Total flavonoids of Mosla scabra leaves attenuates lipopolysaccharide-induced acute lung injury via down-regulation of inflammatory signaling in mice

Journal of Ethnopharmacology 148 (2013) 835–841

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Total flavonoids of Mosla scabra leaves attenuates lipopolysaccharide-induced acute lung injury via down-regulation of inflammatory signaling in mice Jing Chen a,n, Jing-Bo Wang a, Chen-Huan Yu b, Li-Qing Chen b, Pan Xu c, Wen-Ying Yu b a

Zhejiang Chinese Medical University, Hangzhou 310053, China Zhejiang Academy of Medical Sciences, Hangzhou 310013, China c Tongde Hospital of Zhejiang Province, Hangzhou 310013, China b

art ic l e i nf o

a b s t r a c t

Article history: Received 9 January 2013 Received in revised form 2 May 2013 Accepted 14 May 2013 Available online 6 June 2013

Ethnopharmacological relevance: Mosla scabra (Thunb.) C.Y. Wu, belonging to the Labiatae family, is a tomentose and aromatic plant, which is widely used as an antipyretic and antiviral drug for pulmonary diseases and famous for its efficiency in treating colds, fever, pneumonia and chronic bronchitis. To investigate therapeutic effects and possible mechanism of Mosla scabra flavonoids (MF) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Materials and methods: Mice were orally administrated with MF once (30 mg/kg or 90 mg/kg) 1 h before LPS challenge. Lung specimens and the bronchoalveolar lavage fluid (BALF) were isolated for histopathological examinations and biochemical analyses 6 h after LPS challenge. Results: Pretreatment with MF could decrease significantly lung wet-to-dry weight (W/D) ratio, lower myeloperoxidase (MPO) activity and total protein concentrations in the BALF, reduce serum levels of NO, TNF-α, IL-1β and IL-6 in ALI model. Additionally, MF attenuated lung histopathological changes and significantly inhibited the phosphorylation of p38 MAPK and translocation of NF-κB p65. Conclusions: These results showed MF significantly attenuate LPS-induced acute lung injury and production of inflammatory mediators via inhibiting MAPK and NF-κB activation, indicating it as a potential therapeutic agent for ALI. & 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Flavonoids Lipopolysaccharide Inflammatory response NF-κB MAPK

1. Introduction Acute lung injury (ALI), and its more severe form, acute respiratory distress syndrome (ARDS), are common clinical diseases associated with significant morbidity and mortality in shock, sepsis, ischemia reperfusion and viral pneumonia. Both of them are characterized by severe hypoxemia, airway dysfunction and tissue remodeling, such as neutrophil recruitment, interstitial edema, a disruption of epithelial integrity, and lung parenchymal injury (Cross and Matthay, 2011). Despite recent advances in many new strategies for treatment, the mortality of ALI still remains more than 40% (Zambon and Vincent, 2008). The development of new agents is still urgently needed. Due to significant synergic therapeutic effect and relatively low toxicity of the herbal

Abbreviations: MF, flavonoids from Mosla scabra leaves; DEX, dexamethasone; BALF, bronchoalveolar lavage fluid; LPS, lipopolysaccharide; ALI, acute lung injury; MPO, myeloperoxidase; NF-κB, Nuclear factor-kappaB; MAPK, Mitogen activated protein kinases. n Corresponding author. Tel.: +86 571 86613712; fax: +86 571 86613713. E-mail address: [email protected] (J. Chen). 0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.05.020

medicine, increasingly considerable attention has been given to the antioxidant and anti-inflammatory natural products (Lee et al., 2011; Kim et al., 2012; Niu et al., 2012; Wang et al., 2012). Mosla scabra (Thunb.) C.Y. Wu, belonging to the Labiatae family, is a tomentose and aromatic plant. It is widely used as a traditional medicinal herb in East Asian countries for centuries, especially in Korea, Japan and China (Liao et al., 2005). In the folk medicine, it is used as an antimicrobic, antipyretic and antiviral drug for pulmonary diseases and is also found to be effective in colds, fever, cough, pneumonia and chronic bronchitis (Osawa et al., 1991; Wang et al., 2000; Yu et al., 2010). Recently, it is reported to possess a wide range of pharmacological properties such as antibacterial, antioxidant, anti-tumor, anti-inflammatory and antiviral activity as well as immunity-modulation (Osawa et al., 1990; Li et al., 2010; Mazzio and Soliman, 2010; Yu et al., 2010). The main bioactivity compounds, such as apigenin, acacetin, 5-hydroxy-6,7dimethoxyflavone, 5-hydroxy-7,8-dimethoxyflavone, andamanicin, magnosalin and ursolic acid, had been isolated and identified from the herb (Wang et al., 2000; Wu et al., 2010). In our previous studies, the aqueous extract from Mosla scabra plays a role as a suppressor of the production of inflammatory cytokines and

