- Email: [email protected]
Protective effects of syringin against lipopolysaccharide-induced acute lung injury in mice
j o u r n a l o f s u r g i c a l r e s e a r c h m a r c h 2 0 1 7 ( 2 0 9 ) 2 5 2 e2 5 7
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Protective effects of syringin against lipopolysaccharide-induced acute lung injury in mice Ao Zhang, MM,a Zhongmin Liu, MM,a Lulu Sheng, MM,b and Hui Wu, MMb,* a b
Department of Intensive Care Unit, First Hospital of Jilin University, Changchun, China Department of Ophthalmology, First Hospital of Jilin University, Changchun, China
article info
abstract
Article history:
Background: Syringin, a major active substance isolated from Eleutherococcus senticosus, has
Received 25 June 2016
been found to have anti-inflammatory effect. The aim of this study was to investigate the
Received in revised form
effects and underlying mechanisms of syringin on lipopolysaccharide (LPS)-induced acute
27 September 2016
lung injury (ALI) in mice.
Accepted 27 October 2016
Methods: We established an LPS-induced ALI model in mice. We also detected the lung wet-
Available online 5 November 2016
to-dry ratio, myeloperoxidase activity, and inflammatory cytokines tumor necrosis factor alpha, interleukin (IL)-1b, and IL-6 to estimate the index of lung injury in mice. Further-
Keywords:
more, the expression of nuclear factor-erythroid 2-related factor-2 (Nrf2), heme oxygenase-
Syringin
1, and nuclear factor kB (NF-kB) was detected by Western blot analysis.
NF-kB
Results: The results showed that the increases in lung wet-to-dry ratio, myeloperoxidase
LPS
activity, malondialdehyde content, and levels of tumor necrosis factor alpha, IL-1b, and IL-
Lung injury
6 induced by LPS were significantly inhibited by treatment of syringin. The phosphoryla-
Nrf2
tion of IkB-a and p65 NF-kB caused by LPS was inhibited by syringin. Furthermore, syringin was found to upregulate the expression of Nrf2 and heme oxygenase 1. Conclusions: In conclusion, the results suggest that syringin protects against LPS-induced ALI by activating Nrf2 and inhibiting NF-kB signaling pathway. ª 2016 Elsevier Inc. All rights reserved.
Introduction Acute lung injury (ALI) is a critical illness syndrome with high morbidity and mortality in critically ill patients.1 ALI is characterized by severe lung inflammation and increased capillary permeability.2 Lipopolysaccharide (LPS) has been identified as the major factor that leads to ALI.3 In mice with ALI, LPS significantly upregulates the expression of inflammatory cytokines tumor necrosis factor (TNF)-a, interleukin (IL)-1b, and IL-6.4 Studies showed that these cytokines cause the
aggregation of neutrophilic leukocytes and lead to lung injury.5 Therefore, inhibition of these cytokines has drawn a great attention in the treatment of LPS-induced ALI. Nuclear factor kB (NF-kB) is a nuclear transcription factor, which plays an important role in the regulation of inflammatory cytokines.6,7 Inhibition of NF-kB activation has the ability to attenuate LPS-induced ALI.8,9 Syringin is a major active substance isolated from Eleutherococcus senticosus. It has been found that syringin has broad pharmacologic effects, such as anti-inflammatory and
* Corresponding author. Department of Ophthalmology, First Hospital Tel./fax: þ86043188782639. E-mail address: [email protected] (H. Wu). 0022-4804/$ e see front matter ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2016.10.027
of
Jilin
University,
Changchun
130021,
China.
zhang et al protective effects of syringin against lsp-induced ali in mice
253
antinociceptive effects.10 Syringin has been reported to protect against LPS and D-galactosamineeinduced hepatic failure.11 Syringin also inhibits LPS-induced TNF-a production in RAW264.7 cells.12 Furthermore, syringin has been reported to exert sleep-potentiating effects through the nitric oxide synthasesenitric oxide pathway.13 However, the effects of syringin on LPS-induced ALI have not been reported. Therefore, in this study, we investigated the effects of syringin on LPSinduced ALI in mice.
Materials and methods Reagents Mouse TNF-a, IL-6, and IL-1b enzyme-linked immunosorbent assay (ELISA) kits were purchased from BioLegend (San Diego, CA). The myeloperoxidase (MPO) determination kit was purchased by the Jiancheng Bioengineering Institute of Nanjing (Jiangsu, China). Mouse nuclear factor-erythroid 2-related factor-2 (Nrf2), HO-1, NF-kB p65, NF-kB p-p65, IkB, p-IkB, and b-actin were provided from Cell Signaling Technology Inc (Beverly, MA). Syringin was purchased from Chengdu Must Bio-technology Co, Ltd (Chengdu, China). LPS (Escherichia coli 055:B5) was purchased from Sigma Chemical Co (St. Louis, MO). All other chemicals were of reagent grade.
