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Atractylodin attenuates lipopolysaccharide-induced acute lung injury by inhibiting NLRP3 inflammasome and TLR4 pathways Q2
Fayin Tang, Kefeng Fan, Kunli Wang, Chuanzhou Bian* College of Pharmaceutical Engineering, Henan University of Animal Husbandry and Economy, Zhengzhou, 450046, Henan Province, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 August 2017 Received in revised form 16 November 2017 Accepted 27 November 2017 Available online xxx
Acute lung injury (ALI) arises from uncontrolled pulmonary inflammation with high mortality rates. Atractylodin (Atr) is a polyethylene alkynes and has been reported to possess anti-inflammation effect. Thus, we aimed to investigate the protective effect of Atr on lipopolysaccharide (LPS)-induced inflammatory responses ALI. The results indicated that Atr treatment not only significantly attenuated LPSstimulated histopathological changes but also lessened the myeloperoxidase (MPO) activity, the wetto-dry weight ratio of the lungs, protein leakage and infiltration of inflammatory cells. Moreover, Atr inhibited the tumor necrosis factor (TNF)-a, interleukin (IL)-6, IL-1b and monocyte chemoattractant protein (MCP)-1 secretion in BALF. Further study demonstrated that such inhibitory effects of Atr were due to suppression of nucleotide-binding domain-(NOD-) like receptor protein 3 (NLRP3) inflammasome and toll like receptor 4 (TLR4) activation, likely contributing to its anti-inflammatory effects. Collectively, these findings suggest that Atr may be an effective candidate for alleviating LPS-induced inflammatory responses. © 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Atractylodin Inflammation Acute lung injury NLRP3 TLR4
1. Introduction Acute lung injury (ALI) or its more serious form, respiratory distress syndrome (ARDS), is an acute and life-threatening disease, which results from various factors, including aspiration, pneumonia, shock and sepsis, and still keeps high mortality rates.1,2 Increasing experimental or/and clinical evidences shows that inflammation plays a crucial role in the occurrence and development of ALI.3,4 Lung inflammation arises from overwhelming inflammatory cytokines release further leading to tissues of lung injury.5,6 Accordingly, the development of suppressing inflammation may be an effective approach of prevention and treatment ALI. Lipopolysaccharide (LPS), a major cell wall component of the Gram-negative bacteria, results in a disturbance in the immune and inflammatory responses, and is commonly recognized to induce pharmacological research models of ALI.7,8 Abundant researches indicated that LPS could induce toll like receptor 4 (TLR4) activation
* Corresponding author. College of Pharmaceutical Engineering, Henan University of Animal Husbandry and Economy, Longzi Hubei Road 6#, Zhengzhou, 450046, Henan Province, China. E-mail addresses:
[email protected],
[email protected] (C. Bian). Peer review under responsibility of Japanese Pharmacological Society.
and subsequently leads to the activation of factor-kappa B (NF-kB) and mitogen-activated protein kinases (MAPKs) consisted of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38, which cause the overproduction of proinflammatory cytokines such as tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), interleukin-6 (IL-6).9,10 Moreover, previous study suggested monocyte chemoattractant protein (MCP)-1, an activating factor, involved in the LPS-induced ALI animal model.11 On the one hand, NF-kB, a key pro-inflammatory transcription factor, is remained in the cytoplasm by an inhibitor of kB (IkB) protein. However, LPS could induce the phosphorylation and rapid degradation of IkB, which results from NF-kB detaching from IkB, enabling NF-kB dimers to migrate to the nucleus and activate inflammatory responses.12,13 On the other hand, MAPKs also plays a significant role in the regulation of inflammatory responses and its activation induced by LPS could result in excessive TNF-a secretion.14,15 Moreover, nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome has been proved to play an essential role in the inflammation responses during ALI.16 Hence, the inhibition of NLRP3 inflammasome and TLR4-NF-kB and -MAPKs signaling pathway activation may contribute to suppression inflammatory responses for the amelioration of ALI.
https://doi.org/10.1016/j.jphs.2017.11.010 1347-8613/© 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Tang F, et al., Atractylodin attenuates lipopolysaccharide-induced acute lung injury by inhibiting NLRP3 inflammasome and TLR4 pathways, Journal of Pharmacological Sciences (2018), https://doi.org/10.1016/j.jphs.2017.11.010
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Atr attenuation of LPS-induced ALI was associated with the suppression of NLRP3 inflammasome and TLR4 signaling pathways. 2. Materials and methods 2.1. Reagents and chemical
Fig. 1. Chemical structure of Atractylodin (Atr).