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inhibits subsequent viral pneumonia during influenza virus infection; and the flavonoids are the main antiviral ingredients (Wu et al., 2010). However, whether Mosla scabra flavonoids (MF) process an anti-inflammatory property on pulmonary inflammation and the possible molecular mechanisms are still unknown. In the present study, we evaluated the anti-inflammatory effects of MF on lipopolysaccharide (LPS)-induced acute pulmonary inflammation in a mouse model.

2. Materials and methods 2.1. Reagent Dexamethasone (DEX) sodium phosphate injection was purchased from Hui-te pharmaceutical Co., Ltd. (Zhejiang, China). Lipopolysaccharides (from Escherichia coli 055:B5) were purchased from Sigma-Aldrich (St. Louis, USA). All other regents and chemicals used were of analytical grade and obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). 2.2. Herbal extract preparation Mosla scabra leaves in dried powder form were obtained from Chinese Medicine Herbal Factory (Zhejiang, China), and identified by Dr. Wei Cai, Zhejiang Chinese Medical University. A voucher specimen (reference number 121026Y7) has been deposited at the Faculty of Food Science, college of Pharmacy, Zhejiang Chinese Medical University, Zhejiang, China. The extraction and purification of total flavonoids from Mosla scabra leaves (MF) were carried out according to the previous reports (Feng et al., 2011; Zheng et al., 2011). Samples were ground and sieved using a grinder and passed through a 10-mesh sieve. To prepare MF, one kilogram of dry powders were soaked in ten litres of 70% ethanol for 30 min and then boiled heat-reflux extracted for 1 h. The suspension was decompressed and condensed until the ethanol content became approximately 10%. After filtration (to remove chlorophyll and liposoluble impurities), the extract was added into column loading-treated AB-8 macroreticular resin for adsorption for 24 h, and then washed with water and 20% ethanol, respectively, to get rid of polar impurities. MF in the column was eluted with 90% ethanol, and the eluting solution was dried in vacuum condition until yellow powders were achieved. 2.3. Quality control of MF Approximately 20 mg of MF were transferred to a 25 mL volumetric flask and diluted with methanol to volume. The test sample was finally filtered through a 0.45 μm membrane filter before analysis. The total flavonoids content was determined using a colorimetric method described by Jin and Yong-guang Yin (2012) with slightly modified. Briefly, 1.0 mL of NaNO2 solution (5%, w/v), 1.0 mL of AlCl3 solution (10%), and 4.0 mL NaOH solution (1.0 mol/L) were mixed with the same volume of the test sample. The final volume was adjusted to 25 mL with methanol (80%, v/v). The mixture was allowed to stand for 5 min and the absorption was measured at 510 nm against the same mixture, without the sample as a blank. The amount of the total flavonoids was expressed as rutin equivalents (mg rutin/g sample) through the calibration curve of rutin. The calibration curve (y¼2.9101x–0.0008, where x was absorbance value of sample, and y was sample concentration) ranged 10–500 μg/ml (R2 ¼0.9994). The contents of two markers (5-hydroxy-6,7-dimethoxyflavone and 5-hydroxy- 7,8-dimethoxyflavone) in the extract samples were determined by HPLC using the previous method (Xia et al.,

