapy of paraquat-induced ALI. Thus, we undertook this study to investigate lung injury and inflammatory cytokines in paraquat-poisoned mice with the MyD88 gene knockout in order to elucidate the role of MyD88 in the pathogenesis of paraquat-induced ALI.
Materials and methods Materials Paraquat (Sigma-Aldrich, St. Louis, MO, USA), tumor necrosis factor-α (TNF-α) and interleukine-1β (IL-1β) ELISA kits (R&D Systems, Minneapolis, MN, USA), antibodies against TLR4, TLR9, MyD88, and NF-κB p-p65 (Novus, Littleton, CO, USA), and other reagents (analytically pure grade) were used in the present study. 5
Animals Specific-pathogen-free, 8-week-old wild-type and MyD88-/- C57BL/6J mice were purchased from the Animal Institute of Nanjing University and were housed in the Central Animal Center of the Affiliated Shengjing Hospital of China Medical University at a constant temperature (20–25°C) and a relative humidity of 40%–70% with a 12 h/12 h light/dark cycle. Animals were given ad libitum access to water and food. This study was approved by the Ethics Committee of the Affiliated Shengjing Hospital of China Medical University (2015PS302K). Animal model and grouping Wild-type mice were randomly divided into the control group (A group, n=8) and paraquat group (B group, n=8); MyD88-/- mice were randomly divided into the MyD88 KO plus paraquat group (C group, n=6) and MyD88 KO control group (D group, n=6). In Groups B and C, mice were intraperitoneally injected with 30 mg/kg paraquat (10 mg/mL paraquat in saline). In Group A and Group D, mice were injected with an equal volume of normal saline. Sample collection and storage At 48 h after paraquat poisoning, animals were intraperitoneally anesthetized with 10% chloral hydrate at 300 mg/kg. Mice were placed in a supine position, and thoracotomy was performed after sterilization. A 1-mL syringe was used to puncture the right ventricle, followed by blood collection. Blood was transferred to a 1.5-mL tube and centrifuged at 3000 6
rpm for 10 min at 4 °C. The supernatant was collected in a 1.5-mL tube (0.2 mL/tube) and stored at –80 °C. After blood collection, the precipitate was removed, and normal saline was used to flush the lung tissues. When the fluid flowing from the body was clear, both lungs were harvested. The color, nature, and pathological changes in the lungs were observed. After being washed with normal saline, the left lung was fixed in 4% paraformaldehyde at 4 °C overnight. On the second day, tissues were dehydrated in 30% sucrose at 4 °C, then embedded in optimum cutting temperature compound and stored at –80 °C. The right lung was stored in liquid nitrogen at –80 °C for further study. Detection of serum TNF-α and IL-1β Serum was centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatant was collected. Serum IL-1β and TNF-α levels were measured with the appropriate ELISA kits according to the manufacturer’s instructions (R&D Systems). Measurement of wet-to-dry weight ratio The middle lobe of the right lung was washed with normal saline, water was removed with a filter, and the lung tissues were weighed as wet weight. Then, the lung tissues were placed in an oven at 80 °C and weighed 48 h later as dry weight. The wet-to-dry weight ratio was calculated to evaluate lung edema: wet-to-dry weight ratio (W/D) = wet weight/dry weight (both in mg).
7
Scoring of lung injury The left lung was cut into blocks (about 0.5 cm in thickness) and then subjected to dehydration in alcohol, transparentization, embedding, sectioning, and H&E staining. Lung pathology was evaluated under a light microscope, and lung injury was scored according to the method proposed by Mikawa et al. [13] based on (1) alveolar congestion, (2) hemorrhage, (3) neutrophil infiltration or aggregation in the alveolar space or vascular wall, and (4) alveolar wall thickening or hyaline membrane formation. Each was graded 0–4: 0, no lesion or very mild injury; 1, mild injury; 2, moderate injury; 3, severe injury; and 4, extremely severe injury. The sum of each score was used as the final lung injury score. Detection of mRNA expression of MyD88, TNF-α, and IL-1β by RT-PCR The lower lobe of the right lung was homogenized in Trizol reagent for subsequent extraction of total RNA according to the manufacturer’s instructions. RNA was reversely transcribed into
cDNA.
The
primers
5-GTGCCGTCGGATGGTAGT-3
used
in
(forward),
PCR
were
as
follows:
MyD88:
5-CAGTGATGAACCGCAGGAT-3
(reverse); TNF-α: 5-GCAAGCTTCGCTCTTCTGTCTACTGAACTTCGG-3 (forward), 5-GCTCTAGAATGAGATAGCAAATCGGCTGACGG-3 5-CGCAGCAGCACATCAACAAGAGC-3
(reverse);
IL-1β: (forward),
5-TGTCCTCATCCTGGAAGGTCCACG-3 (reverse). PCR was performed in a Model
8
7500 Thermal Cycler (Applied Biosystems, Foster City, CA, USA), and the results were subsequently analyzed. Detection of MyD88 and p-p65 expression by immunofluorescence staining The section prepared from the upper lobe of the right lung was washed in 0.1 mol/L phosphate buffered saline (PBS) twice, followed by antigen retrieval in 0.01 mol/L sodium citrate (pH = 6.0) for 15 min. After incubation with 0.25% Triton X-100 for 10 min, sections were blocked in 10% normal goat serum in PBS for 60 min at room temperature to reduce nonspecific staining. The medium was removed, and sections were then treated with MyD88 or p-p65 antibody (1:500) at 4 °C overnight. After being washed in PBST thrice (10 min for each wash), sections were treated with fluorescence-conjugated secondary antibody at room temperature for 60 min. Following three washes in PBST (10 min for each wash), sections were incubated with 0.5 μg/mL DAPI at room temperature for 10 min. After being washed in PBST thrice (10 min for each wash), sections were mounted with 90% glycerol. Sections were then observed under an inverted laser confocal microscope. Detection of TLR4, TLR9, MyD88, and p-p65 protein expression by Western blotting The lower lobe of the right lung was homogenized, followed by extraction of nuclear proteins and cytoplasmic proteins according to the corresponding kits. The protein concentration was determined by the BCA method. Then, proteins were mixed with loading buffer, followed by boiling for 5 min, and 20 μL of protein solution was loaded for 10% SDS-PAGE at 150 V 9
and 30 mA for 1.5 h. Proteins were transferred onto PVDF membrane at 50 V for 2 h. After blocking in 5% nonfat milk, sections were treated with TLR4, TLR9, MyD88, or p-p65 antibody at 4 °C overnight, and β-actin and TBP served as internal references for cytoplasmic proteins and nuclear proteins, respectively. After being washed in TBST thrice (5 min for each wash), sections were incubated with secondary antibody (1:500) at room temperature for 2 h. Following washing in 0.1% TBST thrice (15 min for each wash), visualization was done with ECL Western blotting detection system(Beyotime Institute of Biotechnology, Haimen, China). Statistical analysis Statistical analysis was performed with SPSS version 17.0 (IBM, Chicago, IL, USA). Data are expressed as means ± standard deviations (SD). One-way analysis of variance was employed for comparisons among groups, and the t test was used for comparisons between two groups. A value of P < 0.05 was considered statistically significant.
