key: cord-339272-trd6rkxw
authors: Chen, Na; Wu, Qianchao; Chi, Gefu; Soromou, Lanan Wassy; Hou, Jinli; Deng, Yanhong; Feng, Haihua
title: Prime-O-glucosylcimifugin attenuates lipopolysaccharide-induced acute lung injury in mice
date: 2013-04-24
journal: Int Immunopharmacol
DOI: 10.1016/j.intimp.2013.04.014
sha: 
doc_id: 339272
cord_uid: trd6rkxw

Prime-O-glucosylcimifugin is an active chromone isolated from Saposhnikovia root which has been reported to have various activities, such as anti-convulsant, anticancer, anti-inflammatory properties. The purpose of this study was to evaluate the effect of prime-O-glucosylcimifugin on acute lung injury (ALI) induced by lipopolysaccharide in mice. BALB/c mice received intraperitoneal injection of Prime-O-glucosylcimifugin 1 h before intranasal instillation (i.n.) of lipopolysaccharide (LPS). Concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and interleukin (IL)-6 in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA). Pulmonary histological changes were evaluated by hematoxylin–eosin, myeloperoxidase (MPO) activity in the lung tissue and lung wet/dry weight ratios were observed. Furthermore, the mitogen-activated protein kinases (MAPK) signaling pathway activation and the phosphorylation of IκBα protein were determined by Western blot analysis. Prime-O-glucosylcimifugin showed promising anti-inflammatory effect by inhibiting the activation of MAPK and NF-κB signaling pathway.

The acute respiratory distress syndrome (ARDS), a clinically important complication of severe acute lung injury (ALI) in humans, highly associated with sepsis pneumonias and severe acute respiratory syndrome (SARS) is a significant cause of morbidity and mortality in critically ill patients [1] [2] [3] . Inflammatory stimuli from microbial pathogens, such as endotoxin (lipopolysaccharide [LPS]), are well recognized for their ability to induce pulmonary inflammation, and experimental administration of LPS, has been used to induce pulmonary inflammation in animal models of ALI [4] [5] [6] . The development of an ALI model by way of i.n. LPS instillation is well suited for preliminary pharmacological studies of new drugs or other therapeutic agents because i.n. instillation of LPS into mice can produce a controlled ALI without causing systemic inflammation and multi-organ failure [4, 7] . LPS-induced ALI is considered a neutrophil-dependent ALI that contributes to local recruitment and activation of neutrophils [8] ; the release of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6; and the formation of reactive oxygen and nitrogen species [9] [10] [11] . Neutrophil recruitment in the lungs is regarded as a histological hallmark in the progression of ALI [12] . Several candidate therapy strategies such as fluid management, surfactants, glucocorticoids, and stem cells have been applied to treat acute lung injury and acute respiratory distress syndrome in the last decade [13, 14] . However, the mortality resulting from these conditions remains high [15] .

Fangfeng, the root of Saposhnikovia divaricata (Turcz) Schischk, is widely applied for headache, febrility, vertigo and arthralgia as an important member of traditional Chinese medicines (TCMs). It has been proved that there are numerous pharmacologic effects of the extract from Fangfeng, such as suppression of adjuvant arthritis, inhibitory effects on the peptic ulcers and analgesic, anti-convulsant, anticancer, anti-inflammatory and anticoagulant activities, etc in modern pharmacological experiments [16] . Abundant compounds were isolated from it, such as chromones, conmarins, and polyacetylenes [17, 18] . Prime-O-glucosylcimifugin is a major active chromone isolated from Fangfeng. In recent years, it has been identified that chromones are the main active components which contribute most to its pharmacological efficacy [19] , but so far, the anti-inflammatory effects of Prime-O-glucosylcimifugin on ALI has not yet been studied. Therefore, with a mouse model of acute lung inflammation, the present study was undertaken to examine the effect of Prime-O-glucosylcimifugin on acute lung injury induced by intranasal instillation of LPS in BALB/c mice and investigate its possible mechanisms.

Prime-O-glucosylcimifugin was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Jilin, China). Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), penicillin and streptomycin for cell culture were purchased from Invitrogen-Gibco (Grand Island, NY, USA). 3-(4, 5dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and lipopolysaccharide (LPS) (Escherichia coli 055:B5) were purchased from Sigma Chemical Co. (San Diego, CA, USA). Rabbit polyclonal anti-p44 ERK, mouse monoclonal phosphospecific p42-p44 ERK antibodies, rabbit polyclonal anti-p54 JNK, mouse monoclonal phospho-specific p46-p54 JNK antibodies, rabbit polyclonal anti-p38, mouse monoclonal phospho-specific p38 antibodies, rabbit mAb IκBα and mouse mAb phospho-IκBα were purchased from Cell Signaling Technology Inc. (Beverly, MA). HRP-conjugated goat antirabbit and goat-mouse antibodies were provided by GE Healthcare (Buckinghamshire, UK). Mouse TNF-α, IL-6 and IL-1β enzyme-linked immunosorbent assay (ELISA) kits were purchased from Biolegend (CA, USA). The myeloperoxidase (MPO) determination kit was purchased from Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu Province, China). All other chemicals were of reagent grade.