2012). HPLC was performed by using a Shimadzu LC-10A HPLCterm, including a vacuum degasser, binary pump, an autosampler and a photodiode array detector, controlled by Class-up station (Shimadzu Co. Ltd., Japan). The HPLC column was a shim-pack CLC2-ODS column (250  4.6 mm2, 5 μm). The isocratic mobile phase consisted of methanol-0.1% phosphoric acid (80:20, v/v) was used to elute for 40 min, and the flow rate was kept at 1.0 mL/min. Column temperature was kept constantly at 28 1C. UV detection: 254 nm. The injection volume was 20 μL. The HPLC analysis of standards (5-hydroxy-6,7-dimethoxyflavone and 5-hydroxy- 7,8-dimethoxyflavone) and all the samples were carried out under the established experimental condition as mentioned above. 2.4. Animals and preparation of acute lung injury (ALI) model Male special pathogen free ICR mice weighing 18–22 g were purchased from Animal Experimental Center, Zhejiang Academy of Medical Sciences, China. Animals were housed in groups of five per standard cage, on 12 h light/dark cycle; and air temperature was maintained at 2272 1C. Experiments reported in this study were carried out in accordance with local guidelines for the care of laboratory animals of Animal Experimental Center, Zhejiang Chinese Medical University, and were approved by the ethics committee for research on laboratory animal use of the institution (No. 201209R033). The mice were divided into six groups: (1) Normal control group, mice were received 50 μL of 0.9% saline by intraperitoneal injections; (2) Model control group, mice were intraperitoneally administered LPS (0.5 mg/kg). (3) Positive control group, dexamethasone (DEX, 10 mg/kg) was intraperitoneally administrated 1 h before LPS challenge. (4) MF10 treated group, MF (10 mg/kg) was orally administrated 1 h before LPS challenge. (5) MF30 treated group, MF (30 mg/kg) was orally administrated 1 h before LPS challenge. (6) MF90 treated group, MF (90 mg/kg) was orally administrated 1 h before LPS challenge. The chosen doses of the drug were based on previous studies (Wu et al., 2010; Bae et al., 2012). Each group has 20 mice. To induced acute lung injury model, mice were intraperitoneally administered with 5 mg/kg LPS (Bae et al., 2012; Sun et al., 2012). Six hours after LPS challenge, mice were all euthanized by cervical dislocation. The whole lungs of ten mice in each group were removed, rinsed twice in PBS, weighed to get the “wet” weight (W), and then placed in an oven at 80 1C for 48 h to obtain the “dry” weight (D). The ratio of the wet weight to the dry weight (i.e. W/D) was calculated to assess tissue edema. Other ten mice in each group were inserted with a plastic cannula into the trachea. After crossing-clamped the hilum of right lung, the bronchoalveolar lavage fluid (BALF) of the left lung was collected by injecting normal saline in a total volume of 1.5 mL (0.5 mL every time). The recovery ratio of the fluid was approximately 90%. The right lung was then harvested and divided into three parts (one for histopathology and the other two for biochemical analysis). 2.5. Biochemical analysis After 10 weeks of treatment, diets were removed from the cages 12 h before the animals were sacrificed. Blood samples were collected and centrifuged at 3000  g for 20 min to obtain serum. The levels of serum IL-1β, IL-6, TNF-α and nitric oxide (NO) were determined by ELISA. BALF samples were centrifuged (4 1C, 2500  g for 10 min). The supernatants were stored at −80 1C for the analyses of protein by bicinchoninic acid assay, and myeloperoxidase (MPO) activity by ELISA. All commercially available kits

J. Chen et al. / Journal of Ethnopharmacology 148 (2013) 835–841

used in the study were purchased from Boster Bioengineering Institute, Wuhan, China. 2.6. Histopathology One lobes of right lung were fixed in 10% formalin and then embedded in paraffin. Six to ten 4-μm-thick sections were prepared in a noncontiguous way and dyed with hematoxylin-eosin; stained areas were viewed using an optical microscope.

2.9. Statistical analysis All parameters were recorded for individuals within all groups. All data were presented as the mean 7SD. Statistical analysis was performed with two-tailed indirect Student tests by using SPSS 13.0 (SPSS, Inc., USA). And statistical analysis for multiple comparisons was performed by a one-way ANOVA test. A value of P o0.05 was considered significant.

2.7. Western blot analysis

3. Results

Lung protein was extracted using nuclear and cytoplasmic protein extraction kits (Boster Bioengineering Institute, Wuhan, China). Protein levels in the supernatants were determined using the BCA assay kits (Boster Bioengineering Institute, Wuhan, China). Each sample (60 μg) was separated by denaturing SDS-PAGE and transferred to a PVDF membrane by electrophoretic transfer (Bio-Rad Laboratories, Inc., USA). The membrane was preblocked with 5% nonfat milk and 0.1% Tween-20 in Tris-buffered saline (TBST), incubated overnight with the primary antibody. Each membrane was washed three times for 15 min and incubated with the secondary horseradish peroxidase-linked antibodies. Quantitative analysis of detected bands was performed with the Scion Image analysis software (Bio-Rad Laboratories, Inc., USA). The GAPDH western blot was performed as an internal control of protein loading.