Results Lung W/D ratio and lung injury In the normal control group, the alveolar structure was complete and clear. At 48 h after paraquat poisoning, the alveolar structure was significantly damaged, there were alveolar hemorrhage, interstitial edema, and infiltration of a large amount of inflammatory cells, and the W/D ratio increased significantly. In MyD88 KO plus paraquat group, lung injury was 10
markedly attenuated, and the W/D ratio also decreased significantly compared to that of paraquat group (Figure 1). Serum TNF-α and IL-1β In paraquat group, serum TNF-α and IL-1β were significantly higher than in the control group. In MyD88 KO plus paraquat group, serum TNF-α tended to decline but was still significantly different from that in paraquat group, and IL-1β in MyD88 KO plus paraquat group was only slightly lower than that in paraquat group. In MyD88 KO group, serum TNF-α and IL-1β were similar to those in the control group (Figure 2). mRNA expression of MyD88, TNF-α, and IL-1β in the lungs In paraquat group and MyD88 KO plus paraquat group, the mRNA expression of MyD88, TNF-α, and IL-1β increased significantly compared to the control group except MyD88 in MyD88 KO plus paraquat group was undetectable. In MyD88 KO plus paraquat group, MyD88, TNF-α and IL-1β mRNA expression tended to decrease compared to that in paraquat group. In MyD88 KO group, TNF-α and IL-1β mRNA expressions were comparable to those in the control group, but MyD88 mRNA expression was significantly lower than in the control group (Figure 3).
11
Expression and distribution of MyD88 and p-p65 MyD88 and p-p65 expressions were low in the lungs of the control group, but their expression increased significantly after paraquat poisoning in paraquat group. In MyD88 KO plus paraquat group, MyD88 was undetectable and p-p65 expression was reduced compared to that of paraquat group (P < 0.05) but was still higher than that in the control group. In MyD88 KO group, MyD88 expression was undetectable, and p-p65 expression was similar to that in the control group (Figure 4). TLR4, TLR9, MyD88, and p-p65 protein expression in the lungs TLR4, TLR9, MyD88, and p-p65 protein expression was low in the control group. In paraquat group, TLR4, TLR9, MyD88, and p-p65 protein expression increased significantly compared to that in the control group. In MyD88 KO plus paraquat group, TLR4 and TLR9 protein expression was similar to that in paraquat group, but MyD88 was undetectable and p-p65 expression was reduced significantly compared to that in paraquat group. In MyD88 KO group, TLR4, TLR9, and p-p65 expression was comparable to that in the control group, but MyD88 expression was undetectable (Figure 5).
12
Discussion Paraquat is extremely toxic, and there is no specific detoxification measure following paraquat poisoning. Thus, the mortality of patients who experience paraquat poisoning is still at a high level [14]. After entering the human body, paraquat is rapidly absorbed into the circulation. The lungs are rich in polyamine transportation systems that may cause the aggregation of paraquat in the lungs. Thus, paraquat poisoning usually results in obvious lung oxidative stress and inflammation [15]. IL-1 is an important cytokine in the inflammatory reaction and may directly reflect its intensity. Our results showed that MyD88 and IL-1 expression were up-regulated after paraquat poisoning and were positively associated with the severity of the lung injury. On the one hand, MyD88, as a downstream adaptor of the TIR pathway, mediates the biological effects of IL-1 [16,17,18]; on the other hand, MyD88 also affects the expression and secretion of IL-1 during inflammation [19,20,21], which is consistent with the reduced expression and secretion of IL-1β and the attenuation of lung injury in MyD88 knockouts. In the innate immune system, TLRs are important receptors that can recognize pathogens. Liu et al. [22] found that TLR4 expression increased after paraquat poisoning, and TLR4 knockout attenuated paraquat-induced lung injury. Qian et al. [23] also revealed that TLR9/NF-κB upregulation in mice was related to the severity of paraquat poisoning. After TLR activation, downstream adaptor proteins are recruited to initiate TLR signal transduction. 13
Of these adaptor proteins, MyD88 is an important member [24,25,26]: After activation, MyD88 may induce the phosphorylation of interleukin receptor–associated kinases 1 and 4 (IRAK-1 and IRAK-4), leading to their activation. Thereafter, in the presence of TNF receptor associated factor 6, the NF-κB complex is phosphorylated, leading to the release of p-p65 into the nucleus, which induces the expression of a series of downstream genes (such as TNF-α and IL-6) [23,24]. In the present study, the results showed that TLR4 and TLR9 protein expression increased significantly after paraquat poisoning, and a similar trend was observed in downstream MyD88/p-p65, which are consistent with those of a previous report [23]. In addition, the changes in lung injury and inflammatory cytokines were similar. These findings indicate that paraquat poisoning activates TLR/MyD88/p-p65, leading to ALI. In addition, in MyD88 KO mice, TLR4 and TLR9 expression was not downregulated, but the expression of downstream MyD88/p-p65 and inflammatory cytokines was markedly downregulated, accompanied by attenuation of ALI. This indicates that, after paraquat poisoning, TLR4 and TLR 9 are activated in mice with MyD88 gene knockout, but the deficiency of MyD88 blocks the activation of downstream factors and fails to induce the release of inflammatory cytokines, including NF-κB, which finally attenuates ALI. MyD88 as an adaptor plays an important role in both innate immunity and acquired immunity [27]. However, in the present study, MyD88
14
gene knockout did not cause adverse effects in mice, which is consistent with the findings of a previous report [28]. Studies have shown that the paraquat concentration peaks in the lungs at about 10–16 h after paraquat
poisoning
[29], and fatal paraquat
poisoning usually causes
obvious
pathophysiological changes within 48 h [30,31]. Thus, assays were conducted at 48 h after paraquat poisoning. Our results also confirmed that mice with paraquat poisoning developed evident ALI by 48 h after poisoning. However, paraquat poisoning causes not only ALI but also may induce pulmonary interstitial fibrosis, which is a major cause of death in the nonacute phase of paraquat poisoning [32]. The present study focused on ALI and did not investigate pulmonary interstitial fibrosis after paraquat poisoning. Thus, the role of MyD88 in pulmonary interstitial fibrosis requires further elucidation in future studies.