Accumulating evidence indicates that abundant natural products were widely accepted to possess various pharmacological activities, such as antioxidant and antiinflammatory properties.17 Atractylodis Rhizoma (Cangzhu), a traditional herbal medicine, contains a verity of essential component, including sesquiterpenes, phenolic acids, and polyethylene alkynes.18 Atractylodin (Atr, Fig. 1), is a polyethylene alkynes extracted from A. Rhizoma and has been reported to ameliorate intestinal inflammation and cooccurring dysmotility in rats and inhibit IL-6 by blocking (nucleophosmin-anaplastic lymphoma kinase) NPM-ALK and MAPKs activation in HMC-1.19,20 In the present study, we explored whether
Atractylodin (Atr), purity >98%, was supplied by the Chengdu Herbpurify CO., LTD (Chengdu, China). LPS (Escherichia coli 055:B5) and dimethyl sulfoxide (DMSO) were purchased from SigmaeAldrich (St. Louis, MO, USA). Penicillin and streptomycin, Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were obtained from Invitrogen-Gibco (Grand Island, NY). Antibodies against NLRP3, caspase-1, ASC, IL-1b, TLR4, P-p65/p65, P-JNK/JNK, P-ERK/ERK, P-p38/p38, P-IkBa/IkBa, Lamin B and b-actin were offered from Cell Signaling (Boston, MA, USA) or Abcam (Cambridge, MA, USA). MPO test kits were supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other chemicals were offered by SigmaeAldrich (St. Louis, MO, USA), if not otherwise indicated. 2.2. Animals Male BALB/c mice, 6e8 weeks, weighing approximately 18e20 g, were obtained from the Zhengzhou University Animal
Fig. 2. Effects of Atr on histopathological changes of lung tissues in LPS-induced ALI mice. Atr (40 or 80 mg/kg) was administered intraperitoneally to mice 1 h prior to an intranasal administration of LPS (0.5 mg/kg). (A) Lungs (n ¼ 5) from each experimental group was processed for histological evaluation at 12 h after LPS challenge: (a) Control, (b) Atr only group, (c) LPS (0.5 mg/kg), (d) LPS þ Atr (40 mg/kg), and (e) LPS þ Atr (80 mg/kg). The lung injury score (B) was graded according to a five-point scale from 0 to 4 as follows: 0, l, 2, 3, and 4 represent no damage, mild damage, moderate damage, severe damage, and very severe damage, respectively. Representative histological section of the lungs was stained by hematoxylin and eosin (H & E staining, magnification 100). All data are presented as means ± SEM (n ¼ 5 in each group). ##p < 0.01 vs. Control group; *p < 0.05 and ** p < 0.01 vs. LPS group.
Please cite this article in press as: Tang F, et al., Atractylodin attenuates lipopolysaccharide-induced acute lung injury by inhibiting NLRP3 inflammasome and TLR4 pathways, Journal of Pharmacological Sciences (2018), https://doi.org/10.1016/j.jphs.2017.11.010
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Fig. 3. Effects of Atr treatment on LPS-induced cytokines generation in BALF. After LPS challenge for 12 h, all mice were euthanized and their BALF was harvested to measure levels of cytokines TNF-a, IL-6, IL-1b and MCP-1 secretion by ELISA. All data are presented as means ± SEM (n ¼ 5 in each group). ##p < 0.01 vs. Control group; *p < 0.05, **p < 0.01 vs. LPS group.