3.1. Quality evaluation of MF

2.8. Acute toxicity After overnight fasting, groups of ten mice were administered MF extract in graded doses up to 1.8 g/kg body weight, while the control group received only the water (Pérez et al., 2012). The groups were observed for 14 days and mortality was recorded for each group at the end of the experiment.

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Using rutin as the standard, the content of total flavonoids in the extract was determined quantitatively as 89.4% by colorimetric method. The contents of 5-hydroxy-6,7-dimethoxyflavone and 5-hydroxy-7,8-dimethoxyflavone in the extract were detected by HPLC analysis (Fig. 1) to be 50.6% and 23.9%, respectively. 3.2. MF reduced pathological damages in ALI mice The mice in the normal control group showed no significant morphologic damages, indicating that intratracheal administration with saline did not induce additional inflammation response in this protocol (Fig. 2). Compared with the normal control group, the lungs of ALI mice in model control group showed marked inflammatory responses characterized by the presence of interstitial edema, hemorrhage, thickening of the alveolar wall and infiltration of inflammatory cells. Histological damage was improved by MF (30 and 90 mg/kg) treated ALI mice and DEX (10 mg/kg). However, treatment with 10 mg/kg of MF was failed to improve those histological damages in lung. It indicated that treatment of MF (30–90 mg/kg) could ameliorate the symptoms of LPS induced ALI in mice.

Fig. 1. HPLC analysis of MF. The isolated compounds were identified in the extract by comparing their retention times. The chromatograms were obtained at a wavelength of 254 nm. (A) 5-hydroxy-6,7-dimethoxyflavone and (B) 5-hydroxy-7,8-dimethoxyflavone.

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Fig. 2. Effects of MF on LPS-induced lung histopathogic changes. Lungs from each experimental group were processed for histological evaluation at 6 h after LPS challenge: (A) the lung section from the normal control group; (B) the lung section from LPS-induced ALI model group; (C) the lung section from the mice exposed to LPS and treated with dexamethasone (10 mg/kg). (D, E and F) the lung section from the mice exposed to LPS and treated with 10, 30 and 90 mg/kg of MF, respectively. Representative histological section of the lungs was stained by hematoxylin and eosin (magnification 200  ).

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Protein contents in BALF (mg/mL)

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Fig. 3. Effects of MF on the lung W/D ratio in the BALF of the LPS-induced ALI mice. Each bar was represented as mean7SD (n¼ 10). NC, normal control group; MC, model control group; PC, positive control group; MF10, ALI mice treated with MF (10 mg/kg); MF30, ALI mice treated with MF (30 mg/kg); MF90, ALI mice treated with MF (90 mg/kg). Asterisks denote the significance levels: nPo0.05, compared with MC.

3.3. MF reduced pulmonary edema in ALI mice challenged with LPS The W/D ratio and total protein concentrations in the BALF were significantly increased after LPS challenged compared with the normal control group (Fig. 3 and Fig. 4). Pretreatment with MF could significantly decrease pulmonary edema, and inhibit the increase of lung W/D ratio and protein concentrations in BALF when orally given once at the doses of 30 and 90 mg/kg, respectively. However, treatment of MF at the dose of 10 mg/kg did not decrease lung W/D ratio induced by LPS challenge. These results were in accordance with histological changes assess above.

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MPO activities in BALF (U/mL)

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Fig. 4. Effects of MF on total protein contents (A) and MPO activity (B) in the BALF of the LPS-induced ALI mice. Each bar was represented as mean 7SD (n¼ 10). NC, normal control group; MC, model control group; PC, positive control group; MF30, ALI mice treated with MF (30 mg/kg); MF90, ALI mice treated with MF (90 mg/kg). Asterisks denote the significance levels: nPo 0.05, compared with MC.

3.4. MF inhibited MPO activity in BALF of ALI mice

3.5. MF reduced LPS-induced inflammatory cytokine production

MPO activity in lungs was determined to assess the effects of MF on neutrophil accumulation. LPS challenge caused the significant increases in lung MPO activity compared with the normal control group. MF (30–90 mg/kg) and DEX (10 mg/kg) pretreatment significantly lowered the MPO activity in lungs compared with the model control group (Po0.05) as shown in Fig. 4.