Conclusions Our results indicate that paraquat poisoning may activate the NF-κB pathway in a MyD88-dependent pathway that increases the expression and release of TNF-α and IL-1, resulting in ALI. MyD88 gene knockout inhibits the activation of NF-κB, and thereafter the expression and release of TNF-α and IL-1 are also reduced, leading to the attenuation of ALI. In sum, MyD88 mediates paraquat poisoning–induced ALI, and MyD88 gene knockout can attenuate paraquat poisoning–induced ALI, which suggests new strategies in the treatment of paraquat poisoning. 15
Conflict of interest The authors have declared no conflicts of interest.
Funding The design of the study and collection, analysis, and interpretation of data and writing of this manuscript were supported by Science Foundation of Liaoning Education Department (No. LK201633& LK201603) and Peking Union Medical Foundation-Ruiyi Emergency Medical Research Fund(No. R2015021) and Provincial Natural Science Foundation of Liaoning(No. 201602879).
Acknowledgements We gratefully acknowledge Hongyu Zhao and Xiaowei Wei for their intellectual support and technical assistance.
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References [1] Kan B, Jian X, Zhang Z, et al. Whole lung lavage therapy: Treatment for lung injury caused by paraquat poisoning [J]. Journal of Toxicology and Environmental Health Sciences. 2014:
6(4): 99-102.
[2] Dunne A, O'Neill LAJ. The interleukin-1 receptor/Toll-like receptor superfamily: signal transduction during inflammation and host defense [J]. Sciences Stke Signal Transduction Knowledge Environment. 2003; 2003(171):re3-re3. [3] Wang NA, Su Y, Che XM, et al. Penehyclidine ameliorates acute lung injury by inhibiting Toll-like receptor 2/4 expression and nuclear factor-κB activation [J]. Experimental & Therapeutic Medicine. 2016; 11(5):1827-1832. [4] Madathilparambil SV, Thomas B, Dolgachev VA, et al. Toll Like Receptor-9 (TLR9) is Requisite for Acute Inflammatory Response and Injury Following Lung Contusion [J]. Shock. 2016; 46(4):412-419. [5] Ferreira TP, Mariano LL, Ghilossobortolini R, et al. Potential of PEGylated Toll-Like Receptor 7 Ligands for Controlling Inflammation and Functional Changes in Mouse Models of Asthma and Silicosis [J]. Frontiers in Immunology. 2016; 7(10):95.
17
[6] Rhee SH. Basic and translational understandings of microbial recognition by toll-like receptors in the intestine [J]. Journal of Neurogastroenterology & Motility. 2011; 17(1):28-34. [7] Herold S, Gabrielli NM, Vadász I. Novel concepts of acute lung injury and alveolar-capillary barrier dysfunction [J]. Am J Physiol Lung Cell Mol Physiol. 2013; 305(10):L665-L681. [8] Hemmi H, Takeuchi O, Kawai T, et al. A Toll-like receptor recognizes bacterial DNA. Nature. 2000; 408(6813):740-745. [9] Ma L, Zhao G, Hua C, et al. Down-Regulation of TLR9 Expression Affects the Maturation and Function of Murine Bone Marrow-Derived Dendritic Cells Induced by CpG [J]. Cellular & molecular immunology. 2009; 6(3):199-205. [10] Nadigel J, Préfontaine D, Baglole C J, et al. Cigarette smoke increases TLR4 and TLR9 expression and induces cytokine production from CD8+ T cells in chronic obstructive pulmonary disease [J]. Respiratory Research. 2011; 12(10):1214-1216. [11] Erroi A, Bianchi M, Ghezzi P. The pneumotoxicant paraquat potentiates IL-1 and TNF production by human mononuclear cells [J]. Agents & Actions. 1992; 36(1-2):66-69.. [12] Li G, Yuzhen L, Yi C, et al. DNaseI protects against Paraquat-induced acute lung injury and pulmonary fibrosis mediated by mitochondrial DNA [J]. BioMed research international. 2015; 2015:386952. 18
[13] Mikawa K, Nishina K, Takao Y, et al. ONO-1714,a nitric oxide synthase inhibitor,attenuates endotoxin-induced acute lung injury in rabbits [J]. Anesthesia & Analgesia. 2003; 97(6):1751-1755． [14]Gawarammana IB, Buckley NA. Medical management of paraquat ingestion [J]. British Journal of Clinical Pharmacology. 2011; 72(5):745-57. [15]Bertram A, Haenel S S, Hadem J, et al. Tissue concentration of paraquat on day 32 after intoxication and failed bridge to transplantation by extracorporeal membrane oxygenation therapy [J]. Bmc Pharmacology & Toxicology. 2013; 14(1): 45. [16]Li X, Qin J. Modulation of Toll–interleukin 1 receptor mediated signaling [J]. Journal of Molecular Medicine. 2005; 83(4):258-266. [17]Ngo VN, Young RM, Schmitz R, et al. Oncogenically active MYD88 mutations in human lymphoma [J]. Nature. 2011; 470(7332):115-9. [18]Takeuchi O, Akira S. MyD88 as a bottle neck in Toll/IL-1 signaling.[M]// Toll-Like Receptor Family Members and Their Ligands. Springer Berlin Heidelberg, 2002:155-167. [19]Bechelli J, Smalley C, Zhao X, et al. MyD88 Mediates Instructive Signaling in Dendritic Cells and Protective Inflammatory Response during Rickettsial Infection [J]. Infection & Immunity. 2016; 84(4):883-93
19
[20]Ye X, Kong D, Wang J, et al. MyD88 contributes to neuroinflammatory responses induced by cerebral ischemia/reperfusion in mice [J]. Biochemical & Biophysical Research Communications. 2016; 480(1):69-74. [21]Poole JA, Wyatt TA, Romberger DJ, et al. MyD88 in lung resident cells governs airway inflammatory and pulmonary function responses to organic dust treatment [J]. Respiratory Research. 2015; 16(1):1-12. [22]Liu W, Shan LP, Dong XS, et al. Toll-like receptor 4 implicated in acute lung injury induced by paraquat poisoning in mice [J]. International Journal of Clinical & Experimental Medicine. 2014; 7(10):3392-3397. [23]Qian J, Liu L, Chen L, et al. Increased toll-like receptor 9 expression is associated with the severity of paraquat-induced lung injury in mice [J]. Human & Experimental Toxicology. 2015; 34(4):430-8. [24]Kayama H, Ramirezcarrozzi VR, Yamamoto M, et al. Class-specific regulation of pro-inflammatory genes by MyD88 pathways and IκBζ [J]. Journal of Biological Chemistry. 2015; 290(8):12468-12477. [25]Jiang
Q,
Yi
M,
Guo
Q,
et
al.
