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Isoalantolactone protects LPS-induced acute lung injury through Nrf2 activation
Microbial Pathogenesis 123 (2018) 213–218
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Isoalantolactone protects LPS-induced acute lung injury through Nrf2 activation
T
Cheng-bo Yuan, Lin Tian, Bo Yang, Hai-yan Zhou∗ Department of Respirology, The Affiliated Hospital of Changchun University of Chinese Medicine, Changchun, 130000, Jilin, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Isoalantolactone LPS Lung injury NF-κB Nrf2
Isoalantolactone (ISO), a sesquiterpene lactone isolated from Inula helenium, is known to have anti-inflammatory activity. Here, using a mouse model of acute lung injury, we investigated the effects of ISO on lung inflammation in vivo. ISO (2.5, 5, 10 mg/kg) was administered 1 h before LPS treatment. Histopathological changes suggested that ISO attenuated the injury of lung tissues induced by LPS. ISO also inhibited LPS-induced MPO activity, MDA content, lung W/D ratio, and the production of inflammatory cytokines TNF-α and IL-1β. LPS decreased the activities of the antioxidant enzymes SOD, GPX, and CAT and the decreases were inhibited by ISO. Further studies were performed to detect the Nrf2 and NF-κB signaling pathway. The results showed that ISO significantly suppressed LPS-induced NF-κB activation, as well as PI3K and AKT phosphorylation. Additionally, the expression of Nrf2 and HO-1 were dose-dependently up-regulated by the treatment of ISO. Taken together, the results indicate the protective action of ISO against LPS-induced ALI were through activation of the Nrf2 signaling pathway.
1. Introduction Acute lung injury (ALI) is a clinical syndrome characterized by inflammatory cell infiltration, diffuse damage of microvascular and alveolar epithelium, pulmonary edema, and interstitial fibrosis of the lung [1]. It can be caused by many factors, such as trauma, pneumonia, and sepsis [2]. Previous studies showed that up to 40% of patients with sepsis had associated ALI [3]. LPS, the major component of gram negative bacteria, is known as one of the important pathogenic factors of sepsis [4]. LPS can lead to lung injury by releasing inflammatory cytokine production [5]. Inhibition of inflammatory cytokines could attenuate the injury of lung tissues induced by LPS [6]. ALI is an important cause of death in patients with sepsis [7]. Therefore, seeking effective strategies for the prevention and treatment of ALI has important clinical significance. Isoalantolactone (ISO), a sesquiterpene lactone isolated from Inula helenium, has been known to have anti-inflammatory activity. ISO has been known to inhibit LPS-induced inflammatory cytokines production in peritoneal macrophages and protect mice against sepsis [8]. Also, ISO has been reported to induce ROS-dependent apoptosis in U2OS cells via a novel mechanism involving inhibition of NF-κB activation [9]. Furthermore, ISO was found to induce apoptosis in SGC-7901 cells via regulating PI3K/AKT signaling pathway [10]. In addition, ISO was
∗
found to induce the detoxifying enzymes in HepG2-C8 cells [11]. However, whether ISO had anti-inflammatory effects against lung injury induced by LPS had not been reported. The purpose of this study was to investigate the protective effects of ISO on LPS-induced lung injury in mice. 2. Materials and methods 2.1. Materials ISO (purity > 98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). ELISA kits for TNF-α and IL-1β were purchased from BioLegend (CA, USA). GPX, SOD, CAT, MPO and MDA assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). LPS and dexamethasone (DEX) were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). 2.2. Experimental design BALB/c mice, weighing approximately 20–25 g, were obtained from the Center of Experiment Animals of Jilin University (Changchun, China). All animal experimental procedures followed the guidelines of
Corresponding author. Department of Respirology, The Affiliated Hospital of Changchun University of Chinese Medicine, Changchun, 130000, Jilin, China. E-mail address: [email protected] (H.-y. Zhou).
https://doi.org/10.1016/j.micpath.2018.07.010 Received 1 May 2018; Received in revised form 9 July 2018; Accepted 11 July 2018 0882-4010/ © 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. Effects of ISO on histopathological changes in lung tissues in LPS-induced ALI mice. Representative histological changes of lung obtained from mice of different groups. A: Control group, B: LPS group, C: LPS + ISO (2.5 mg/kg) group, D: LPS + ISO (5 mg/kg) group, E: LPS + ISO (10 mg/kg) group, F: LPS + DEX group (Hematoxylin and eosin staining, magnification 200×).