Animal
Fig. 1 e Effects of syringin on the lung W/D ratio of LPSinduced ALI mice. The values presented are the means ± standard error of the mean of three independent experiments. #P < 0.01 versus control group, *P < 0.05 and **P < 0.01 versus LPS group.
Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.
Hematoxylin and eosin staining Twelve hour after LPS treatment, the lung tissues were collected and fixed in 4% formaldehyde. The samples were
Male BALB/c mice of 18-22 g weight (Animals Experiment Center, Baiqiuen Medical College of Jilin University, Jilin, China) were housed in microisolator cages with controlled environment at a temperature of 22 C-25 C. All studies involving animals were performed according to the National Institutes of Health guide for the Care and Use of Laboratory Animal.
Study design and grouping Sixty mice were randomly divided into three groups: control group, LPS group, and LPS þ syringin (25, 50, and 100 mg/kg) groups. The mice of LPS group were received 50-mL LPS given intranasally to induce ALI. The mice of LPS þ syringin (25, 50, and 100 mg/kg) groups were given intraperitoneally to the mice 1 h after LPS treatment. The mice of control group were given equal amount of phosphate-buffered saline.
Lung wet-to-dry weight ratio Twelve hour after LPS treatment, the mice were condemned to death, and the lungs were collected to obtain the wet weight. Next, we obtained dry weight after keeping the lungs at 80 C for 48 h. The lung wet-to-dry (W/D) ratio was calculated by dividing the wet weight by the dry weight.
MPO and malondialdehyde assay Twelve hour after LPS treatment, the lung tissues were homogenized and the MPO and malondialdehyde (MDA) contents were measured by MPO determination kit (Nanjing
Fig. 2 e Effects of syringin on MPO and MDA contents. The values presented are the mean ± standard error of the mean of three independent experiments. #P < 0.01 versus control group, **P < 0.01 versus LPS group.
254
j o u r n a l o f s u r g i c a l r e s e a r c h m a r c h 2 0 1 7 ( 2 0 9 ) 2 5 2 e2 5 7
Fig. 3 e Effects of syringin on histopathologic changes in lung tissues in LPS-induced ALI mice. Representative histologic changes of lung obtained from mice of different groups. (A) Control group. (B) LPS group. (C) LPS D syringin (25 mg/kg) group. (D) LPS D syringin (50 mg/kg) group. (E) LPS D syringin (100 mg/kg) group. (F) Lung injury score (hematoxylin and eosin staining, magnification 2003). (Color version of figure is available online.)
dehydrated and embedded in paraffin. Then the samples were cut into 5 mm and stained with hematoxylin and eosin staining. Finally, the sections were observed under an Olympus microscope. The histologic changes in the lung tissues were scored as previously described.14 The percentages of neutrophil infiltration, interstitial edema, hemorrhage, hyaline membrane formation, necrosis, and congestion were scored as follows: 0 (normal) to 4 (severe). The total score was calculated by adding the individual scores of each category.
Statistical analysis All values are expressed as means standard error of the mean. Differences between mean values of normally distributed data were analyzed using one-way analysis of variance (Dunnett’s t-test) and two-tailed Student’s t-test. Statistical significance was accepted at P < 0.05 or P < 0.01.
Results Syringin reduces LPS-induced lung edema in mice
ELISA assay The levels of inflammatory cytokines TNF-a, IL-6, and IL-1b in the bronchoalveolar lavage fluid were measured by commercially available ELISA kits (BioLegend) according to the manufacturer’s instructions.
Lung W/D ratio was used to assess the effects of syringin on lung edema. As shown in Figure 1, the lung W/D ratio was dramatically higher after LPS challenged compared with the control group. However, the increase induced by LPS was dose dependently inhibited by treatment of syringin.
Western blot analysis
Syringin ameliorates LPS-induced MPO activity and MDA content
Total proteins of lung tissues were extracted using Protein Extraction Reagent Kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instruction. Then, the proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with primary antibodies at 4 C overnight and probed with secondary antibodies for 2 h at room temperature. After washing three times, the immunostained bands were detected using an ECL kit (Amersham Biosciences, Buckinghamshire, UK). Lamin B was used as the marker of nucleus and b-actin was used as the marker of cytoplasm.
MPO activity and MDA content were used to assess the neutrophil accumulation and the oxidative stress within lung tissues. In this study, our results showed that the MPO activity and MDA content increased significantly in LPS-treated group. However, the increases induced by LPS were dose dependently inhibited by treatment of syringin (Fig. 2).