Protective
effects
of
polydatin
on
lipopolysaccharide-induced acute lung injury through TLR4-MyD88-NF-κB pathway [J]. International Immunopharmacology. 2015; 29(2):370-6.
20
[26]Adachi O, Kawai T, Takeda K, et al. Targeted Disruption of the MyD88, Gene Results in Loss of IL-1- and IL-18-Mediated Function [J]. Immunity. 1998; 9(1):143–150. [27]Huang X, Yang Y. Targeting the TLR9-MyD88 pathway in the regulation of adaptive immune responses [J]. Expert Opinion on Therapeutic Targets. 2010; 14(8):787-96. [28]Janssens S, Beyaert R. A universal role for MyD88 in TLR/IL-1R-mediated signaling [J]. Trends in Biochemical Sciences. 2002; 27(9):474-482. [29] Senarathna L, Eddleston M, Wilks MF, et al. Prediction of outcome after paraquat poisoning by measurement of the plasma paraquat concentration [J]. Qjm Monthly Journal of the Association of Physicians. 2009; 102(4):251-259. [30]Mohammadi-Bardbori A, Ghazi-Khansari M. Alternative electron acceptors: Proposed mechanism of paraquat mitochondrial toxicity [J]. Environmental Toxicology & Pharmacology. 2008; 26(1):1-5. [31]Wu N, Shen H, Liu H, et al. Acute blood glucose fluctuation enhances rat aorta endothelial cell apoptosis, oxidative stress and pro-inflammatory cytokine expression in vivo [J]. Cardiovascular Diabetology. 2016; 15(1):109. [32]Eddleston M, Wilks MF, Buckley NA. Prospects for teatment of paraquat-induced lung fibrosis with immunosuppressive drugs and the need for better prediction of outcome: A systematic review [J]. Qjm Monthly Journal of the Association of Physicians. 2003; 96(11):809-24. 21
Figure 1 Lung wet/dry (W/D) ratio and lung pathology score. A: control group; B: paraquat group; C: MyD88 knockout (KO) plus paraquat group; D: MyD88 KO group. #P < 0.05 vs. Group A; *P < 0.05 vs. Group B. n=6-8.
22
Figure 2 Serum TNF-α and IL-1β contents (ELISA). A: control group; B: paraquat group; C: MyD88 knockout (KO) plus paraquat group; D: MyD88 KO group. #P < 0.05 vs. Group A; *P < 0.05 vs. Group B. n=6-8.
23
Figure 3 mRNA expression of MyD88, TNF-α, and IL-1β in the lungs (RT-PCR). A: control group; B: paraquat group; C: MyD88 knockout (KO) plus paraquat group; D: MyD88 KO group. #P < 0.05 vs. Group A; *P < 0.05 vs. Group B. n=6-8.
24
Figure 4 Expression and distribution of MyD88 and p-p65 in the lungs (immunofluorescence staining). A: control group; B: paraquat group; C: MyD88 knockout (KO) plus paraquat group; D: MyD88 KO group. n=6-8.
25
Figure 5 TLR4, TLR9, MyD88, and p-p65 expression in the lungs (Western blotting). A: control group; B: paraquat group; C: MyD88 knockout (KO) plus paraquat group; D: MyD88 KO group. n =6- 8.
26
S0378-4274(17)30025-5 http://dx.doi.org/doi:10.1016/j.toxlet.2017.01.015 TOXLET 9686
To appear in:
Toxicology Letters
Received date: Revised date: Accepted date:
23-11-2016 17-1-2017 21-1-2017
Please cite this article as: Shen, Haitao, Wu, Na, Wang, Yu, Guo, Feng, Chen, Lianghong, Zhang, Zhigang, Jia, Dong, Zhao, Min, MyD88 gene knockout attenuates paraquat-induced acute lung injury.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2017.01.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
MyD88 gene knockout attenuates paraquat-induced acute lung injury
Haitao Shena, Na Wub, Yu Wanga, Feng Guoa, Lianghong Chena, Zhigang Zhang, Dong Jiaa, Min Zhaoa, *
a
Department of Emergency Medicine, Shengjing Hospital of China Medical University,
Shenyang 110004, People’s Republic of China b
Department of Endocrinology, Shengjing Hospital of China Medical University, Shenyang
110004, People’s Republic of China
*
Corresponding author: Dr. Min Zhao, E-mail: [email protected]. Department of
Emergency Medicine, Shengjing Hospital of China Medical University, 36 Sanhao Street, Shenyang 110004, People’s Republic of China Tel: +86-24-96615-64131
1
HIGHLIGHT
1 Paraquat poisoning activates the MyD88-dependent pathway causing ALI
2 MyD88 gene knockout attenuates paraquat-induced ALI
3 MyD88 gene knockout reduces the levels of serum inflammatory cytokines in paraquat poisoning
Abstract Objective: This study investigated the role of myeloid differentiation factor 88 (MyD88) in paraquat-induced acute lung injury (ALI). Methods: C57BL mice were divided into the control group, paraquat group, MyD88 knockout (KO) group, and MyD88 KO plus paraquat group. At 48 h after paraquat poisoning, serum and lung tissues were collected. ELISA was employed to detect tumor necrosis factor-α (TNF-α) and interleukine-1β (IL-1β) contents in serum. Lung tissues were processed for hematoxylin-eosin staining, followed by histological scoring. PCR was performed to detect the mRNA expression of MyD88, TNF-α, and IL-1β in the lungs. Immunofluorescence staining was done to evaluate the expression and distribution of MyD88 and nuclear factor κB (NF-κB) in the lungs. Western blotting was conducted to detect the protein level of toll-like receptor (TLR) 4, TLR9, MyD88, and NF-κB in the lungs.