To determine the inflammatory cytokine levels in response to LPS stimulation, IL-1β, IL-6, TNF-α and NO levels were measured in the serum. As shown in Fig. 5, those cytokines in the model control group were found to be dramatically increased compared with those in the normal control group. MF intervention prevented the release of those cytokines in a dose-dependent manner (P o0.05).

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Fig. 5. Effects of MF on the serum levels of inflammatory cytokines in the LPS-induced ALI mice. Each bar was represented as mean 7 SD (n¼10). NC, normal control group; MC, model control group; PC, positive control group; MF30, ALI mice treated with MF (30 mg/kg); MF90, ALI mice treated with MF (90 mg/kg). Asterisks denote the significance levels: nPo 0.05, compared with MC.

3.6. MF inhibited LPS-induced lung NF-κB and MAPK activation in ALI mice As showed in Fig. 6, LPS group showed significant IκB-α degradation and NF-κB p65 decrease in cytoplasm protein extracts but increase of NF-κB p65 expression in cytoplasm compared with the normal control group. In contrast, MF significantly inhibited translocation of p65 from cytoplasm to nuclear (P o0.05) and decreased IκB-α degradation, compared with those levels in the model control group. Compared with the normal control group, intravenous administration of LPS increased p38 MAPK activation in the lung tissue of the ALI mice. As shown in Fig. 7, MF inhibited p38MAPK significantly with total p38 unchanged compared with the model control group (P o0.05). 3.7. MF was low toxic Oral administration of MF in a dosage of 1.8 g/kg did not lead to toxic effects in mice when given daily for 14 days observation.

4. Discussion Mosla scabra is one of the main antipyretic of Chinese herbs, which has been commonly used in respiratory diseases for relieving cough and reducing sputum for thousands of years. Flavonoids as the main components of this herbal extracts possess a wide range of pharmacological properties such as antioxidant, antimicrobial and antiviral activities as well as immunity-modulation (Peterson and Dwyer, 1998; Wang et al., 2000; Wu et al., 2010; Agati et al., 2012). In our last study (published in Chinese), MF dose-dependently showed significant anti-nociceptive activity in the acetic acid-induced writhing model, and also showed significant anti-inflammatory activity in the acetic acid-induced vascular permeability and air pouch models in rats. However, there is no

information regarding the effects of MF on LPS-induced lung inflammation. To further expand the application of the agent, in this study, anti-inflammatory effects of MF had been investigated by using LPS-induced ALI model. The results indicated that MF could prevent LPS-induced pulmonary inflammatory responses and suppress secretion of pro-inflammatory mediators via inhibition of MAPK and NF-κB pathways. Endotoxin or LPS derived from Gram-negative bacteria has been well recognized as the most important pathogen that leads to the development of ALI. Intratracheal administration of LPS has been used as a common experimental model of ALI characterized by increased levels of neutrophils, protein content, cytokines and chemokines in the BALF, associating with the severity of disease (Jerala, 2007; Lu et al., 2008). Edema is a typical symptom of inflammation not only in systemic inflammation, but also in local inflammation. Widespread destruction of alveolar epithelium and neutrophils infiltration in the alveolar spaces with large amounts of proteinaceous exudates represent the typical lesion in ALI (Redl et al., 1993). MPO is an enzyme located mainly in the primary granules of neutrophils, and thus MPO activity in the parenchyma reflects the adhesion and margination of neutrophils in the lungs. To quantify the magnitude of pulmonary vascular permeability, the lung W/D ratio, and total protein concentrations and MPO activity in the BALF were evaluated. In this study, mice exposed to LPS showed obvious pulmonary edema including increased MPO activity and total protein concentrations in the lungs. But, the symptoms were significantly reduced in MF-treated group. Pretreatment with MF markedly reduced the inflammatory histological changes in lung tissues produced by LPS challenge, as well as suppressed LPS-induced neutrophil migration into the lung and MPO activity. These findings indicated that MF significantly decreased the high lung vascular permeability and has the protective effects on LPS-induced ALI. The pathogenesis of ALI/ARDS involves disorders of oxidant/ anti-oxidant and inflammation/anti-inflammation, the upregulation of adhesion molecules, and the increased production