for 48 h. Then, the tissues were exposed to a series of graded ethanol for dehydration, and embedded in paraffin. Finally, the tissues were cut into sections (5 μm), and stained with hematoxylin and eosin (H & E). Pathologic examination was assessed using a light microscope (Olympus, Japan). 2.6. ELISA assay The production of TNF-α and IL-1β in the BALF was measured in this study using the commercially available ELISA kits (BioLegend, CA, USA) according to the instructions of the manufacturer. Fig. 2. Effects of ISO on the lung W/D ratio of LPS-induced ALI mice. The values presented are the means ± SEM of three independent experiments. #p < 0.01 vs. control group, *p < 0.05 and **p < 0.01 vs. LPS group.
2.7. Western blot analysis
the US National Institute of Health. The mice were divided into six groups: the control group, LPS group, LPS + ISO (2.5, 5, 10 mg/kg) groups, and LPS + DEX (5 mg/kg) group. LPS-induced ALI was induced by giving 10 μg of LPS dissolved in 50 μl of PBS by intratracheal instillation. ISO (2.5, 5, 10 mg/kg) or DEX (5 mg/kg) was given intraperitoneal 1 h before LPS treatment. The doses of ISO used in this study were based on a previous study [8].
Lung tissues were collected and lysed with RIPA buffer to collect proteins. The protein concentration was measured by BCA kit. Subsequently, the proteins were separated on 10% SDS-PAGE and transferred onto PVDF membranes. After blocking, the membranes were incubated with primary antibodies: NF-κB p65, NF-κB p-p65, IκBα, p-IκBα, Nrf2, and HO-1. Then, the membranes were washed three times and incubated with secondary antibodies. Finally, the proteins were visualized using enhanced chemiluminescence reagents (ECL) (Thermo, IL, USA).
2.3. Lung wet-to-dry weight (W/D) ratio 2.8. Statistical analysis The lung tissues were collected and the wet weight was recorded. Subsequently, the tissues were placed in an incubator at 80 °C for 48 h. Then, the lung tissues were weighted to obtain the ‘dry’ weight. The ratio of wet lung to dry lung was calculated to assess tissue edema.
All data were expressed as means ± SEM and analyzed using SPSS software (Chicago, IL, USA). The differences among multiple groups were analyzed using one-way ANOVA and the LSD method. Statistical significance was defined as p < 0.05.
2.4. MPO and MDA assay 3. Results Lung tissues were weighted and homogenized in cold PBS. The supernatants were collected and the MPO activity and MDA content in lung tissues were detected by the assay kits according to the manufacturer's instructions.
3.1. Effects of ISO on LPS-induced lung histopathological changes The effects of ISO on lung histopathology were tested by H&E staining. The results demonstrated that the lung tissues of the control group exhibited normal alveolar structure. However, the lung tissues of the LPS group exhibited severe pathological damage, such as inflammatory cell infiltration, lung edema, and obvious alveolar wall
2.5. Histopathological evaluation of the lung tissue The right lobes of the lung tissues were fixed in 10% formaldehyde 214
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Fig. 5. Effects of ISO on TNF-α and IL-1β production in the BALF of LPS-induced ALI mice. The values presented are mean ± SEM of three independent experiments. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group. Fig. 3. Effects of ISO on MPO activity and MDA content. The values presented are the mean ± SEM of three independent experiments. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group.
In contrast, the level of the lung W/D ratio decreased significantly with the treatment of ISO and this decrease occurred in a dose-dependent manner (Fig. 2).
thickening. These changes induced by LPS were attenuated by the treatment of ISO (Fig. 1).
3.3. ISO attenuates LPS-induced MPO activity and MDA content The effects of ISO on lung MPO activity and MDA content induced by LPS were detected in the present study. As shown in Fig. 3, LPS treatment increased the lung MPO activity and MDA content as compared with the normal control group. In contrast, the levels of lung MPO activity and MDA content decreased significantly when treated with ISO (Fig. 3).