2
Results: Paraquat poisoning significantly increased serum inflammatory cytokines, as well as MyD88, TLR4, TLR9, and NF-κB, and resulted in ALI. After MyD88 KO, the levels of inflammatory cytokines and NF-κB decreased markedly, and ALI was also attenuated although TLR4 and TLR9 expression continued at an elevated level. Conclusion: MyD88 mediates paraquat-induced ALI, and MyD88 gene knockout may attenuate paraquat-induced ALI and reduce the production of proinflammatory cytokines.
Keywords: paraquat; acute lung injury; MyD88; proinflammatory cytokines
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Introduction Paraquat is a quick-acting organic heterocyclic herbicide that can kill green plant tissue on contact and has been widely used worldwide. Although lots of measures have been taken to treat paraquat poisoning, the mortality is as high as 70%–80%. Death is usually caused by multiple organ failure based on acute lung injury (ALI) for the lungs are target organs of paraquat. Alveolar epithelial cells may actively transport paraquat into cells, which elevates the paraquat concentration in lung tissues 10–90 times than in plasma. Thus, paraquat cause more severe lung injury than other organs, so ALI become the major cause of death in cases of paraquat poisoning [1]. Although a large number of studies have investigated paraquat poisoning, the molecular mechanism underlying the pathogenesis of paraquat-induced ALI is still poorly understood. Myeloid differentiation factor 88 (MyD88) is a key adaptor in the Toll-like receptor/interleukin-1 (TLR/IL-1) receptor (TIR) pathway, and TLRs are important components in the innate immune system that are responsible for the recognition of pathogen-associated molecular patterns and the subsequent activation of the innate immune system. IL-1 is a representative inflammatory cytokine in the acquired immune response and plays a key role in the specific immune response and inflammatory reaction [2]. In recent years, evidence has shown that TIR is able to facilitate the progression of ALI [3–5]. At least two TIR pathways have been identified: the MyD88-dependent and the MyD88-independent 4
pathways. All TIR-related signal transduction depends on MyD88 except for the TLR3-mediated one [6]. MyD88-dependent TIR activation may induce the activation of the downstream molecule nuclear factor κB (NF-κB) via MyD88, leading to the release of inflammatory cytokines that promote lung inflammation and cause ALI [7,8]. Inhibition of TLR activation attenuates ALI and suppresses the production of inflammatory cytokines [9,10]. The severity of paraquat-induced ALI is positively related to the IL-1 level [11], indicating that interventions targeting the TIR/MyD88 pathway may relieve ALI after paraquat poisoning, and the key adaptor in the MyD88-dependent TIR pathway may become a new target in the therapy of paraquat-induced ALI. Thus, we undertook this study to investigate lung injury and inflammatory cytokines in paraquat-poisoned mice with the MyD88 gene knockout in order to elucidate the role of MyD88 in the pathogenesis of paraquat-induced ALI.
Materials and methods Materials Paraquat (Sigma-Aldrich, St. Louis, MO, USA), tumor necrosis factor-α (TNF-α) and interleukine-1β (IL-1β) ELISA kits (R&D Systems, Minneapolis, MN, USA), antibodies against TLR4, TLR9, MyD88, and NF-κB p-p65 (Novus, Littleton, CO, USA), and other reagents (analytically pure grade) were used in the present study. 5
Animals Specific-pathogen-free, 8-week-old wild-type and MyD88-/- C57BL/6J mice were purchased from the Animal Institute of Nanjing University and were housed in the Central Animal Center of the Affiliated Shengjing Hospital of China Medical University at a constant temperature (20–25°C) and a relative humidity of 40%–70% with a 12 h/12 h light/dark cycle. Animals were given ad libitum access to water and food. This study was approved by the Ethics Committee of the Affiliated Shengjing Hospital of China Medical University (2015PS302K). Animal model and grouping Wild-type mice were randomly divided into the control group (A group, n=8) and paraquat group (B group, n=8); MyD88-/- mice were randomly divided into the MyD88 KO plus paraquat group (C group, n=6) and MyD88 KO control group (D group, n=6). In Groups B and C, mice were intraperitoneally injected with 30 mg/kg paraquat (10 mg/mL paraquat in saline). In Group A and Group D, mice were injected with an equal volume of normal saline. Sample collection and storage At 48 h after paraquat poisoning, animals were intraperitoneally anesthetized with 10% chloral hydrate at 300 mg/kg. Mice were placed in a supine position, and thoracotomy was performed after sterilization. A 1-mL syringe was used to puncture the right ventricle, followed by blood collection. Blood was transferred to a 1.5-mL tube and centrifuged at 3000 6
rpm for 10 min at 4 °C. The supernatant was collected in a 1.5-mL tube (0.2 mL/tube) and stored at –80 °C. After blood collection, the precipitate was removed, and normal saline was used to flush the lung tissues. When the fluid flowing from the body was clear, both lungs were harvested. The color, nature, and pathological changes in the lungs were observed. After being washed with normal saline, the left lung was fixed in 4% paraformaldehyde at 4 °C overnight. On the second day, tissues were dehydrated in 30% sucrose at 4 °C, then embedded in optimum cutting temperature compound and stored at –80 °C. The right lung was stored in liquid nitrogen at –80 °C for further study. Detection of serum TNF-α and IL-1β Serum was centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatant was collected. Serum IL-1β and TNF-α levels were measured with the appropriate ELISA kits according to the manufacturer’s instructions (R&D Systems). Measurement of wet-to-dry weight ratio The middle lobe of the right lung was washed with normal saline, water was removed with a filter, and the lung tissues were weighed as wet weight. Then, the lung tissues were placed in an oven at 80 °C and weighed 48 h later as dry weight. The wet-to-dry weight ratio was calculated to evaluate lung edema: wet-to-dry weight ratio (W/D) = wet weight/dry weight (both in mg).
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Scoring of lung injury The left lung was cut into blocks (about 0.5 cm in thickness) and then subjected to dehydration in alcohol, transparentization, embedding, sectioning, and H&E staining. Lung pathology was evaluated under a light microscope, and lung injury was scored according to the method proposed by Mikawa et al. [13] based on (1) alveolar congestion, (2) hemorrhage, (3) neutrophil infiltration or aggregation in the alveolar space or vascular wall, and (4) alveolar wall thickening or hyaline membrane formation. Each was graded 0–4: 0, no lesion or very mild injury; 1, mild injury; 2, moderate injury; 3, severe injury; and 4, extremely severe injury. The sum of each score was used as the final lung injury score. Detection of mRNA expression of MyD88, TNF-α, and IL-1β by RT-PCR The lower lobe of the right lung was homogenized in Trizol reagent for subsequent extraction of total RNA according to the manufacturer’s instructions. RNA was reversely transcribed into
cDNA.