Effects of syringin on LPS-induced lung histologic changes In this study, lung histologic changes were examined by hematoxylin and eosin staining. As shown in Figure 3, lung
zhang et al protective effects of syringin against lsp-induced ali in mice
255
Effect of syringin on LPS-induced NF-kB activation NF-kB plays an important role in the regulation of inflammatory cytokines production. In this study, we detected the effects of syringin on LPS-induced NF-kB activation. The results showed that NF-kB activation was dramatically increased by LPS administration. However, LPS-induced NF-kB activation was significantly suppressed by syringin (Fig. 5).
Effects of syringin on Nrf2 and HO-1 expression The effects of syringin on Nrf2 and HO-1 expression were measured by Western blot analysis. The results showed that the expression of Nrf2 and HO-1 in lung tissues was upregulated by LPS. However, the increases in Nrf2 and HO-1 expression induced by LPS were augmented by syringin (Fig. 6).
Discussion
Fig. 4 e Effects of syringin on TNF-a, IL-1b, and IL-6 production in the BALF of LPS-induced ALI mice. The values presented are mean ± standard error of the mean of three independent experiments. BALF [ bronchoalveolar lavage fluid. #P < 0.01 versus control group, **P < 0.01 versus LPS group.
tissues of control group exhibited normal structure of lung (Fig. 3A). Lung tissues of LPS group showed marked pathologic changes, such as increase in alveolar wall thickness, interstitial edema, and inflammatory cell infiltration (Fig. 3B). However, these pathologic changes were significantly inhibited by treatment of syringin (25, 50, and 100 mg/kg; Fig. 3C-F).
Syringin inhibits LPS-induced inflammatory cytokines production The effects of syringin on LPS-induced TNF-a, IL-1b, and IL-6 production were detected in this study. As shown in Figure 4, the levels of TNF-a, IL-1b, and IL-6 in LPS group were evidently increased compared with the control group. However, the increases were inhibited by treatment of syringin.
LPS-induced ALI is characterized by severe inflammatory response in lung tissues. Syringin has been reported to have anti-inflammatory effects. However, whether syringin has anti-inflammatory effects on LPS-induced ALI remains unclear. Here, we attempted to investigate the effects of syringin on LPS-induced ALI in mice. LPS-induced ALI model in mice is characterized by the intra-alveolar and interstitial edema, alveolar hemorrhage, and overwhelming lung inflammation, which ideally imitate the pathophysiological events observed in ALI patients.15 LPS-induced injury is a very useful experimental in vivo model closely resembling acute lung injury and/or acute respiratory distress syndrome in humans.16 Therefore, in this study, we investigated the effects of syringin in LPSinduced ALI in mice. It is well known that inflammation and oxidative stress are major causes of lung injury.17,18 Previous studies showed that inflammatory cytokines, such as TNF-a, IL-6, and IL-1b played a critical role in the pathogenesis of lung injury.2 Evidence from several studies indicate that the inflammatory cytokine networks play an important role in the initiating and amplifying the lung injury process.19,20 The cytokines could induce the infiltration of neutrophils.21 And stimulating of neutrophils with these cytokines leads to the release of other inflammatory cytokines and oxidant production.22 It is well known that oxidant production within lung also leads to lung injury.23 Inhibition of the release of inflammatory cytokines and oxidant production could inhibit LPS-induced ALI.24 In this study, our results showed that the production of inflammatory cytokines significantly inhibited by syringin. In addition, LPS-induced MDA, the markers of oxidative stress, was also inhibited by treatment of syringin. These results indicated that syringin protected against LPS-induced ALI by inhibiting inflammatory response and oxidative stress. NF-kB is one of the important transcription factors that act as the regulator of inflammatory cytokines.25 In normal conditions, NF-kB is restricted in the cytoplasm with inhibitory protein IkB.26 Once stimulating by LPS, NF-kB p65 dissociates from IkB and translocates into the nucleus to regulate the expression of inflammatory cytokines.27 Inhibition of NF-kB
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j o u r n a l o f s u r g i c a l r e s e a r c h m a r c h 2 0 1 7 ( 2 0 9 ) 2 5 2 e2 5 7
Fig. 5 e Syringin inhibits LPS-induced NF-kB activation. The values presented are the means ± standard error of the mean of three independent experiments. #P < 0.01 versus control group, **P < 0.01 versus LPS group.
