Inflammation ( # 2014) DOI: 10.1007/s10753-014-9932-z
Wogonoside Ameliorates Lipopolysaccharide-Induced Acute Lung Injury in Mice Liang Zhang,1 Yi Ren,1 Chengliang Yang,1 Yue Guo,2 Xiaojing Zhang,3 Gang Hou,4 Xinjin Guo,5 Nan Sun,1 and Yongyu Liu1,6
Abstract—Wogonoside has been reported to have anti-inflammatory properties. In this study, we evaluated the effect of wogonoside on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Male BALB/c mice with ALI, induced by intranasal instillation of LPS, were treated with wogonoside 1 h prior to LPS exposure. Mice treated with LPS alone showed significantly increased TNF-α, IL-6, and IL-1β levels in the bronchoalveolar lavage fluid (BALF). When pretreated with wogonoside, the TNF-α, IL-6, and IL-1β levels were significantly decreased. Meanwhile, wogonoside significantly inhibited LPS-induced increases in the macrophage and neutrophil infiltration of lung tissues and markedly attenuated myeloperoxidase activity. Furthermore, wogonoside inhibited the TLR4 expression and the phosphorylation of NF-κB p65, and IκB induced by LPS. In conclusion, our results indicate that wogonoside exhibits a protective effect on LPS-induced ALI via suppression of TLR4-mediated NF-κB signaling pathways. KEY WORDS: wogonoside; lipopolysaccharide (LPS); acute lung injury (ALI); nuclear factor-kappaB (NF-κB); TLR4.
INTRODUCTION Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), were characterized by severe hypoxemia, pulmonary edema, and neutrophil accumulation in the lungs [1]. It can be induced by many extreme conditions
1
Department of Thoracic Surgery, Liaoning Cancer Hospital and Institute, Shenyang, 110042, Liaoning Province, People’s Republic of China 2 Shenyang Maternity Hospital, Shenyang, 110042, Liaoning Province, People’s Republic of China 3 Department of Bone and Soft tissue Surgery, Liaoning Cancer Hospital and Institute, Shenyang, 110042, Liaoning Province, People’s Republic of China 4 Department of Respiratory Medicine, The First Affiliated Hospital of China Medical University, Shenyang, 110042, Liaoning Province, People’s Republic of China 5 School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, SAR 999077, People’s Republic of China 6 To whom correspondence should be addressed at Department of Thoracic Surgery, Liaoning Cancer Hospital and Institute, Shenyang, 110042, Liaoning Province, People’s Republic of China. E-mail: [email protected]
including severe sepsis, severe bacterial pneumonia, trauma, and burn [2–4]. Lipopolysaccharide (LPS) has been referred to be an important risk factor of acute lung injury [5]. LPS activates TLR4 signal pathway and triggers an inflammatory response, resulting in acute lung injury [6]. In the last decade, ALI is a major clinical problem that has a high mortality rate of 30 % to 40 % [7]. There are few effective therapies in clinic. Therefore, the development of novel therapies for acute respiratory distress syndrome is urgently needed. Wogonoside, one flavonoid derived from the root of Scutellaria baicalensis Georgi, has been shown to have antioxidant, anti-tumor, and antithrombotic activities [8– 10]. Recently, wogonoside has been reported to have antiinflammatory effects. Wogonoside was found to inhibit TNF-α and IL-6 production in LPS-activated RAW264.7 cells [11], to inhibit LPS-induced angiogenesis in vitro and in vivo [12]. However, the effect of wogonoside on LPSinduced acute lung injury remains unclear. In this study, we sought to assess the preventive effects of wogonoside on LPS-induced mouse acute lung injury and elucidated the potential anti-inflammatory mechanism.
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Zhang, Ren, Yang, Guo, Zhang, Hou, Guo, Sun, and Liu MATERIALS AND METHODS Materials Wogonoside (purity >99 %) was purchased from Shanghai Winherb Medical S&T Development Co. Ltd. (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits of TNF-α, IL-6, and IL-1β were purchased from BioLegend (CA, USA). LPS was purchased from Sigma (St. Louis, MO, USA). Anti-pNF-κB p65, antiNF-κB p65, anti-TLR4, and anti-β-actin monoclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The myeloperoxidase (MPO) determination kit was provided by the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). All other reagents were of analytical grade.