The
primers
5-GTGCCGTCGGATGGTAGT-3
used
in
(forward),
PCR
were
as
follows:
MyD88:
5-CAGTGATGAACCGCAGGAT-3
(reverse); TNF-α: 5-GCAAGCTTCGCTCTTCTGTCTACTGAACTTCGG-3 (forward), 5-GCTCTAGAATGAGATAGCAAATCGGCTGACGG-3 5-CGCAGCAGCACATCAACAAGAGC-3
(reverse);
IL-1β: (forward),
5-TGTCCTCATCCTGGAAGGTCCACG-3 (reverse). PCR was performed in a Model
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7500 Thermal Cycler (Applied Biosystems, Foster City, CA, USA), and the results were subsequently analyzed. Detection of MyD88 and p-p65 expression by immunofluorescence staining The section prepared from the upper lobe of the right lung was washed in 0.1 mol/L phosphate buffered saline (PBS) twice, followed by antigen retrieval in 0.01 mol/L sodium citrate (pH = 6.0) for 15 min. After incubation with 0.25% Triton X-100 for 10 min, sections were blocked in 10% normal goat serum in PBS for 60 min at room temperature to reduce nonspecific staining. The medium was removed, and sections were then treated with MyD88 or p-p65 antibody (1:500) at 4 °C overnight. After being washed in PBST thrice (10 min for each wash), sections were treated with fluorescence-conjugated secondary antibody at room temperature for 60 min. Following three washes in PBST (10 min for each wash), sections were incubated with 0.5 μg/mL DAPI at room temperature for 10 min. After being washed in PBST thrice (10 min for each wash), sections were mounted with 90% glycerol. Sections were then observed under an inverted laser confocal microscope. Detection of TLR4, TLR9, MyD88, and p-p65 protein expression by Western blotting The lower lobe of the right lung was homogenized, followed by extraction of nuclear proteins and cytoplasmic proteins according to the corresponding kits. The protein concentration was determined by the BCA method. Then, proteins were mixed with loading buffer, followed by boiling for 5 min, and 20 μL of protein solution was loaded for 10% SDS-PAGE at 150 V 9
and 30 mA for 1.5 h. Proteins were transferred onto PVDF membrane at 50 V for 2 h. After blocking in 5% nonfat milk, sections were treated with TLR4, TLR9, MyD88, or p-p65 antibody at 4 °C overnight, and β-actin and TBP served as internal references for cytoplasmic proteins and nuclear proteins, respectively. After being washed in TBST thrice (5 min for each wash), sections were incubated with secondary antibody (1:500) at room temperature for 2 h. Following washing in 0.1% TBST thrice (15 min for each wash), visualization was done with ECL Western blotting detection system(Beyotime Institute of Biotechnology, Haimen, China). Statistical analysis Statistical analysis was performed with SPSS version 17.0 (IBM, Chicago, IL, USA). Data are expressed as means ± standard deviations (SD). One-way analysis of variance was employed for comparisons among groups, and the t test was used for comparisons between two groups. A value of P < 0.05 was considered statistically significant.
Results Lung W/D ratio and lung injury In the normal control group, the alveolar structure was complete and clear. At 48 h after paraquat poisoning, the alveolar structure was significantly damaged, there were alveolar hemorrhage, interstitial edema, and infiltration of a large amount of inflammatory cells, and the W/D ratio increased significantly. In MyD88 KO plus paraquat group, lung injury was 10
markedly attenuated, and the W/D ratio also decreased significantly compared to that of paraquat group (Figure 1). Serum TNF-α and IL-1β In paraquat group, serum TNF-α and IL-1β were significantly higher than in the control group. In MyD88 KO plus paraquat group, serum TNF-α tended to decline but was still significantly different from that in paraquat group, and IL-1β in MyD88 KO plus paraquat group was only slightly lower than that in paraquat group. In MyD88 KO group, serum TNF-α and IL-1β were similar to those in the control group (Figure 2). mRNA expression of MyD88, TNF-α, and IL-1β in the lungs In paraquat group and MyD88 KO plus paraquat group, the mRNA expression of MyD88, TNF-α, and IL-1β increased significantly compared to the control group except MyD88 in MyD88 KO plus paraquat group was undetectable. In MyD88 KO plus paraquat group, MyD88, TNF-α and IL-1β mRNA expression tended to decrease compared to that in paraquat group. In MyD88 KO group, TNF-α and IL-1β mRNA expressions were comparable to those in the control group, but MyD88 mRNA expression was significantly lower than in the control group (Figure 3).
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Expression and distribution of MyD88 and p-p65 MyD88 and p-p65 expressions were low in the lungs of the control group, but their expression increased significantly after paraquat poisoning in paraquat group. In MyD88 KO plus paraquat group, MyD88 was undetectable and p-p65 expression was reduced compared to that of paraquat group (P < 0.05) but was still higher than that in the control group. In MyD88 KO group, MyD88 expression was undetectable, and p-p65 expression was similar to that in the control group (Figure 4). TLR4, TLR9, MyD88, and p-p65 protein expression in the lungs TLR4, TLR9, MyD88, and p-p65 protein expression was low in the control group. In paraquat group, TLR4, TLR9, MyD88, and p-p65 protein expression increased significantly compared to that in the control group. In MyD88 KO plus paraquat group, TLR4 and TLR9 protein expression was similar to that in paraquat group, but MyD88 was undetectable and p-p65 expression was reduced significantly compared to that in paraquat group. In MyD88 KO group, TLR4, TLR9, and p-p65 expression was comparable to that in the control group, but MyD88 expression was undetectable (Figure 5).