activation has the ability to protect against LPS-induced ALI.28 Thus, we investigated the possibility that syringin suppresses the production of inflammatory cytokines by inhibiting NF-kB activation. The results suggested that syringin inhibits LPSinduced inflammatory response by inhibiting NF-kB activation. The Nrf2 signaling pathway plays an important role in inducing phase II detoxifying enzymes, particularly the HO1.29 Nrf2 has been considered as a potential target for lung injury.30 Previous studies showed that activating Nrf2 could inhibit LPS-induced NF-kB activation.31 Furthermore, studies showed that activating Nrf2 could protect against LPS-induced ALI.32 Several studies have confirmed that many compounds
exhibited its antioxidant effects by activating Nrf2 signaling pathway.33,34 To investigate the therapeutic mechanism of syringin, the effects of syringin on Nrf2 signaling pathway were measured. The results showed that syringin exhibited therapeutic effects by activating Nrf2/HO-1 signaling pathway. In conclusion, our results suggest that syringin protects against LPS-induced ALI through inhibiting inflammatory cytokines production, lung edema, and infiltration of inflammatory cells. The mechanism underlying these effects of syringin may be mediated by the inhibition of NF-kB and activating Nrf2 signaling pathways.
Fig. 6 e Effects of syringing on Nrf2 and HO-1 expression. The values presented are the means ± standard error of the mean of three independent experiments. #P < 0.01 versus control group, **P < 0.01 versus LPS group.
zhang et al protective effects of syringin against lsp-induced ali in mice
Acknowledgment Authors’ contributions: A.Z. and H.W. contributed to the conception and design. A.Z. and Z.L. did the analysis and interpretation. L.S. did the data collection. A.Z. and H.W. wrote the article and did the critical revision of the article.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article. The authors have no conflict of interest to declare.
references
1. Gajic O, Rana R, Winters JL, et al. Transfusion-related acute lung injury in the critically ill: prospective nested case-control study. Am J Respir Crit Care Med. 2007;176:886e891. 2. Goodman RB, Pugin J, Lee JS, Matthay MA. Cytokine-mediated inflammation in acute lung injury. Cytokine Growth Factor Rev. 2003;14:523e535. 3. Lee JW, Fang XH, Gupta N, Serikov V, Matthay MA. Allogeneic human mesenchymal stem cells for treatment of E. coli endotoxin-induced acute lung injury in the ex vivo perfused human lung. P Natl Acad Sci U S A. 2009;106:16357e16362. 4. Fu K, Piao TK, Wang MZ, et al. Protective effect of catalpol on lipopolysaccharide-induced acute lung injury in mice. Int Immunopharmacol. 2014;23:400e406. 5. Abraham E, Carmody A, Shenkar R, Arcaroli J. Neutrophils as early immunologic effectors in hemorrhage-or endotoxemiainduced acute lung injury. Am J Physiol Lung Cell Mol Physiol. 2000;279:L1137eL1145. 6. Vallabhapurapu S, Karin M. Regulation and function of NFkappaB transcription factors in the immune system. Annu Rev Immunol. 2009;27:693e733. 7. Lawrence T. The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harbor Perspect Biol. 2009;1:a001651. 8. Everhart MB, Han W, Sherrill TP, et al. Duration and intensity of NF-kappaB activity determine the severity of endotoxininduced acute lung injury. J Immunol. 2006;176:4995e5005. 9. Liu G, Park YJ, Tsuruta Y, Lorne E. Abraham E p53 attenuates lipopolysaccharide-induced NF-kappaB activation and acute lung injury. J Immunol. 2009;182:5063e5071. 10. Choi J, Shin KM, Park HJ, et al. Anti-inflammatory and antinociceptive effects of sinapyl alcohol and its glucoside syringin. Planta Med. 2004;70:1027e1032. 11. Gong X, Zhang L, Jiang R, et al. Hepatoprotective effects of syringin on fulminant hepatic failure induced by D-galactosamine and lipopolysaccharide in mice. J Appl Toxicol. 2014;34:265e271. 12. Cho JY, Nam KH, Kim AR, et al. In-vitro and in-vivo immunomodulatory effects of syringin. J Pharm Pharmacol. 2001;53:1287e1294. 13. Cui Y, Zhang Y, Liu G. Syringin may exert sleep-potentiating effects through the NOS/NO pathway. Fundam Clin Pharmacol. 2015;29:178e184. 14. Lv HM, Zhu C, Liao YJ, et al. Tenuigenin ameliorates acute lung injury by inhibiting NF-kappa B and MAPK signalling pathways. Respir Physiol Neurobiol. 2015;216:43e51. 15. Bakowitz M, Bruns B, McCunn M. Acute lung injury and the acute respiratory distress syndrome in the injured patient. Scand J Trauma Resusc Emerg Med. 2012;20:54.