3.2. ISO attenuates the LPS-induced lung W/D ratio The effects of ISO on lung edema induced by LPS were detected by measuring the lung W/D ratio. As shown in Fig. 2, LPS treatment increased the lung W/D ratio as compared with the normal control group.
Fig. 4. Effects of ISO on LPS-induced antioxidant enzymes SOD, GPX, and CAT activities. The values presented are mean ± SEM of three independent experiments. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group. 215
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Fig. 6. ISO inhibits LPS-induced NF-κB activation. The values presented are the means ± SEM of three independent experiments. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group.
Fig. 7. ISO inhibits LPS-induced PI3K and AKT phosphorylation. The values presented are the means ± SEM of three independent experiments. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group.
Fig. 8. Effects of ISO on Nrf2 signaling pathway. The values presented are the means ± SEM of three independent experiments. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group.
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role in the regulation of the inflammatory response [16]. NF-κB is involved in the regulation of inflammatory cytokine production and is a known target for the treatment of ALI [16,17]. Inhibition of NF-κB could attenuate the injury of lung tissues induced by LPS [18,19]. We found ISO dose-dependently inhibited LPS-induced NF-κB activation. Previous reports suggested that activation of P13 K/AKT led to an increase in the nuclear translocation and transcriptional activity of NF-κB [20]. P13K and AKT are up-stream molecules of NF-κB. In this study, our result showed that ISO significantly inhibited LPS-induced P13K and AKT phosphorylation. Nrf2 is a transcription factor essential for protection against oxidative injury [21,22]. Previous studies showed that Nrf2 was essential for protection against lung injury [21]. Furthermore, many natural herbal medicines protected lung injury through activation of the Nrf2 signaling pathway [23,24]. In this study, the expression levels of Nrf2 and HO-1 were up-regulated by the treatment of ISO. In conclusion, the data of this study demonstrated that the protective effects of ISO were attributed to the suppression of inflammatory and oxidative responses. We also showed that ISO could inhibit LPSinduced NF-κB activation and activate Nrf2 signaling pathway. ISO may be considered as a potential therapeutic agent for ALI.
3.4. Effects of ISO on the activities of LPS-induced antioxidant enzymes SOD, GPX, and CAT The effects of ISO on the activities of LPS-induced antioxidant enzymes SOD, GPX, and CAT were ascertained. As shown in Fig. 4, LPS treatment decreased lung SOD, GPX, and CAT activities as compared with the normal control group. In contrast, the decreases were reversed by the treatment of ISO (Fig. 4). 3.5. ISO attenuates LPS-induced TNF-α and IL-1β production in the BALF The effects of ISO on TNF-α and IL-1β production in the BALF induced by LPS were detected in the present study. As shown in Fig. 5, LPS treatment increased TNF-α and IL-1β production in the BALF as compared with the normal control group. In contrast, the levels of TNFα and IL-1β production in the BALF decreased significantly when treatment of ISO (Fig. 5). 3.6. ISO attenuates LPS-induced NF-κB activation The effects of ISO on NF-κB activation induced by LPS were detected in the present study. As shown in Fig. 6, LPS treatment increased phosphorylation levels of p65 and IκBα as compared with the normal control group. In contrast, the phosphorylation levels of p65 and IκBα decreased significantly when treatment of ISO (Fig. 6).
Conflicts of interest All authors declare that they have no conflict of interest.
3.7. ISO attenuates LPS-induced PI3K and AKT phosphorylation
References
The effects of ISO on LPS-induced PI3K and AKT phosphorylation were detected in the present study. As shown in Fig. 7, LPS treatment increased phosphorylation levels of PI3K and AKT as compared with the normal control group. In contrast, the phosphorylation levels of PI3K and AKT decreased significantly when treatment of ISO (Fig. 7).