Experiment Center (Henan, China). All animals were housed in a room with temperature at 24 ± 1 C, a 12 h lightedark cycle and relative humidity about 40e80%. All studies were in accordance with the International Guiding Principles for Biomedical Research Involving Animals, which was published by the Council for the International Organizations of Medical Sciences. 2.3. Experimental protocol To induce the ALI model, the mice (n ¼ 25, 5/group) were randomly divided into five groups: Control (saline þ 0.5% DMSO), Atr only (80 mg/kg, dissolved in 0.5% DMSO), LPS only (0.5 mg/kg, dissolved in saline) and Atr (40 or 80 mg/kg) þ LPS, were administered intraperitoneally. After 1 h, the mice were anesthetized with diethyl ether, and LPS was administered intranasally (i.n.) to induce lung injury. After LPS administration for 12 h, the animals were euthanized. Subsequently, lung tissue samples and the bronchoalveolar lavage fluid (BALF) were collected and used for hematoxylin and eosin (H & E) staining, Western blot assay, enzymelinked immunosorbent assay (ELISA) and flow cytometry assay.
2.5. Histopathological evaluation The left lungs of mice were excised at 12 h after the LPS challenge. A histopathological examination was performed on the mice that were not subjected to BALF collection. The lung tissue samples were immersed in normal 10% neutral buffered formalin and fixed for 48 h, dehydrated in a series of graded ethanol, embedded in paraffin wax, and cut into 5-mm-thick sections. The paraffinembedded sections were stained with hematoxylin and eosin (H & E) for pathological analysis. The histological changes were evaluated by a point-counting method for severity of lung injury using an ordinal scales in accordance with the methods as previous described.21 Briefly, the sections were assessed by the airway epithelial necrosis, intra-alveolar edema, hyaline membranes, hemorrhage, and the recruitment of inflammatory cells to the air space. The lung injury score was graded according to a four-point scale from 0 to 3 as follows: 0, l, 2, and 3 represent no damage, mild damage, moderate damage, and very severe damage, respectively. 2.6. Measurement of MPO in lung tissues
2.4. Cell counting and protein concentration assay in bronchoalveolar lavage fluid (BALF) After LPS administration for 12 h, all mice were euthanized before their BALF was collected; lavages were performed three times in each animal through a tracheal cannula with autoclaved PBS to obtain a total volume of up to 1.3 ml. The BALF samples were centrifuged to pellet the cells. The sedimented cells were resuspended in PBS to obtain the total cell, neutrophil and macrophage counts using a hemocytometer and for cytosine staining using the WrighteGiemsa staining method. In addition, the BALF samples were centrifuged, and their protein concentrations were determined using a BCA protein assay kit (Beyotime, China).
All mice were sacrificed using diethyl ether anesthesia, and the right lungs were excised after 12 h of LPS-administration. The lung tissues were homogenized and dissolved in extraction buffer for the analysis of MPO activities. To examine the accumulation of neutrophils and level of lipid peroxidation in the lung tissue, MPO content was assessed using commercially available assay kits in accordance with the respective manufacturer's instructions. 2.7. Lung wet/dry (W/D) ratios Lung samples were obtained 12 h after LPS stimulation, blotted dry and weighed immediately after removal (wet weight) before
Please cite this article in press as: Tang F, et al., Atractylodin attenuates lipopolysaccharide-induced acute lung injury by inhibiting NLRP3 inflammasome and TLR4 pathways, Journal of Pharmacological Sciences (2018), https://doi.org/10.1016/j.jphs.2017.11.010
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being subjected to desiccation in an oven at 80 C for 48 h to obtain the ‘dry’ weight. The ratio of wet lung weight to dry lung weight was calculated to assess tissue edema.
compared with the control group. In contrast, Atr (40 or 80 mg/kg) pretreatment remarkably inhibited TNF-a, IL-1b, IL-6, and MCP-1 secretion in a dose-dependent manner (Fig. 3).