BALB/c male mice, 8 weeks old and weighing approximately 18 to 20 g, were purchased from the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China), and maintained at an animal facility under pathogen free conditions. The mice were fed a standard diet and water ad libitum and housed in microisolator cages under standard conditions (temperature: 24 ± 1°C, relative humidity: 40%-80%). The mice were allowed to adapt themselves to their environment for 2-3 days before experimentation. All animal experiments were performed in accordance with the National Institutes of Health (NIH) guide for the Care and Use of Laboratory Animals and approved by the Jilin University animal administration committee.

The RAW 264.7 mouse macrophage cell line was obtained from the China Cell Line Bank (Beijing, China). Cells were cultured in complete medium (DMEM supplemented with 10% heat-inactivated FBS, 3 mM glutamine and antibiotics (100 U/mL penicillin and 100 U/mL streptomycin)) at 37°C in a humidified incubator containing 5% CO 2 and 95% air. Cells were treated with various concentrations of Prime-O-glucosylcimifugin for 1 h followed by stimulation with LPS (1 mg/L).

Cytotoxicity studies induced by Prime-O-glucosylcimifugin were evaluated by the MTT assay. RAW 264.7 macrophages were seeded in 96-well plates at a density of 4 × 10 5 cells/mL in complete medium and incubated for 1 h (100 μL/well). Then the cells were treated with different concentrations of Prime-O-glucosylcimifugin (0-100 μg/mL, 50 μL/well) for 1 h, followed by stimulation with LPS (1 mg/L, 50 μL/well) for 18 h. After 18 h, 10 μL MTT (5 g/L) was added to each well and the cells were further incubated for 4 h. The supernatant was removed and the cells were lysed with 150 μL/well DMSO. The optical density was measured at 570 nm on a microplate reader.

RAW 264.7 cells were seeded in 24-well plates (4 × 10 5 cells/mL) and treated with 12.5, 25 or 50 μg/mL of Prime-O-glucosylcimifugin for 1 h prior to stimulation of 1 mg/L LPS for 24 h in a 37°C, 5% CO 2 incubator. Cell-free supernatants were collected and assayed. The concentrations of TNF-α, IL-1β and IL-6 in the supernatants of RAW 264.7 cell cultures were measured by using an ELISA kit, according to the manufacturer's instructions (BioLegend, Inc., Camino Santa Fe, Suite E, San Diego, CA, USA).

RAW 264.7 cells (4 × 10 5 cells/mL) plated onto 6-well plates were incubated for 24 h and treated with 12.5, 25 or 50 μg/mL of Prime-O-glucosylcimifugin for 1 h and then stimulated with 1 mg/L of LPS for 30 min. The cells were collected and washed three times with ice-cold PBS. The cells were treated with a cell lysis buffer [50 mM Tris (pH 7.6), 150 mM NaCl, 5 mM EDTA (pH 8.0), 0.6% NP-40, 1 mM Na3VO4, 20 mM β-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 2 mM p-nitrophenyl phosphate, and 1:25 Complete Mini Protease Inhibitor cocktail (Boehringer, Mannheim, Germany)] and kept on ice for 30 min. The cell lysates were centrifuged (12,000 g at 4°C) for 5 min to obtain a cytosolic fraction. The protein concentration was determined by BCA protein assay kit (Beyotime, Haimen, China). Aliquots of the lysates were separated on 10% sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) and then electroblotted onto a polyvinylidene difluoride (PVDF) membrane. The blots were blocked with 5% (w/v) non-fat dry milk for 2 h at 37°C, followed by incubation with specific primary antibody at 4°C overnight. Blots were washed with Tween 20/Tris-buffered saline [TTBS, 20 mM Tris-HCl buffer, pH 7.6, containing 137 mM NaCl and 0.05% (vol/vol) Tween 20] and incubated with a peroxidase-conjugated secondary antibody for 1 h. Blots were again washed with TTBS and the immunoactive proteins were detected using ECL plus (Thermo, USA).

The mice were randomly divided into five groups: Control group; LPS group; LPS + Prime-O-glucosylcimifugin (2.5, 5 or 10 mg/kg bodyweight). Prime-O-glucosylcimifugin was given intraperitoneally. One hour later, LPS group and LPS + Prime-O-glucosylcimifugin group mice were given 50 μL LPS intranasally (i.n) (200 mg/L) to induce acute lung injury. Control mice were given 50 μL PBS intranasally (i.n) without LPS.