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p-p38

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p38 Cytoplasm-p65

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Fig. 6. Effects of MF on LPS-induced NF-κB activation. MF (30 mg/kg or 90 mg/kg) was administrated orally 1 h prior to LPS administration. Nuclear and cytoplasm extracts were prepared from the lung tissues and subjected to western blotting. Blots normalized to GAPDH expression were shown. NC, normal control group; MC, model control group; PC, positive control group; MF30, ALI mice treated with MF (30 mg/kg); MF90, ALI mice treated with MF (90 mg/kg). All data were presented as mean 7SD (n¼ 6). Asterisks denote the significance levels: nPo 0.05, compared with MC.

of inflammatory cytokines (Kovach and Standiford, 2011; Kuo et al., 2011; Bhargava and Wendt, 2012). NO and the potent proinflammatory cytokines (such as TNF-α, IL-1β and IL-6) are that play a role in the occurrence and development of systemic inflammatory responses (Su et al., 2006; Wang et al., 2009). Some cytokines are the significant predictors of morbidity and mortality in patients with ARDS. Since they have been found to be the potent mediators of potentially damaging tissue responses, several mechanisms exist to ensure that the effects of these cytokines are restricted (Gando et al., 2003; Galani et al., 2010). In this study, serum levels of the cytokines NO, TNF-α, IL-1β and IL-6 were

Fig. 7. Effects of MF on LPS-induced p38 MAPK activation. MF (30 mg/kg or 90 mg/ kg) was administrated orally 1 h prior to LPS administration. Blots normalized to GAPDH expression were shown. NC, normal control group; MC, model control group; PC, positive control group; MF30, ALI mice treated with MF (30 mg/kg); MF90, ALI mice treated with MF (90 mg/kg). All data were presented as mean7 SD (n ¼6). Asterisks denote the significance levels: nPo 0.05, compared with MC.MF prevents the LPS-induced pulmonary histopathological changes and suppresses secretion of pro-inflammatory mediators via inhibition of NF-κB and MAPK pathways.

dramatically increased after LPS administration. However, pretreatment of MF (30 or 90 mg/kg) significantly lowered LPSinduced over-production of those cytokines with the lessening of pulmonary injury. These results appear to correlate well with our previous reports that MF decreased levels of pro-inflammatory cytokines in influenza A virus-induced pneumonia (Wu et al., 2010). Therefore, these results suggested that MF may be effective in treating systemic inflammatory response syndrome induced by LPS. It is well-known that expression of NO, TNF-α and IL-6 gene is dependent on the activation of transcription factor NF-κB, which plays a crucial role in immune and inflammatory responses (Lawrence and Fong, 2010). Activation of NF-κB requires phosphorylation and proteolytic degradation of the inhibitory protein IκB-α, an endogenous inhibitor that binds to NF-κB in the cytoplasm (Hayden and Ghosh, 2008). Stimulated with LPS, NF-κB is activated and translocated into the nucleus as a result of phosphorylation-mediated degradation of IκB proteins in the lung of ALI mice. However, pretreatment with MF could decrease the degradation of IκB-α and nuclear translocation of p65 NF-κB. These data demonstrate that MF specifically attenuates activation of NF-κB and downstream TNF-α, IL-8 and IL-6 production. On the other hand, MAPK, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38MAPK, also plays an important role in signal transduction pathways and regulates cytokine release (Kim and Choi, 2010). Among all of those, p38 signaling pathway is reported to be activated by pro-inflammatory cytokines such as TNF-α. In this study, western blot analysis showed that MAPK was activated in LPS-induced lung injury.

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However, MF treatment markedly suppressed LPS-induced phosphorylation of p38. The inhibition of NO, TNF-α, IL-1β and IL-6 productions by MF occurs through pathways that converge on p38 and IκB activation since these kinases have been shown to regulate cytokine production in LPS-induced acute lung injury. Some studies also showed that the extracts of Mosla dianthera, belonging to the Mosla genus, inhibits mast cell-mediated allergic reactions through inhibiting degradation of IκB-α and nuclear translocation of NF-κB and downstream TNF-α, NO and IL-6 secretion (Lee et al., 2006). Therefore, these results suggested that the activity of MF was partly dependent on the inhibition of MAPK and NF-κB signaling pathways.

5. Conclusion In summary, our results showed that pretreatment of MF significantly attenuated pulmonary inflammation in mice with LPS-induced ALI, and the protective effect of MF in ALI might be related to its suppression of NF-κB and MAPK activation, and subsequently caused a remarkable reduction in inflammatory cell infiltration, and inflammatory cytokine secretion in lung tissues. These findings suggested that MF had a protective effect on LPSinduced ALI in mice.

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