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Discussion Paraquat is extremely toxic, and there is no specific detoxification measure following paraquat poisoning. Thus, the mortality of patients who experience paraquat poisoning is still at a high level [14]. After entering the human body, paraquat is rapidly absorbed into the circulation. The lungs are rich in polyamine transportation systems that may cause the aggregation of paraquat in the lungs. Thus, paraquat poisoning usually results in obvious lung oxidative stress and inflammation [15]. IL-1 is an important cytokine in the inflammatory reaction and may directly reflect its intensity. Our results showed that MyD88 and IL-1 expression were up-regulated after paraquat poisoning and were positively associated with the severity of the lung injury. On the one hand, MyD88, as a downstream adaptor of the TIR pathway, mediates the biological effects of IL-1 [16,17,18]; on the other hand, MyD88 also affects the expression and secretion of IL-1 during inflammation [19,20,21], which is consistent with the reduced expression and secretion of IL-1β and the attenuation of lung injury in MyD88 knockouts. In the innate immune system, TLRs are important receptors that can recognize pathogens. Liu et al. [22] found that TLR4 expression increased after paraquat poisoning, and TLR4 knockout attenuated paraquat-induced lung injury. Qian et al. [23] also revealed that TLR9/NF-κB upregulation in mice was related to the severity of paraquat poisoning. After TLR activation, downstream adaptor proteins are recruited to initiate TLR signal transduction. 13
Of these adaptor proteins, MyD88 is an important member [24,25,26]: After activation, MyD88 may induce the phosphorylation of interleukin receptor–associated kinases 1 and 4 (IRAK-1 and IRAK-4), leading to their activation. Thereafter, in the presence of TNF receptor associated factor 6, the NF-κB complex is phosphorylated, leading to the release of p-p65 into the nucleus, which induces the expression of a series of downstream genes (such as TNF-α and IL-6) [23,24]. In the present study, the results showed that TLR4 and TLR9 protein expression increased significantly after paraquat poisoning, and a similar trend was observed in downstream MyD88/p-p65, which are consistent with those of a previous report [23]. In addition, the changes in lung injury and inflammatory cytokines were similar. These findings indicate that paraquat poisoning activates TLR/MyD88/p-p65, leading to ALI. In addition, in MyD88 KO mice, TLR4 and TLR9 expression was not downregulated, but the expression of downstream MyD88/p-p65 and inflammatory cytokines was markedly downregulated, accompanied by attenuation of ALI. This indicates that, after paraquat poisoning, TLR4 and TLR 9 are activated in mice with MyD88 gene knockout, but the deficiency of MyD88 blocks the activation of downstream factors and fails to induce the release of inflammatory cytokines, including NF-κB, which finally attenuates ALI. MyD88 as an adaptor plays an important role in both innate immunity and acquired immunity [27]. However, in the present study, MyD88
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gene knockout did not cause adverse effects in mice, which is consistent with the findings of a previous report [28]. Studies have shown that the paraquat concentration peaks in the lungs at about 10–16 h after paraquat
poisoning
[29], and fatal paraquat
poisoning usually causes
obvious
pathophysiological changes within 48 h [30,31]. Thus, assays were conducted at 48 h after paraquat poisoning. Our results also confirmed that mice with paraquat poisoning developed evident ALI by 48 h after poisoning. However, paraquat poisoning causes not only ALI but also may induce pulmonary interstitial fibrosis, which is a major cause of death in the nonacute phase of paraquat poisoning [32]. The present study focused on ALI and did not investigate pulmonary interstitial fibrosis after paraquat poisoning. Thus, the role of MyD88 in pulmonary interstitial fibrosis requires further elucidation in future studies.
Conclusions Our results indicate that paraquat poisoning may activate the NF-κB pathway in a MyD88-dependent pathway that increases the expression and release of TNF-α and IL-1, resulting in ALI. MyD88 gene knockout inhibits the activation of NF-κB, and thereafter the expression and release of TNF-α and IL-1 are also reduced, leading to the attenuation of ALI. In sum, MyD88 mediates paraquat poisoning–induced ALI, and MyD88 gene knockout can attenuate paraquat poisoning–induced ALI, which suggests new strategies in the treatment of paraquat poisoning. 15
Conflict of interest The authors have declared no conflicts of interest.
Funding The design of the study and collection, analysis, and interpretation of data and writing of this manuscript were supported by Science Foundation of Liaoning Education Department (No. LK201633& LK201603) and Peking Union Medical Foundation-Ruiyi Emergency Medical Research Fund(No. R2015021) and Provincial Natural Science Foundation of Liaoning(No. 201602879).
Acknowledgements We gratefully acknowledge Hongyu Zhao and Xiaowei Wei for their intellectual support and technical assistance.
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References [1] Kan B, Jian X, Zhang Z, et al. Whole lung lavage therapy: Treatment for lung injury caused by paraquat poisoning [J]. Journal of Toxicology and Environmental Health Sciences. 2014:
6(4): 99-102.
[2] Dunne A, O'Neill LAJ. The interleukin-1 receptor/Toll-like receptor superfamily: signal transduction during inflammation and host defense [J]. Sciences Stke Signal Transduction Knowledge Environment. 2003; 2003(171):re3-re3. [3] Wang NA, Su Y, Che XM, et al. Penehyclidine ameliorates acute lung injury by inhibiting Toll-like receptor 2/4 expression and nuclear factor-κB activation [J]. Experimental & Therapeutic Medicine. 2016; 11(5):1827-1832. [4] Madathilparambil SV, Thomas B, Dolgachev VA, et al. Toll Like Receptor-9 (TLR9) is Requisite for Acute Inflammatory Response and Injury Following Lung Contusion [J]. Shock. 2016; 46(4):412-419. [5] Ferreira TP, Mariano LL, Ghilossobortolini R, et al. Potential of PEGylated Toll-Like Receptor 7 Ligands for Controlling Inflammation and Functional Changes in Mouse Models of Asthma and Silicosis [J]. Frontiers in Immunology. 2016; 7(10):95.