[1] M. Bhatia, S. Moochhala, Role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome, J. Pathol. 202 (2004) 145–156. [2] E. Abraham, M.A. Matthay, C.A. Dinarello, J.L. Vincent, J. Cohen, S.M. Opal, et al., Consensus conference definitions for sepsis, septic shock, acute lung injury, and acute respiratory distress syndrome: time for a reevaluation, Crit. Care Med. 28 (2000) 232–235. [3] A.P. Wheeler, G.R. Bernard, Acute lung injury and the acute respiratory distress syndrome: a clinical review, Lancet 369 (2007) 1553–1564. [4] R.J. Ulevitch, P.S. Tobias, Recognition of gram-negative bacteria and endotoxin by the innate immune system, Curr. Opin. Immunol. 11 (1999) 19–22. [5] K. Murakami, K. Okajima, M. Uchiba, M. Johno, T. Nakagaki, H. Okabe, et al., Activated protein C prevents LPS-induced pulmonary vascular injury by inhibiting cytokine production, Am. J. Physiol. 272 (1997) L197–L202. [6] S. Liu, G. Feng, G.L. Wang, G.J. Liu, p38MAPK inhibition attenuates LPS-induced acute lung injury involvement of NF-kappaB pathway, Eur. J. Pharmacol. 584 (2008) 159–165. [7] L.B. Ware, M.D. Eisner, B.T. Thompson, P.E. Parsons, M.A. Matthay, Significance of von Willebrand factor in septic and nonseptic patients with acute lung injury, Am. J. Respir. Crit. Care Med. 170 (2004) 766–772. [8] G. He, X. Zhang, Y. Chen, J. Chen, L. Li, Y. Xie, Isoalantolactone inhibits LPS-induced inflammation via NF-kappaB inactivation in peritoneal macrophages and improves survival in sepsis, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 90 (2017) 598–607. [9] W. Di, M. Khan, A. Rasul, M. Sun, Y. Sui, L. Zhong, et al., Isoalantolactone inhibits constitutive NF-kappaB activation and induces reactive oxygen species-mediated apoptosis in osteosarcoma U2OS cells through mitochondrial dysfunction, Oncol. Rep. 32 (2014) 1585–1593. [10] A. Rasul, M. Khan, B. Yu, M. Ali, Y.J. Bo, H. Yang, et al., Isoalantolactone, a sesquiterpene lactone, induces apoptosis in SGC-7901 cells via mitochondrial and phosphatidylinositol 3-kinase/Akt signaling pathways, Arch Pharm. Res. (Seoul) 36 (2013) 1262–1269. [11] J.Y. Seo, J. Park, H.J. Kim, I.A. Lee, J.S. Lim, S.S. Lim, et al., Isoalantolactone from Inula helenium caused Nrf2-mediated induction of detoxifying enzymes, J. Med. Food 12 (2009) 1038–1045. [12] H. Makita, M. Nishimura, K. Miyamoto, T. Nakano, Y. Tanino, J. Hirokawa, et al., Effect of anti-macrophage migration inhibitory factor antibody on lipopolysaccharide-induced pulmonary neutrophil accumulation, Am. J. Respir. Crit. Care Med. 158 (1998) 573–579. [13] X. Zhou, Q. Dai, X. Huang, Neutrophils in acute lung injury, Front. Biosci. 17 (2012) 2278–2283. [14] W.L. Lee, G.P. Downey, Neutrophil activation and acute lung injury, Curr. Opin. Crit. Care 7 (2001) 1–7. [15] K.Y. Yang, J.J. Arcaroli, E. Abraham, Early alterations in neutrophil activation are associated with outcome in acute lung injury, Am. J. Respir. Crit. Care Med. 167 (2003) 1567–1574. [16] J. Wang, C. Guo, Z. Wei, X. He, J. Kou, E. Zhou, et al., Morin suppresses inflammatory cytokine expression by downregulation of nuclear factor-kappaB and mitogen-activated protein kinase (MAPK) signaling pathways in lipopolysaccharide-stimulated primary bovine mammary epithelial cells, J. Dairy Sci. 99
3.8. Effects of ISO on Nrf2 and HO-1 expression The effects of ISO on Nrf2 and HO-1 expression induced by LPS were detected in the present study. As shown in Fig. 8, LPS treatment increased the expression of Nrf2 and HO-1 as compared with the normal control group. In contrast, the expression of Nrf2 and HO-1 were further increased when treatment of ISO (Fig. 8). 