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16. Mittal N, Sanyal SN. In vivo effect of surfactant on inflammatory cytokines during endotoxin-induced lung injury in rodents. J immunotoxicol. 2011;8:274e283. 17. Imai Y, Kuba K, Neely GG, et al. Identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury. Cell. 2008;133:235e249. 18. MacNee W. Oxidative stress and lung inflammation in airways disease. Eur J Pharmacol. 2001;429:195e207. 19. Ishii H, Fujii T, Hogg JC, et al. Contribution of IL-1 beta and TNF-alpha to the initiation of the peripheral lung response to atmospheric particulates (PM10). Am J Physiol Lung Cell Mol Physiol. 2004;287:L176eL183. 20. Song Y, Ao L, Raeburn CD, et al. A low level of TNF-alpha mediates hemorrhage-induced acute lung injury via p55 TNF receptor. Am J Physiol Lung Cell Mol Physiol. 2001;281:L677eL684. 21. Driscoll KE, Carter JM, Hassenbein DG, Howard B. Cytokines and particle-induced inflammatory cell recruitment. Environ Health Perspect. 1997;105 Suppl 5:1159e1164. 22. Mantovani A, Cassatella MA, Costantini C, Jaillon S. Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol. 2011;11:519e531. 23. Butterfield TA, Best TM, Merrick MA. The dual roles of neutrophils and macrophages in inflammation: a critical balance between tissue damage and repair. J Athl Train. 2006;41:457e465. 24. Gong Q, Yin H, Fang M, et al. Heme oxygenase-1 upregulation significantly inhibits TNF-alpha and Hmgb1 releasing and attenuates lipopolysaccharide-induced acute lung injury in mice. Int Immunopharmacol. 2008;8:792e798. 25. Oeckinghaus A, Ghosh S. The NF-kappaB family of transcription factors and its regulation. Cold Spring Harbor Perspect Biol. 2009;1:a000034. 26. Sun Y, Zhao Y, Yao J, et al. Wogonoside protects against dextran sulfate sodium-induced experimental colitis in mice by inhibiting NF-kappaB and NLRP3 inflammasome activation. Biochem Pharmacol. 2015;94:142e154. 27. Lu YC, Yeh WC, Ohashi PS. LPS/TLR4 signal transduction pathway. Cytokine. 2008;42:145e151. 28. Shen W, Gan J, Xu S, Jiang G, Wu H. Penehyclidine hydrochloride attenuates LPS-induced acute lung injury involvement of NF-kappaB pathway. Pharmacol Res. 2009;60:296e302. 29. Chen XL, Kunsch C. Induction of cytoprotective genes through Nrf2/antioxidant response element pathway: a new therapeutic approach for the treatment of inflammatory diseases. Curr Pharm Des. 2004;10:879e891. 30. Boutten A, Goven D, Artaud-Macari E, Boczkowski J, Bonay M. NRF2 targeting: a promising therapeutic strategy in chronic obstructive pulmonary disease. Trends Mol Med. 2011;17:363e371. 31. Ren H, Hao J, Liu T, et al. Hesperetin suppresses inflammatory responses in lipopolysaccharide-induced RAW 264.7 cells via the inhibition of NF-kappaB and activation of Nrf2/HO-1 pathways. Inflammation. 2016;39:964e973. 32. Kong M, Mao J, Luo B, Qin Z. Role of transcriptional factor Nrf2 in the acute lung injury of mice. Int J Clin Exp Pathol. 2015;8:10929e10934. 33. Lyu JH, Kim KH, Kim HW, et al. Dangkwisoo-san, an herbal medicinal formula, ameliorates acute lung inflammation via activation of Nrf2 and suppression of NF-kappaB. J ethnopharmacol. 2012;140:107e116. 34. Han CW, Kwun MJ, Kim KH, et al. Ethanol extract of Alismatis Rhizoma reduces acute lung inflammation by suppressing NF-kappaB and activating Nrf2. J ethnopharmacol. 2013;146:402e410.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Protective effects of syringin against lipopolysaccharide-induced acute lung injury in mice Ao Zhang, MM,a Zhongmin Liu, MM,a Lulu Sheng, MM,b and Hui Wu, MMb,* a b
Department of Intensive Care Unit, First Hospital of Jilin University, Changchun, China Department of Ophthalmology, First Hospital of Jilin University, Changchun, China
article info
abstract
Article history:
Background: Syringin, a major active substance isolated from Eleutherococcus senticosus, has
Received 25 June 2016
been found to have anti-inflammatory effect. The aim of this study was to investigate the
Received in revised form
effects and underlying mechanisms of syringin on lipopolysaccharide (LPS)-induced acute
27 September 2016
lung injury (ALI) in mice.
Accepted 27 October 2016
Methods: We established an LPS-induced ALI model in mice. We also detected the lung wet-
Available online 5 November 2016
to-dry ratio, myeloperoxidase activity, and inflammatory cytokines tumor necrosis factor alpha, interleukin (IL)-1b, and IL-6 to estimate the index of lung injury in mice. Further-
Keywords:
more, the expression of nuclear factor-erythroid 2-related factor-2 (Nrf2), heme oxygenase-
Syringin
1, and nuclear factor kB (NF-kB) was detected by Western blot analysis.