(5 mg/kg) were given with an intraperitoneal injection (i.p.) 1 h before LPS administration. Then, the mice were slightly anesthetized with an inhalation of diethyl ether; 10 μg of LPS in 50 μl PBS was instilled intranasal (i.n.) to induce lung injury. Control mice were given 50 μl PBS without LPS. After infusion of LPS 7 h, the mice were killed using
Animals Male BALB/c mice, weighing approximately 18 to 22 g, were purchased from the Experimental Animal Center of the China Medical University (Shenyang, China). The mice were housed in a room maintained at 24±1 °C with 40–80 % humidity. All mice received food and water ad libitum. All animal experiments were performed in accordance with the guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Experimental Design After adjustment to the environment, seventy-two mice were randomly divided into six groups and each group contained twelve mice: control group, LPS group, wogonoside (10, 20, and 40 mg/kg) + LPS group, DEX + LPS group. Wogonoside (10, 20, and 40 mg/kg) and DEX
Fig. 1. Effects of wogonoside on the lung W/D ratio of LPS-induced ALI mice. The values presented are the means ± SEM (n=6 in each group). # p
Wogonoside Ameliorates Lipopolysaccharide-Induced Acute Lung Injury in Mice Liang Zhang,1 Yi Ren,1 Chengliang Yang,1 Yue Guo,2 Xiaojing Zhang,3 Gang Hou,4 Xinjin Guo,5 Nan Sun,1 and Yongyu Liu1,6
Abstract—Wogonoside has been reported to have anti-inflammatory properties. In this study, we evaluated the effect of wogonoside on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Male BALB/c mice with ALI, induced by intranasal instillation of LPS, were treated with wogonoside 1 h prior to LPS exposure. Mice treated with LPS alone showed significantly increased TNF-α, IL-6, and IL-1β levels in the bronchoalveolar lavage fluid (BALF). When pretreated with wogonoside, the TNF-α, IL-6, and IL-1β levels were significantly decreased. Meanwhile, wogonoside significantly inhibited LPS-induced increases in the macrophage and neutrophil infiltration of lung tissues and markedly attenuated myeloperoxidase activity. Furthermore, wogonoside inhibited the TLR4 expression and the phosphorylation of NF-κB p65, and IκB induced by LPS. In conclusion, our results indicate that wogonoside exhibits a protective effect on LPS-induced ALI via suppression of TLR4-mediated NF-κB signaling pathways. KEY WORDS: wogonoside; lipopolysaccharide (LPS); acute lung injury (ALI); nuclear factor-kappaB (NF-κB); TLR4.
INTRODUCTION Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), were characterized by severe hypoxemia, pulmonary edema, and neutrophil accumulation in the lungs [1]. It can be induced by many extreme conditions
1
Department of Thoracic Surgery, Liaoning Cancer Hospital and Institute, Shenyang, 110042, Liaoning Province, People’s Republic of China 2 Shenyang Maternity Hospital, Shenyang, 110042, Liaoning Province, People’s Republic of China 3 Department of Bone and Soft tissue Surgery, Liaoning Cancer Hospital and Institute, Shenyang, 110042, Liaoning Province, People’s Republic of China 4 Department of Respiratory Medicine, The First Affiliated Hospital of China Medical University, Shenyang, 110042, Liaoning Province, People’s Republic of China 5 School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, SAR 999077, People’s Republic of China 6 To whom correspondence should be addressed at Department of Thoracic Surgery, Liaoning Cancer Hospital and Institute, Shenyang, 110042, Liaoning Province, People’s Republic of China. E-mail: [email protected]
including severe sepsis, severe bacterial pneumonia, trauma, and burn [2–4]. Lipopolysaccharide (LPS) has been referred to be an important risk factor of acute lung injury [5]. LPS activates TLR4 signal pathway and triggers an inflammatory response, resulting in acute lung injury [6]. In the last decade, ALI is a major clinical problem that has a high mortality rate of 30 % to 40 % [7]. There are few effective therapies in clinic. Therefore, the development of novel therapies for acute respiratory distress syndrome is urgently needed. Wogonoside, one flavonoid derived from the root of Scutellaria baicalensis Georgi, has been shown to have antioxidant, anti-tumor, and antithrombotic activities [8– 10]. Recently, wogonoside has been reported to have antiinflammatory effects. Wogonoside was found to inhibit TNF-α and IL-6 production in LPS-activated RAW264.7 cells [11], to inhibit LPS-induced angiogenesis in vitro and in vivo [12]. However, the effect of wogonoside on LPSinduced acute lung injury remains unclear. In this study, we sought to assess the preventive effects of wogonoside on LPS-induced mouse acute lung injury and elucidated the potential anti-inflammatory mechanism.
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Zhang, Ren, Yang, Guo, Zhang, Hou, Guo, Sun, and Liu MATERIALS AND METHODS Materials Wogonoside (purity >99 %) was purchased from Shanghai Winherb Medical S&T Development Co. Ltd. (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits of TNF-α, IL-6, and IL-1β were purchased from BioLegend (CA, USA). LPS was purchased from Sigma (St. Louis, MO, USA). Anti-pNF-κB p65, antiNF-κB p65, anti-TLR4, and anti-β-actin monoclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The myeloperoxidase (MPO) determination kit was provided by the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). All other reagents were of analytical grade.
(5 mg/kg) were given with an intraperitoneal injection (i.p.) 1 h before LPS administration. Then, the mice were slightly anesthetized with an inhalation of diethyl ether; 10 μg of LPS in 50 μl PBS was instilled intranasal (i.n.) to induce lung injury. Control mice were given 50 μl PBS without LPS. After infusion of LPS 7 h, the mice were killed using
Animals Male BALB/c mice, weighing approximately 18 to 22 g, were purchased from the Experimental Animal Center of the China Medical University (Shenyang, China). The mice were housed in a room maintained at 24±1 °C with 40–80 % humidity. All mice received food and water ad libitum. All animal experiments were performed in accordance with the guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Experimental Design After adjustment to the environment, seventy-two mice were randomly divided into six groups and each group contained twelve mice: control group, LPS group, wogonoside (10, 20, and 40 mg/kg) + LPS group, DEX + LPS group. Wogonoside (10, 20, and 40 mg/kg) and DEX
Fig. 1. Effects of wogonoside on the lung W/D ratio of LPS-induced ALI mice. The values presented are the means ± SEM (n=6 in each group). # p