4. Discussion In this study, we showed for the first time that administration of ISO dose-dependently attenuated LPS-induced lung injury, as confirmed by decreased inflammatory and oxidative mediators. The mechanism underlying these effects may be through inhibiting NF-κB activation and activating Nrf2 signaling pathways. LPS treatment lead to a large number of neutrophils accumulating in the lungs of rats, and the pulmonary vascular permeability increased significantly [12]. Previous studies showed that attenuation of neutrophils infiltration could inhibit LPS-induced ALI [13]. In this study, ISO significantly inhibited LPS-induced neutrophil infiltration, as confirmed by decreased MPO activity. The neutrophil infiltration has the ability to release inflammatory and oxidative mediators [14,15]. We found that ISO significantly suppressed LPS-induced TNF-α and IL-1β production, as well as MDA content. This was consistent with previous studies which showed ISO could inhibit inflammatory cytokine production [8]. SOD, CAT and GPX are important antioxidant enzymes that play critical roles in the oxidative response. In this study, we found that LPS decreased the activities of these antioxidant enzymes and this decrease was prevented by ISO. The results showed that ISO protected mice against LPS-induced ALI via suppressing inflammatory and oxidative responses. The transcription factor NF-κB has been reported to play a critical 217
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[21] H.Y. Cho, A.E. Jedlicka, S.P. Reddy, T.W. Kensler, M. Yamamoto, L.Y. Zhang, et al., Role of NRF2 in protection against hyperoxic lung injury in mice, Am. J. Respir. Cell Mol. Biol. 26 (2002) 175–182. [22] J.M. Lee, J.A. Johnson, An important role of Nrf2-ARE pathway in the cellular defense mechanism, J. Biochem. Mol. Biol. 37 (2004) 139–143. [23] W. Jiang, F. Luo, Q. Lu, J. Liu, P. Li, X. Wang, et al., The protective effect of Trillin LPS-induced acute lung injury by the regulations of inflammation and oxidative state, Chem. Biol. Interact. 243 (2016) 127–134. [24] S.P. Guan, W. Tee, D.S. Ng, T.K. Chan, H.Y. Peh, W.E. Ho, et al., Andrographolide protects against cigarette smoke-induced oxidative lung injury via augmentation of Nrf2 activity, Br. J. Pharmacol. 168 (2013) 1707–1718.
(2016) 3016–3022. [17] M.B. Everhart, W. Han, T.P. Sherrill, M. Arutiunov, V.V. Polosukhin, J.R. Burke, et al., Duration and intensity of NF-kappaB activity determine the severity of endotoxin-induced acute lung injury, J. Immunol. 176 (2006) 4995–5005. [18] Y. Liu, H. Wu, Y.C. Nie, J.L. Chen, W.W. Su, P.B. Li, Naringin attenuates acute lung injury in LPS-treated mice by inhibiting NF-kappaB pathway, Int. Immunopharm. 11 (2011) 1606–1612. [19] Y. Fu, B. Liu, N. Zhang, Z. Liu, D. Liang, F. Li, et al., Magnolol inhibits lipopolysaccharide-induced inflammatory response by interfering with TLR4 mediated NFkappaB and MAPKs signaling pathways, J. Ethnopharmacol. 145 (2013) 193–199. [20] B.T. Hennessy, D.L. Smith, P.T. Ram, Y. Lu, G.B. Mills, Exploiting the PI3K/AKT pathway for cancer drug discovery, Nat. Rev. Drug Discov. 4 (2005) 988–1004.
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Isoalantolactone protects LPS-induced acute lung injury through Nrf2 activation
T
Cheng-bo Yuan, Lin Tian, Bo Yang, Hai-yan Zhou∗ Department of Respirology, The Affiliated Hospital of Changchun University of Chinese Medicine, Changchun, 130000, Jilin, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Isoalantolactone LPS Lung injury NF-κB Nrf2
Isoalantolactone (ISO), a sesquiterpene lactone isolated from Inula helenium, is known to have anti-inflammatory activity. Here, using a mouse model of acute lung injury, we investigated the effects of ISO on lung inflammation in vivo. ISO (2.5, 5, 10 mg/kg) was administered 1 h before LPS treatment. Histopathological changes suggested that ISO attenuated the injury of lung tissues induced by LPS. ISO also inhibited LPS-induced MPO activity, MDA content, lung W/D ratio, and the production of inflammatory cytokines TNF-α and IL-1β. LPS decreased the activities of the antioxidant enzymes SOD, GPX, and CAT and the decreases were inhibited by ISO. Further studies were performed to detect the Nrf2 and NF-κB signaling pathway. The results showed that ISO significantly suppressed LPS-induced NF-κB activation, as well as PI3K and AKT phosphorylation. Additionally, the expression of Nrf2 and HO-1 were dose-dependently up-regulated by the treatment of ISO. Taken together, the results indicate the protective action of ISO against LPS-induced ALI were through activation of the Nrf2 signaling pathway.