NF-kB
Results: The results showed that the increases in lung wet-to-dry ratio, myeloperoxidase
LPS
activity, malondialdehyde content, and levels of tumor necrosis factor alpha, IL-1b, and IL-
Lung injury
6 induced by LPS were significantly inhibited by treatment of syringin. The phosphoryla-
Nrf2
tion of IkB-a and p65 NF-kB caused by LPS was inhibited by syringin. Furthermore, syringin was found to upregulate the expression of Nrf2 and heme oxygenase 1. Conclusions: In conclusion, the results suggest that syringin protects against LPS-induced ALI by activating Nrf2 and inhibiting NF-kB signaling pathway. ª 2016 Elsevier Inc. All rights reserved.
Introduction Acute lung injury (ALI) is a critical illness syndrome with high morbidity and mortality in critically ill patients.1 ALI is characterized by severe lung inflammation and increased capillary permeability.2 Lipopolysaccharide (LPS) has been identified as the major factor that leads to ALI.3 In mice with ALI, LPS significantly upregulates the expression of inflammatory cytokines tumor necrosis factor (TNF)-a, interleukin (IL)-1b, and IL-6.4 Studies showed that these cytokines cause the
aggregation of neutrophilic leukocytes and lead to lung injury.5 Therefore, inhibition of these cytokines has drawn a great attention in the treatment of LPS-induced ALI. Nuclear factor kB (NF-kB) is a nuclear transcription factor, which plays an important role in the regulation of inflammatory cytokines.6,7 Inhibition of NF-kB activation has the ability to attenuate LPS-induced ALI.8,9 Syringin is a major active substance isolated from Eleutherococcus senticosus. It has been found that syringin has broad pharmacologic effects, such as anti-inflammatory and
* Corresponding author. Department of Ophthalmology, First Hospital Tel./fax: þ86043188782639. E-mail address: [email protected] (H. Wu). 0022-4804/$ e see front matter ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2016.10.027
of
Jilin
University,
Changchun
130021,
China.
zhang et al protective effects of syringin against lsp-induced ali in mice
253
antinociceptive effects.10 Syringin has been reported to protect against LPS and D-galactosamineeinduced hepatic failure.11 Syringin also inhibits LPS-induced TNF-a production in RAW264.7 cells.12 Furthermore, syringin has been reported to exert sleep-potentiating effects through the nitric oxide synthasesenitric oxide pathway.13 However, the effects of syringin on LPS-induced ALI have not been reported. Therefore, in this study, we investigated the effects of syringin on LPSinduced ALI in mice.
Materials and methods Reagents Mouse TNF-a, IL-6, and IL-1b enzyme-linked immunosorbent assay (ELISA) kits were purchased from BioLegend (San Diego, CA). The myeloperoxidase (MPO) determination kit was purchased by the Jiancheng Bioengineering Institute of Nanjing (Jiangsu, China). Mouse nuclear factor-erythroid 2-related factor-2 (Nrf2), HO-1, NF-kB p65, NF-kB p-p65, IkB, p-IkB, and b-actin were provided from Cell Signaling Technology Inc (Beverly, MA). Syringin was purchased from Chengdu Must Bio-technology Co, Ltd (Chengdu, China). LPS (Escherichia coli 055:B5) was purchased from Sigma Chemical Co (St. Louis, MO). All other chemicals were of reagent grade.
Animal
Fig. 1 e Effects of syringin on the lung W/D ratio of LPSinduced ALI mice. The values presented are the means ± standard error of the mean of three independent experiments. #P < 0.01 versus control group, *P < 0.05 and **P < 0.01 versus LPS group.
Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.
Hematoxylin and eosin staining Twelve hour after LPS treatment, the lung tissues were collected and fixed in 4% formaldehyde. The samples were
Male BALB/c mice of 18-22 g weight (Animals Experiment Center, Baiqiuen Medical College of Jilin University, Jilin, China) were housed in microisolator cages with controlled environment at a temperature of 22 C-25 C. All studies involving animals were performed according to the National Institutes of Health guide for the Care and Use of Laboratory Animal.
Study design and grouping Sixty mice were randomly divided into three groups: control group, LPS group, and LPS þ syringin (25, 50, and 100 mg/kg) groups. The mice of LPS group were received 50-mL LPS given intranasally to induce ALI. The mice of LPS þ syringin (25, 50, and 100 mg/kg) groups were given intraperitoneally to the mice 1 h after LPS treatment. The mice of control group were given equal amount of phosphate-buffered saline.