2.8. ELISA assay
3.3. Atr treatment lessened lung dry-to-wet (W/D) ratio and amount of protein in BALF of LPS-induced ALI mice
The BALF was obtained from each sample in vivo, centrifuged, collected supernatants for measurement of the MCP-1, TNF-a, IL-6, and IL-1b secretion using an enzyme-linked immunosorbent assay (ELISA) kit as the manufacturer's instructions (BioLegend, Inc., CA, USA), respectively. The optical density from each well was detected at 450 nm. 2.9. Isolation of nuclear and cytosolic fractions The cytoplasmic and the nuclear extracts were prepared using an NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Pierce Biotechnology, Rockford, IL, USA), following the manufacturer's instructions. All steps were performed from on ice or at 4 C. 2.10. Western blot analysis Lung tissue samples or cells were lysed in a RIPA buffer with protease and phosphatase inhibitors for 30 min. The protein concentrations were measured using a BCA protein assay kit (Beyotime, China), and 40 mg of proteins were electrophoretically transferred onto a PVDF membrane following separation on a 10% SDS-polyacrylamide gel. The membrane was blocked with blocking solution (5% (w/v) nonfat dry milk) for 1 h, followed by an overnight incubation at 4 C with a specific primary antibody. The following day, the membrane was incubated for an additional 1 h with HRP-conjugated secondary antibody (1:3000 dilution) at room temperature after thoroughly washing three times with PBST.
Due to W/D ratios were regarded as reflecting pulmonary edema in lung tissue, the W/D ratio of lung was measured in our studies. As shown in Fig. 4A, the W/D ratio of LPS-induced lungs was significantly higher than that of the control group. However, pretreatment with Atr (40 or 80 mg/kg) dramatically decreased the W/D ration when administered intraperitoneally 1 h prior to LPS challenge. Additionally, the protein leakage in BALF is a character of capillary permeability increase resulted from LPS. Indeed, a dramatical rise of total protein concentration in BALF was found in LPS group, whereas such rise was inhibited by Atr treatment (Fig. 4B). 3.4. Atr treatment decreased the total cell number, neutrophils and MPO levels in LPS-induced ALI mice Furthermore, to explore the effects of Atr on inflammatory cell infiltration, including neutrophils and macrophages, the numbers of inflammatory cells of BALF were observed by WrighteGiemsa
2.11. Statistical analysis All data referenced above were expressed as the means ± SEM and analyzed using SPSS19.0 (IBM). Comparisons between experimental groups were conducted using one-way ANOVA, whereas multiple comparisons were made using Turkey's test. Statistical significance was defined as p < 0.05 or p < 0.01. 3. Results 3.1. Atr treatment improved histological changes of lung tissues in LPS-induced ALI mice The histological changes were observed to evaluate lung tissue damage induced by LPS. As illustrated in Fig. 2A, LPS induced obviously pathologic changes by increasing tissue interstitial edema, accumulation of inflammatory cells and pulmonary hemorrhage (Fig. 2c), when compared with control group (Fig. 2a) or Atr treatment alone group (Fig. 2b). However, LPS-induced severe histopathological changes were significantly weakened by pretreatment of Atr (40 or 80 mg/kg) (Fig. 2d and e), which was assessed by the lung injury score (Fig. 2B). 3.2. Atr treatment inhibited LPS-induced pro-inflammatory cytokines secretion in BALF To further assess whether Atr possesses an anti-inflammation activity in LPS-induced ALI, we used ELISA kits to detect the secretion of TNF-a, IL-1b, IL-6 and MCP-1. In consistent with abundant previous reports, our results indicated that LPS challenge effectively induced the generation of TNF-a, IL-1b, IL-6 and MCP-1
Fig. 4. Effects of Atr treatment on LPS-induced lung W/D ratio and amount of protein in BALF. Atr (40 or 80 mg/kg) was administered intraperitoneally to mice 1 h prior to an intranasal administration of LPS, BALF and lungs of mice were harvested. (A) Effects of Atr on levels of the lung W/D ratio and (B) total protein concentration in the BALF. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n ¼ 5 in each group). ##p < 0.01 vs. Control group; * p < 0.05 and **p < 0.01 vs. LPS group.