1. Introduction Acute lung injury (ALI) is a clinical syndrome characterized by inflammatory cell infiltration, diffuse damage of microvascular and alveolar epithelium, pulmonary edema, and interstitial fibrosis of the lung [1]. It can be caused by many factors, such as trauma, pneumonia, and sepsis [2]. Previous studies showed that up to 40% of patients with sepsis had associated ALI [3]. LPS, the major component of gram negative bacteria, is known as one of the important pathogenic factors of sepsis [4]. LPS can lead to lung injury by releasing inflammatory cytokine production [5]. Inhibition of inflammatory cytokines could attenuate the injury of lung tissues induced by LPS [6]. ALI is an important cause of death in patients with sepsis [7]. Therefore, seeking effective strategies for the prevention and treatment of ALI has important clinical significance. Isoalantolactone (ISO), a sesquiterpene lactone isolated from Inula helenium, has been known to have anti-inflammatory activity. ISO has been known to inhibit LPS-induced inflammatory cytokines production in peritoneal macrophages and protect mice against sepsis [8]. Also, ISO has been reported to induce ROS-dependent apoptosis in U2OS cells via a novel mechanism involving inhibition of NF-κB activation [9]. Furthermore, ISO was found to induce apoptosis in SGC-7901 cells via regulating PI3K/AKT signaling pathway [10]. In addition, ISO was
∗
found to induce the detoxifying enzymes in HepG2-C8 cells [11]. However, whether ISO had anti-inflammatory effects against lung injury induced by LPS had not been reported. The purpose of this study was to investigate the protective effects of ISO on LPS-induced lung injury in mice. 2. Materials and methods 2.1. Materials ISO (purity > 98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). ELISA kits for TNF-α and IL-1β were purchased from BioLegend (CA, USA). GPX, SOD, CAT, MPO and MDA assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). LPS and dexamethasone (DEX) were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). 2.2. Experimental design BALB/c mice, weighing approximately 20–25 g, were obtained from the Center of Experiment Animals of Jilin University (Changchun, China). All animal experimental procedures followed the guidelines of
Corresponding author. Department of Respirology, The Affiliated Hospital of Changchun University of Chinese Medicine, Changchun, 130000, Jilin, China. E-mail address: [email protected] (H.-y. Zhou).
https://doi.org/10.1016/j.micpath.2018.07.010 Received 1 May 2018; Received in revised form 9 July 2018; Accepted 11 July 2018 0882-4010/ © 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. Effects of ISO on histopathological changes in lung tissues in LPS-induced ALI mice. Representative histological changes of lung obtained from mice of different groups. A: Control group, B: LPS group, C: LPS + ISO (2.5 mg/kg) group, D: LPS + ISO (5 mg/kg) group, E: LPS + ISO (10 mg/kg) group, F: LPS + DEX group (Hematoxylin and eosin staining, magnification 200×).
for 48 h. Then, the tissues were exposed to a series of graded ethanol for dehydration, and embedded in paraffin. Finally, the tissues were cut into sections (5 μm), and stained with hematoxylin and eosin (H & E). Pathologic examination was assessed using a light microscope (Olympus, Japan). 2.6. ELISA assay The production of TNF-α and IL-1β in the BALF was measured in this study using the commercially available ELISA kits (BioLegend, CA, USA) according to the instructions of the manufacturer. Fig. 2. Effects of ISO on the lung W/D ratio of LPS-induced ALI mice. The values presented are the means ± SEM of three independent experiments. #p < 0.01 vs. control group, *p < 0.05 and **p < 0.01 vs. LPS group.