Lung wet-to-dry weight ratio Twelve hour after LPS treatment, the mice were condemned to death, and the lungs were collected to obtain the wet weight. Next, we obtained dry weight after keeping the lungs at 80 C for 48 h. The lung wet-to-dry (W/D) ratio was calculated by dividing the wet weight by the dry weight.
MPO and malondialdehyde assay Twelve hour after LPS treatment, the lung tissues were homogenized and the MPO and malondialdehyde (MDA) contents were measured by MPO determination kit (Nanjing
Fig. 2 e Effects of syringin on MPO and MDA contents. The values presented are the mean ± standard error of the mean of three independent experiments. #P < 0.01 versus control group, **P < 0.01 versus LPS group.
254
j o u r n a l o f s u r g i c a l r e s e a r c h m a r c h 2 0 1 7 ( 2 0 9 ) 2 5 2 e2 5 7
Fig. 3 e Effects of syringin on histopathologic changes in lung tissues in LPS-induced ALI mice. Representative histologic changes of lung obtained from mice of different groups. (A) Control group. (B) LPS group. (C) LPS D syringin (25 mg/kg) group. (D) LPS D syringin (50 mg/kg) group. (E) LPS D syringin (100 mg/kg) group. (F) Lung injury score (hematoxylin and eosin staining, magnification 2003). (Color version of figure is available online.)
dehydrated and embedded in paraffin. Then the samples were cut into 5 mm and stained with hematoxylin and eosin staining. Finally, the sections were observed under an Olympus microscope. The histologic changes in the lung tissues were scored as previously described.14 The percentages of neutrophil infiltration, interstitial edema, hemorrhage, hyaline membrane formation, necrosis, and congestion were scored as follows: 0 (normal) to 4 (severe). The total score was calculated by adding the individual scores of each category.
Statistical analysis All values are expressed as means standard error of the mean. Differences between mean values of normally distributed data were analyzed using one-way analysis of variance (Dunnett’s t-test) and two-tailed Student’s t-test. Statistical significance was accepted at P < 0.05 or P < 0.01.
Results Syringin reduces LPS-induced lung edema in mice
ELISA assay The levels of inflammatory cytokines TNF-a, IL-6, and IL-1b in the bronchoalveolar lavage fluid were measured by commercially available ELISA kits (BioLegend) according to the manufacturer’s instructions.
Lung W/D ratio was used to assess the effects of syringin on lung edema. As shown in Figure 1, the lung W/D ratio was dramatically higher after LPS challenged compared with the control group. However, the increase induced by LPS was dose dependently inhibited by treatment of syringin.
Western blot analysis
Syringin ameliorates LPS-induced MPO activity and MDA content
Total proteins of lung tissues were extracted using Protein Extraction Reagent Kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instruction. Then, the proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with primary antibodies at 4 C overnight and probed with secondary antibodies for 2 h at room temperature. After washing three times, the immunostained bands were detected using an ECL kit (Amersham Biosciences, Buckinghamshire, UK). Lamin B was used as the marker of nucleus and b-actin was used as the marker of cytoplasm.
MPO activity and MDA content were used to assess the neutrophil accumulation and the oxidative stress within lung tissues. In this study, our results showed that the MPO activity and MDA content increased significantly in LPS-treated group. However, the increases induced by LPS were dose dependently inhibited by treatment of syringin (Fig. 2).
Effects of syringin on LPS-induced lung histologic changes In this study, lung histologic changes were examined by hematoxylin and eosin staining. As shown in Figure 3, lung
zhang et al protective effects of syringin against lsp-induced ali in mice
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Effect of syringin on LPS-induced NF-kB activation NF-kB plays an important role in the regulation of inflammatory cytokines production. In this study, we detected the effects of syringin on LPS-induced NF-kB activation. The results showed that NF-kB activation was dramatically increased by LPS administration. However, LPS-induced NF-kB activation was significantly suppressed by syringin (Fig. 5).
Effects of syringin on Nrf2 and HO-1 expression The effects of syringin on Nrf2 and HO-1 expression were measured by Western blot analysis. The results showed that the expression of Nrf2 and HO-1 in lung tissues was upregulated by LPS. However, the increases in Nrf2 and HO-1 expression induced by LPS were augmented by syringin (Fig. 6).
Discussion
Fig. 4 e Effects of syringin on TNF-a, IL-1b, and IL-6 production in the BALF of LPS-induced ALI mice. The values presented are mean ± standard error of the mean of three independent experiments. BALF [ bronchoalveolar lavage fluid. #P < 0.01 versus control group, **P < 0.01 versus LPS group.
tissues of control group exhibited normal structure of lung (Fig. 3A). Lung tissues of LPS group showed marked pathologic changes, such as increase in alveolar wall thickness, interstitial edema, and inflammatory cell infiltration (Fig. 3B). However, these pathologic changes were significantly inhibited by treatment of syringin (25, 50, and 100 mg/kg; Fig. 3C-F).