Please cite this article in press as: Tang F, et al., Atractylodin attenuates lipopolysaccharide-induced acute lung injury by inhibiting NLRP3 inflammasome and TLR4 pathways, Journal of Pharmacological Sciences (2018), https://doi.org/10.1016/j.jphs.2017.11.010
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staining method. In our studies, LPS challenge obviously enhanced the number of total cells, macrophages and neutrophils compared with the control group. Conversely, pretreatment with Atr (40 or 80 mg/kg) efficiently reduced the number of total cells and neutrophils, whereas this effect did not exhibit in macrophages (Fig. 5A). Moreover, MPO, a functional biomarker of neutrophils, was measured to evaluate the neutrophil accumulation in lung. Our results indicated that the MPO activity was evidently enhanced in the LPS group when compared with the control group. However, Atr treatment effectively reduced the level of MPO (Fig. 5B).
Atr, its roles in TLR4-MAPK and -NF-kB signaling pathway activation were measured by Western blot analysis. As shown in Fig. 6A and B, pretreatment with Atr remarkably inhibited the expression of TLR4 protein induced by LPS challenge for 12 h. Moreover, Atr pretreatment significantly suppressed JNK, ERK and p38 phosphorylation (Fig. 6CeH) and apparently blocked IkBa phosphorylation and degradation, decreased the nuclear translocation of NFkB (p65) in LPS-induced ALI (Fig. 7). Taken together, the above findings determined involvement of the TLR4-MAPK and -NF-kB pathways in the anti-inflammatory effect of Atr in LPS-induced ALI.
3.5. Atr treatment suppressed LPS-activated TLR4-MAPK and -NFkB signaling pathways in ALI mice
3.6. Atr treatment inhibited LPS-induced NLRP3 inflammasome activation in ALI mice
TLR4, which is a key regulator, could regulate MAPK and NF-kB signaling pathway involved in inflammatory responses. Accordingly, to further investigated the anti-inflammatory mechanism of
Importantly, NLRP3 inflammasome activation also is essential for regulating inflammation mediators' expression, which is related to LPS-induced ALI. In the present study, Western blot analysis
Fig. 5. Effects of Atr treatment on LPS-induced inflammatory cells and MPO activity. Atr (40 or 80 mg/kg) was administered intraperitoneally to mice 1 h prior to an intranasal administration of LPS (0.5 mg/kg), BALF and lungs of mice were harvested. (A) The total counts of cells, macrophages and neutrophils from the BALF were counted using a hemocytometer, and the WrighteGiemsa staining method was used for cytosine staining. (B) MPO activity in lung tissues was measured at 12 h after LPS challenge. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n ¼ 5 in each group). ##p < 0.01 vs. Control group; *p < 0.05 and **p < 0.01 vs. LPS group.
Please cite this article in press as: Tang F, et al., Atractylodin attenuates lipopolysaccharide-induced acute lung injury by inhibiting NLRP3 inflammasome and TLR4 pathways, Journal of Pharmacological Sciences (2018), https://doi.org/10.1016/j.jphs.2017.11.010
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Fig. 6. Effects of Atr treatment on LPS-activated TLR4-MAPK signal pathways in ALI mice. Atr (40 or 80 mg/kg) was administered intraperitoneally to mice 1 h prior to an intranasal administration of LPS (0.5 mg/kg) and lungs of mice were harvested. Protein samples were extracted from the lungs and analyzed by Western blot. Quantification of relative expression of (A and B) TLR4/b-actin, (C and D) P-JNK/JNK, (E and F) P-ERK/ERK, and (G and H) P-p38/p38 were performed by densitometric analysis. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n ¼ 5 in each group). ##p < 0.01 vs. Control group; *p < 0.05 and **p < 0.01 vs. LPS group.