2.7. Western blot analysis
the US National Institute of Health. The mice were divided into six groups: the control group, LPS group, LPS + ISO (2.5, 5, 10 mg/kg) groups, and LPS + DEX (5 mg/kg) group. LPS-induced ALI was induced by giving 10 μg of LPS dissolved in 50 μl of PBS by intratracheal instillation. ISO (2.5, 5, 10 mg/kg) or DEX (5 mg/kg) was given intraperitoneal 1 h before LPS treatment. The doses of ISO used in this study were based on a previous study [8].
Lung tissues were collected and lysed with RIPA buffer to collect proteins. The protein concentration was measured by BCA kit. Subsequently, the proteins were separated on 10% SDS-PAGE and transferred onto PVDF membranes. After blocking, the membranes were incubated with primary antibodies: NF-κB p65, NF-κB p-p65, IκBα, p-IκBα, Nrf2, and HO-1. Then, the membranes were washed three times and incubated with secondary antibodies. Finally, the proteins were visualized using enhanced chemiluminescence reagents (ECL) (Thermo, IL, USA).
2.3. Lung wet-to-dry weight (W/D) ratio 2.8. Statistical analysis The lung tissues were collected and the wet weight was recorded. Subsequently, the tissues were placed in an incubator at 80 °C for 48 h. Then, the lung tissues were weighted to obtain the ‘dry’ weight. The ratio of wet lung to dry lung was calculated to assess tissue edema.
All data were expressed as means ± SEM and analyzed using SPSS software (Chicago, IL, USA). The differences among multiple groups were analyzed using one-way ANOVA and the LSD method. Statistical significance was defined as p < 0.05.
2.4. MPO and MDA assay 3. Results Lung tissues were weighted and homogenized in cold PBS. The supernatants were collected and the MPO activity and MDA content in lung tissues were detected by the assay kits according to the manufacturer's instructions.
3.1. Effects of ISO on LPS-induced lung histopathological changes The effects of ISO on lung histopathology were tested by H&E staining. The results demonstrated that the lung tissues of the control group exhibited normal alveolar structure. However, the lung tissues of the LPS group exhibited severe pathological damage, such as inflammatory cell infiltration, lung edema, and obvious alveolar wall
2.5. Histopathological evaluation of the lung tissue The right lobes of the lung tissues were fixed in 10% formaldehyde 214
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Fig. 5. Effects of ISO on TNF-α and IL-1β production in the BALF of LPS-induced ALI mice. The values presented are mean ± SEM of three independent experiments. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group. Fig. 3. Effects of ISO on MPO activity and MDA content. The values presented are the mean ± SEM of three independent experiments. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group.
In contrast, the level of the lung W/D ratio decreased significantly with the treatment of ISO and this decrease occurred in a dose-dependent manner (Fig. 2).
thickening. These changes induced by LPS were attenuated by the treatment of ISO (Fig. 1).
3.3. ISO attenuates LPS-induced MPO activity and MDA content The effects of ISO on lung MPO activity and MDA content induced by LPS were detected in the present study. As shown in Fig. 3, LPS treatment increased the lung MPO activity and MDA content as compared with the normal control group. In contrast, the levels of lung MPO activity and MDA content decreased significantly when treated with ISO (Fig. 3).
3.2. ISO attenuates the LPS-induced lung W/D ratio The effects of ISO on lung edema induced by LPS were detected by measuring the lung W/D ratio. As shown in Fig. 2, LPS treatment increased the lung W/D ratio as compared with the normal control group.
Fig. 4. Effects of ISO on LPS-induced antioxidant enzymes SOD, GPX, and CAT activities. The values presented are mean ± SEM of three independent experiments. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group. 215
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Fig. 6. ISO inhibits LPS-induced NF-κB activation. The values presented are the means ± SEM of three independent experiments. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group.
Fig. 7. ISO inhibits LPS-induced PI3K and AKT phosphorylation. The values presented are the means ± SEM of three independent experiments. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group.
Fig. 8. Effects of ISO on Nrf2 signaling pathway. The values presented are the means ± SEM of three independent experiments. p# < 0.01 vs. control group, p* < 0.05, p** < 0.01 vs. LPS group.