Syringin inhibits LPS-induced inflammatory cytokines production The effects of syringin on LPS-induced TNF-a, IL-1b, and IL-6 production were detected in this study. As shown in Figure 4, the levels of TNF-a, IL-1b, and IL-6 in LPS group were evidently increased compared with the control group. However, the increases were inhibited by treatment of syringin.
LPS-induced ALI is characterized by severe inflammatory response in lung tissues. Syringin has been reported to have anti-inflammatory effects. However, whether syringin has anti-inflammatory effects on LPS-induced ALI remains unclear. Here, we attempted to investigate the effects of syringin on LPS-induced ALI in mice. LPS-induced ALI model in mice is characterized by the intra-alveolar and interstitial edema, alveolar hemorrhage, and overwhelming lung inflammation, which ideally imitate the pathophysiological events observed in ALI patients.15 LPS-induced injury is a very useful experimental in vivo model closely resembling acute lung injury and/or acute respiratory distress syndrome in humans.16 Therefore, in this study, we investigated the effects of syringin in LPSinduced ALI in mice. It is well known that inflammation and oxidative stress are major causes of lung injury.17,18 Previous studies showed that inflammatory cytokines, such as TNF-a, IL-6, and IL-1b played a critical role in the pathogenesis of lung injury.2 Evidence from several studies indicate that the inflammatory cytokine networks play an important role in the initiating and amplifying the lung injury process.19,20 The cytokines could induce the infiltration of neutrophils.21 And stimulating of neutrophils with these cytokines leads to the release of other inflammatory cytokines and oxidant production.22 It is well known that oxidant production within lung also leads to lung injury.23 Inhibition of the release of inflammatory cytokines and oxidant production could inhibit LPS-induced ALI.24 In this study, our results showed that the production of inflammatory cytokines significantly inhibited by syringin. In addition, LPS-induced MDA, the markers of oxidative stress, was also inhibited by treatment of syringin. These results indicated that syringin protected against LPS-induced ALI by inhibiting inflammatory response and oxidative stress. NF-kB is one of the important transcription factors that act as the regulator of inflammatory cytokines.25 In normal conditions, NF-kB is restricted in the cytoplasm with inhibitory protein IkB.26 Once stimulating by LPS, NF-kB p65 dissociates from IkB and translocates into the nucleus to regulate the expression of inflammatory cytokines.27 Inhibition of NF-kB
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j o u r n a l o f s u r g i c a l r e s e a r c h m a r c h 2 0 1 7 ( 2 0 9 ) 2 5 2 e2 5 7
Fig. 5 e Syringin inhibits LPS-induced NF-kB activation. The values presented are the means ± standard error of the mean of three independent experiments. #P < 0.01 versus control group, **P < 0.01 versus LPS group.
activation has the ability to protect against LPS-induced ALI.28 Thus, we investigated the possibility that syringin suppresses the production of inflammatory cytokines by inhibiting NF-kB activation. The results suggested that syringin inhibits LPSinduced inflammatory response by inhibiting NF-kB activation. The Nrf2 signaling pathway plays an important role in inducing phase II detoxifying enzymes, particularly the HO1.29 Nrf2 has been considered as a potential target for lung injury.30 Previous studies showed that activating Nrf2 could inhibit LPS-induced NF-kB activation.31 Furthermore, studies showed that activating Nrf2 could protect against LPS-induced ALI.32 Several studies have confirmed that many compounds
exhibited its antioxidant effects by activating Nrf2 signaling pathway.33,34 To investigate the therapeutic mechanism of syringin, the effects of syringin on Nrf2 signaling pathway were measured. The results showed that syringin exhibited therapeutic effects by activating Nrf2/HO-1 signaling pathway. In conclusion, our results suggest that syringin protects against LPS-induced ALI through inhibiting inflammatory cytokines production, lung edema, and infiltration of inflammatory cells. The mechanism underlying these effects of syringin may be mediated by the inhibition of NF-kB and activating Nrf2 signaling pathways.
Fig. 6 e Effects of syringing on Nrf2 and HO-1 expression. The values presented are the means ± standard error of the mean of three independent experiments. #P < 0.01 versus control group, **P < 0.01 versus LPS group.
zhang et al protective effects of syringin against lsp-induced ali in mice
Acknowledgment Authors’ contributions: A.Z. and H.W. contributed to the conception and design. A.Z. and Z.L. did the analysis and interpretation. L.S. did the data collection. A.Z. and H.W. wrote the article and did the critical revision of the article.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article. The authors have no conflict of interest to declare.
references
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