Fig. 7. Effects of Atr treatment on LPS-activated TLR4-NF-kB signal pathways in ALI mice. Atr (40 or 80 mg/kg) was administered intraperitoneally to mice 1 h prior to an intranasal administration of LPS (0.5 mg/kg) and lungs of mice were harvested. (A and B) The protein levels of IkBaand P-IkBa, as well as (C and D) the nuclear and cytoplasmic levels of NF-kB (p65) were analyzed by Western blot. The relative densities of protein were performed by densitometric analysis; b-actin and Lamin B were used acted as an internal control, respectively. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n ¼ 5 in each group). ##p < 0.01 vs. Control group; **p < 0.01 vs. LPS group.
Please cite this article in press as: Tang F, et al., Atractylodin attenuates lipopolysaccharide-induced acute lung injury by inhibiting NLRP3 inflammasome and TLR4 pathways, Journal of Pharmacological Sciences (2018), https://doi.org/10.1016/j.jphs.2017.11.010
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discovered that LPS significantly induced NLRP3, ASC, caspase-1 and IL-1b protein expression, which was inhibited by Atr pretreatment (Fig. 8). Accordingly, Atr treatment reduced mature IL-1b levels via inhibition of the NLRP3 inflammasome, which plays a vital role in inhibiting LPS-induced inflammation. 4. Discussion Inflammation is a critical component of the innate immune responses protecting from tissue damage, whereas inflammation gives rise to tissue damage in an unbalanced condition.22 Importantly, the pathogenesis of acute lung injury (ALI) also attributes to prolonged or overwhelmed inflammatory processes which results in disruption of the lung tissues.23 Accordingly, the agent that controls overdevelopment of inflammatory responses may be an effective candidate for prevention and/or treatment ALI. Interestingly, phytochemicals have been regarded as possessing a variety of pharmacological activities, including anti-inflammatory, antioxidative, anti-rheumatic, anti-cancer and anti-microbial, particularly anti-inflammatory.24,25 Atractylodin (Atr), as one of
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phytochemicals extracted from A. Rhizoma, has been reported to inhibit inflammatory responses in vivo and in vitro.19,20 In the present study, our results demonstrated that the protective effect of Atr on ALI induced by LPS occurs likely due to its anti-inflammatory property. Accumulating evidences indicates that ALI is characterized by extensive infiltration of neutrophils, release of inflammatory mediators, and disruption of endothelial and epithelial integrity and edema of the lungs.26 LPS-induced mouse ALI model is ideally accepted to imitate the pathophysiological events observed in ALI patients.27 In our studies, pulmonary morphological examination showed that LPS obviously induced pulmonary edema, coagulation and inflammatory cell infiltration, whereas these histopathologic changes of lung tissues were significantly alleviated by Atr pretreatment. On the one hand, because an increased pulmonary permeability and lung edema are the representative symptoms of lungs in LPS-induced ALI,28 we further evaluated the lung W/D ratio and the amount of protein leakage in the BALF. Our investigation showed that Atr effectively decreased the lung W/D ratio, providing evidence of the protective effect of Atr in ALI. On the other hand, a
Fig. 8. Effects of Atr treatment on LPS-induced NLRP3 inflammasome activation in ALI mice. Atr (40 or 80 mg/kg) was administered intraperitoneally to mice 1 h prior to an intranasal administration of LPS (0.5 mg/kg) and lungs of mice were harvested. Protein samples were extracted from the lungs and analyzed by Western blot with specific antibodies. (AeF) Protein expressions of NLRP3, ASC, caspase-1 and IL-1b were measured by Western blot analysis. Quantification of relative protein expression was performed by densitometric analysis and b-actin was acted as an internal control. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n ¼ 5 in each group). ##p < 0.01 vs. Control group; *p < 0.05 and **p < 0.01 vs. LPS group.