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role in the regulation of the inflammatory response [16]. NF-κB is involved in the regulation of inflammatory cytokine production and is a known target for the treatment of ALI [16,17]. Inhibition of NF-κB could attenuate the injury of lung tissues induced by LPS [18,19]. We found ISO dose-dependently inhibited LPS-induced NF-κB activation. Previous reports suggested that activation of P13 K/AKT led to an increase in the nuclear translocation and transcriptional activity of NF-κB [20]. P13K and AKT are up-stream molecules of NF-κB. In this study, our result showed that ISO significantly inhibited LPS-induced P13K and AKT phosphorylation. Nrf2 is a transcription factor essential for protection against oxidative injury [21,22]. Previous studies showed that Nrf2 was essential for protection against lung injury [21]. Furthermore, many natural herbal medicines protected lung injury through activation of the Nrf2 signaling pathway [23,24]. In this study, the expression levels of Nrf2 and HO-1 were up-regulated by the treatment of ISO. In conclusion, the data of this study demonstrated that the protective effects of ISO were attributed to the suppression of inflammatory and oxidative responses. We also showed that ISO could inhibit LPSinduced NF-κB activation and activate Nrf2 signaling pathway. ISO may be considered as a potential therapeutic agent for ALI.
3.4. Effects of ISO on the activities of LPS-induced antioxidant enzymes SOD, GPX, and CAT The effects of ISO on the activities of LPS-induced antioxidant enzymes SOD, GPX, and CAT were ascertained. As shown in Fig. 4, LPS treatment decreased lung SOD, GPX, and CAT activities as compared with the normal control group. In contrast, the decreases were reversed by the treatment of ISO (Fig. 4). 3.5. ISO attenuates LPS-induced TNF-α and IL-1β production in the BALF The effects of ISO on TNF-α and IL-1β production in the BALF induced by LPS were detected in the present study. As shown in Fig. 5, LPS treatment increased TNF-α and IL-1β production in the BALF as compared with the normal control group. In contrast, the levels of TNFα and IL-1β production in the BALF decreased significantly when treatment of ISO (Fig. 5). 3.6. ISO attenuates LPS-induced NF-κB activation The effects of ISO on NF-κB activation induced by LPS were detected in the present study. As shown in Fig. 6, LPS treatment increased phosphorylation levels of p65 and IκBα as compared with the normal control group. In contrast, the phosphorylation levels of p65 and IκBα decreased significantly when treatment of ISO (Fig. 6).
Conflicts of interest All authors declare that they have no conflict of interest.
3.7. ISO attenuates LPS-induced PI3K and AKT phosphorylation
References
The effects of ISO on LPS-induced PI3K and AKT phosphorylation were detected in the present study. As shown in Fig. 7, LPS treatment increased phosphorylation levels of PI3K and AKT as compared with the normal control group. In contrast, the phosphorylation levels of PI3K and AKT decreased significantly when treatment of ISO (Fig. 7).
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3.8. Effects of ISO on Nrf2 and HO-1 expression The effects of ISO on Nrf2 and HO-1 expression induced by LPS were detected in the present study. As shown in Fig. 8, LPS treatment increased the expression of Nrf2 and HO-1 as compared with the normal control group. In contrast, the expression of Nrf2 and HO-1 were further increased when treatment of ISO (Fig. 8). 4. Discussion In this study, we showed for the first time that administration of ISO dose-dependently attenuated LPS-induced lung injury, as confirmed by decreased inflammatory and oxidative mediators. The mechanism underlying these effects may be through inhibiting NF-κB activation and activating Nrf2 signaling pathways. LPS treatment lead to a large number of neutrophils accumulating in the lungs of rats, and the pulmonary vascular permeability increased significantly [12]. Previous studies showed that attenuation of neutrophils infiltration could inhibit LPS-induced ALI [13]. In this study, ISO significantly inhibited LPS-induced neutrophil infiltration, as confirmed by decreased MPO activity. The neutrophil infiltration has the ability to release inflammatory and oxidative mediators [14,15]. We found that ISO significantly suppressed LPS-induced TNF-α and IL-1β production, as well as MDA content. This was consistent with previous studies which showed ISO could inhibit inflammatory cytokine production [8]. SOD, CAT and GPX are important antioxidant enzymes that play critical roles in the oxidative response. In this study, we found that LPS decreased the activities of these antioxidant enzymes and this decrease was prevented by ISO. The results showed that ISO protected mice against LPS-induced ALI via suppressing inflammatory and oxidative responses. The transcription factor NF-κB has been reported to play a critical 217
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