17
[6] Rhee SH. Basic and translational understandings of microbial recognition by toll-like receptors in the intestine [J]. Journal of Neurogastroenterology & Motility. 2011; 17(1):28-34. [7] Herold S, Gabrielli NM, Vadász I. Novel concepts of acute lung injury and alveolar-capillary barrier dysfunction [J]. Am J Physiol Lung Cell Mol Physiol. 2013; 305(10):L665-L681. [8] Hemmi H, Takeuchi O, Kawai T, et al. A Toll-like receptor recognizes bacterial DNA. Nature. 2000; 408(6813):740-745. [9] Ma L, Zhao G, Hua C, et al. Down-Regulation of TLR9 Expression Affects the Maturation and Function of Murine Bone Marrow-Derived Dendritic Cells Induced by CpG [J]. Cellular & molecular immunology. 2009; 6(3):199-205. [10] Nadigel J, Préfontaine D, Baglole C J, et al. Cigarette smoke increases TLR4 and TLR9 expression and induces cytokine production from CD8+ T cells in chronic obstructive pulmonary disease [J]. Respiratory Research. 2011; 12(10):1214-1216. [11] Erroi A, Bianchi M, Ghezzi P. The pneumotoxicant paraquat potentiates IL-1 and TNF production by human mononuclear cells [J]. Agents & Actions. 1992; 36(1-2):66-69.. [12] Li G, Yuzhen L, Yi C, et al. DNaseI protects against Paraquat-induced acute lung injury and pulmonary fibrosis mediated by mitochondrial DNA [J]. BioMed research international. 2015; 2015:386952. 18
[13] Mikawa K, Nishina K, Takao Y, et al. ONO-1714,a nitric oxide synthase inhibitor,attenuates endotoxin-induced acute lung injury in rabbits [J]. Anesthesia & Analgesia. 2003; 97(6):1751-1755． [14]Gawarammana IB, Buckley NA. Medical management of paraquat ingestion [J]. British Journal of Clinical Pharmacology. 2011; 72(5):745-57. [15]Bertram A, Haenel S S, Hadem J, et al. Tissue concentration of paraquat on day 32 after intoxication and failed bridge to transplantation by extracorporeal membrane oxygenation therapy [J]. Bmc Pharmacology & Toxicology. 2013; 14(1): 45. [16]Li X, Qin J. Modulation of Toll–interleukin 1 receptor mediated signaling [J]. Journal of Molecular Medicine. 2005; 83(4):258-266. [17]Ngo VN, Young RM, Schmitz R, et al. Oncogenically active MYD88 mutations in human lymphoma [J]. Nature. 2011; 470(7332):115-9. [18]Takeuchi O, Akira S. MyD88 as a bottle neck in Toll/IL-1 signaling.[M]// Toll-Like Receptor Family Members and Their Ligands. Springer Berlin Heidelberg, 2002:155-167. [19]Bechelli J, Smalley C, Zhao X, et al. MyD88 Mediates Instructive Signaling in Dendritic Cells and Protective Inflammatory Response during Rickettsial Infection [J]. Infection & Immunity. 2016; 84(4):883-93
19
[20]Ye X, Kong D, Wang J, et al. MyD88 contributes to neuroinflammatory responses induced by cerebral ischemia/reperfusion in mice [J]. Biochemical & Biophysical Research Communications. 2016; 480(1):69-74. [21]Poole JA, Wyatt TA, Romberger DJ, et al. MyD88 in lung resident cells governs airway inflammatory and pulmonary function responses to organic dust treatment [J]. Respiratory Research. 2015; 16(1):1-12. [22]Liu W, Shan LP, Dong XS, et al. Toll-like receptor 4 implicated in acute lung injury induced by paraquat poisoning in mice [J]. International Journal of Clinical & Experimental Medicine. 2014; 7(10):3392-3397. [23]Qian J, Liu L, Chen L, et al. Increased toll-like receptor 9 expression is associated with the severity of paraquat-induced lung injury in mice [J]. Human & Experimental Toxicology. 2015; 34(4):430-8. [24]Kayama H, Ramirezcarrozzi VR, Yamamoto M, et al. Class-specific regulation of pro-inflammatory genes by MyD88 pathways and IκBζ [J]. Journal of Biological Chemistry. 2015; 290(8):12468-12477. [25]Jiang
Q,
Yi
M,
Guo
Q,
et
al.
Protective
effects
of
polydatin
on
lipopolysaccharide-induced acute lung injury through TLR4-MyD88-NF-κB pathway [J]. International Immunopharmacology. 2015; 29(2):370-6.
20
[26]Adachi O, Kawai T, Takeda K, et al. Targeted Disruption of the MyD88, Gene Results in Loss of IL-1- and IL-18-Mediated Function [J]. Immunity. 1998; 9(1):143–150. [27]Huang X, Yang Y. Targeting the TLR9-MyD88 pathway in the regulation of adaptive immune responses [J]. Expert Opinion on Therapeutic Targets. 2010; 14(8):787-96. [28]Janssens S, Beyaert R. A universal role for MyD88 in TLR/IL-1R-mediated signaling [J]. Trends in Biochemical Sciences. 2002; 27(9):474-482. [29] Senarathna L, Eddleston M, Wilks MF, et al. Prediction of outcome after paraquat poisoning by measurement of the plasma paraquat concentration [J]. Qjm Monthly Journal of the Association of Physicians. 2009; 102(4):251-259. [30]Mohammadi-Bardbori A, Ghazi-Khansari M. Alternative electron acceptors: Proposed mechanism of paraquat mitochondrial toxicity [J]. Environmental Toxicology & Pharmacology. 2008; 26(1):1-5. [31]Wu N, Shen H, Liu H, et al. Acute blood glucose fluctuation enhances rat aorta endothelial cell apoptosis, oxidative stress and pro-inflammatory cytokine expression in vivo [J]. Cardiovascular Diabetology. 2016; 15(1):109. [32]Eddleston M, Wilks MF, Buckley NA. Prospects for teatment of paraquat-induced lung fibrosis with immunosuppressive drugs and the need for better prediction of outcome: A systematic review [J]. Qjm Monthly Journal of the Association of Physicians. 2003; 96(11):809-24. 21
Figure 1 Lung wet/dry (W/D) ratio and lung pathology score. A: control group; B: paraquat group; C: MyD88 knockout (KO) plus paraquat group; D: MyD88 KO group. #P < 0.05 vs. Group A; *P < 0.05 vs. Group B. n=6-8.
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Figure 2 Serum TNF-α and IL-1β contents (ELISA). A: control group; B: paraquat group; C: MyD88 knockout (KO) plus paraquat group; D: MyD88 KO group. #P < 0.05 vs. Group A; *P < 0.05 vs. Group B. n=6-8.
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Figure 3 mRNA expression of MyD88, TNF-α, and IL-1β in the lungs (RT-PCR). A: control group; B: paraquat group; C: MyD88 knockout (KO) plus paraquat group; D: MyD88 KO group. #P < 0.05 vs. Group A; *P < 0.05 vs. Group B. n=6-8.
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Figure 4 Expression and distribution of MyD88 and p-p65 in the lungs (immunofluorescence staining). A: control group; B: paraquat group; C: MyD88 knockout (KO) plus paraquat group; D: MyD88 KO group. n=6-8.
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Figure 5 TLR4, TLR9, MyD88, and p-p65 expression in the lungs (Western blotting). A: control group; B: paraquat group; C: MyD88 knockout (KO) plus paraquat group; D: MyD88 KO group. n =6- 8.
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