Please cite this article in press as: Tang F, et al., Atractylodin attenuates lipopolysaccharide-induced acute lung injury by inhibiting NLRP3 inflammasome and TLR4 pathways, Journal of Pharmacological Sciences (2018), https://doi.org/10.1016/j.jphs.2017.11.010
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Fig. 9. Scheme summarizing the protective effects of Atr on LPS-induced acute lung injury via suppressing activation of TLR4-NF-kB and -MAPK pathway and the NLRP3 inflammasome.
well-characterized model of LPS-stimulated ALI is related to accumulation of neutrophils and activation of macrophages in the lungs, which promote release of inflammatory cytokines and cells, resulting in increase of alveolar-capillary permeability.29 In the current study, Atr could dramatically inhibit the increases in total cells and neutrophils in the BALF that were present in LPSchallenged lungs. MPO, which exists mainly in the primary granules of neutrophils, has been reported to involve in tissues damage in various diseases, including ALI.30 Our data revealed that pretreatment with Atr efficiently lessened the LPS-induced increase in MPO levels in lung tissues. Moreover, numerous studies have suggested that the inflammatory cytokine networks are crucial for initiating and exaggerating the lung injury process.31 TNF-a, IL-1b, IL-6, and MCP-1, as three important pro-inflammatory cytokines, have been reported to be associated with the development of ALI.11,32,33 Taken together, these investigations indicated that Atr is able to attenuate LPS-induced lung injury via inhibiting inflammatory responses. More importantly, these inflammatory cytokines secretion are involved in two major signal pathway activation, including TLR4 and NLRP3 inflammasome, which are generally required for the development of ALI.34,35 On the one hand, in monocytes, LPS, as the main ligand, binds to TLR4 to result in inflammatory responses by coordinating sequence of binding events between soluble and cell membrane proteins including myeloid differentiation-2 (MD-2) protein, LPS-binding protein (LBP), and CD14. Meanwhile, TLR4 signaling needs to other binding partners, such as myeloid differentiation factor 88 (MyD88), an adapter and NF-kB, a transcription factor.36 In addition, CD14, as an important LPS co-receptor, plays a
pivotal role in the initial binding of LPS and transferring of LPS to the MD2/TLR4 complex to initiate signal signaling cascades.37 Previous reports have suggested that LPS could induce TLR4 activation to further motivate NF-kB and MAPK signaling pathways, resulting in overproduction of inflammatory cytokines.33 Indeed, our results discovered that LPS could significantly activated TLR4, NF-kB, and MAPK signaling pathways, whereas these effects were obviously inhibited by Atr treatment via inhibiting TLR4 protein expression, NF-kB nuclear transcription, blocking the phosphorylation and subsequent degradation of IkB, and suppressing phosphorylation of JNK, ERK and p38. On the other hand, Tamura et al indicated that the NLRP3/ASC/caspase-1 signaling pathway is downstream of TLR4.38 NLRP3 inflammasome, which is comprised of NOD like receptor (NLRP3), the adapter protein ASC and Caspase1, is an important intracellular multiprotein inflammatory pathway.39 Upon diverse stimuli (microbial- and stress-substances, etc.), NLRP3 mediates the maturation of proinflammatory cytokines IL-1b by a cascade process of NLRP3 activation, ASC and procaspase-1 recruitment, caspase-1 activation and the subsequent process of pro-IL-1b.23,40,41 Consequently, to further explore the anti-inflammatory mechanism of Atr on LPS-induced ALI, the effects of Atr on NLRP3 signaling pathway were analyzed by western blotting. Our results revealed that Atr treatment efficiently inhibited NLRP3 signaling pathway. These results indicated that Atr exhibited its anti-inflammatory effects by inhibition of NLRP3 inflammasome and TLR4-mediated NF-kB and MAPK signaling pathways activation. In summary, as illustrated in Fig. 9, the results of this study firstly demonstrated that Atr alleviated LPS-induced ALI by the suppression of inflammatory cytokines generation, which was largely dependent on the inhibition of NLRP3 inflammasome and TLR4 signaling pathways activation. This study provides beneficial evidence that Atr might be useful as a potential therapeutic medication for the treatment and prevention of the LPS-induced ALI.
Conflict of interest The authors report no conflicts of interest.
Acknowledgements This work supported by Henan Province Natural Science Planning Fund Projects (No. 162300410128); Henan Province Science and Technology Open Cooperation Project (No. 172106000009) and Henan Province outstanding young teacher project (No. 2013GGJS191).
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