All the mice were alive after 7 h LPS stimulation. Collection of BALF was performed three times through a tracheal cannula with 0.5 mL of autoclaved PBS, instilled up to a total volume of 1.3 mL. BALF was centrifuged (4°C, 3000 rpm, 10 min) to pellet the cells. The concentrations of TNF-α, IL-1β and IL-6 in the BALF were measured using sandwich enzyme-linked immunosorbent assay (ELISA) kits according to the protocol recommended by the manufacturer.

The cell pellets were resuspended in PBS, and the total cell number was counted using a standard hemocytometer. Differences in cell numbers were examined by counting on a smear prepared by Wright-Giemsa staining.

Seven hours after intranasal instillation of LPS, the mouse lungs were excised, and immediately weighed to obtain the wet weight. The dry weight was determined after heating the lungs at 80°C for 48 h. The W/D ratio was calculated by dividing the wet weight by the dry weight.

MPO activity, which reflects the parenchymal infiltration of neutrophils and macrophages, was measured as described previously [20, 21] . Mice were killed 7 h after LPS administration under diethyl ether anesthesia. Lung tissues were homogenized in 50 mM hydroxyethyl piperazine ethanesulfonic acid (HEPES) (pH 8.0) containing 0.5% cetyltrimethyl ammonium bromide (CTAB) and subjected to three freeze-thaw cycles. The homogenate was centrifuged at 13,000 ×g for 30 min at 4°C, and the cell-free extracts were stored at − 20°C until further use. The MPO activity was assayed using a mouse MPO ELISA kit. Samples were diluted in phosphate citrate buffer (pH 5.0).

Histopathologic examination was performed on mice that were not subjected to BALF collection. The lungs were excised and fixed in 10% buffered formalin. Then the tissues were dehydrated with graded alcohol, embedded in paraffin and sliced. The sections were stained with hematoxylin and eosin (H&E) and pathological changes of lung tissues were observed under a light microscope. 

All values are presented as mean ± SD. Data were entered into a database and analyzed using SPSS software (SPSS for Windows version 13.0, Chicago, USA) and comparison between groups was made with one-way ANOVA (Dunnett's t-test) and Student's t-test. P-values of 0.05 or less were considered statistically significant.

To assess the suitable concentration of Prime-O-glucosylcimifugin for the study, RAW 264.7 cells were incubated with Prime-Oglucosylcimifugin at concentrations ranging from 12.5 to 100 mg/L in the absence or presence of LPS and cell viability was measured by MTT test 18 h later. The results showed that Prime-O-glucosylcimifugin at concentrations from 12.5 to 100 mg/L had no cytotoxic effect on RAW 264.7 cells (Fig. 1) .

TNF-α, IL-1β, IL-6 and IL-10 concentrations in the culture supernatant of RAW 264.7 macrophages were measured by ELISA kits (Fig. 2) . RAW 264.7 macrophages treated with LPS alone produced significant amounts of TNF-α, IL-1β, IL-6 and IL-10 compared to the control group. However, the production of TNF-α was slightly decreased while the levels of IL-1β and IL-6 were significantly inhibited in a dose-dependent manner when the cells were treated with 12.5, 25 or 50 mg/L of Prime-O-glucosylcimifugin ( ⁎ P b 0.05, ⁎⁎ P b 0.01). In contrast, the concentrations of IL-10 was significantly increased when the cells were treated with 50 mg/L of Prime-O-glucosylcimifugin (P b 0.05 ⁎ ).

In order to investigate the mechanism by which Prime-Oglucosylcimifugin inhibits LPS-induced cytokine production, we examined the levels of LPS-induced phosphorylation of ERK1/2, JNK and p38 MAPK in the cytoplasm by Western blotting analysis using three different phosphor-special antibodies. In our study, Fig. 3A showed that LPS stimulation significantly increased the phosphorylation of ERK1/2, JNK and p38. However, Prime-O-glucosylcimifugin inhibited the phosphorylation of ERK1/2, JNK and p38 in LPS-induced cells. There were no changes in the expression of non-phosphorylated MAPKs among groups. Furthermore, we examined the effect of Prime-O-glucosylcimifugin on IκBα phosphorylation and degradation. The results showed that LPS-induced IκBα degradation was inhibited after pretreatment with Prime-O-glucosylcimifugin in a dose-dependent manner (Fig. 3B ).

BALF was collected at 7 h after LPS administration and the cytokine levels in BALF were measured by ELISA according to the manufacturer's instructions and as described in the Materials and methods section. The levels of TNF-α, IL-1β and IL-6 in BALF were increased dramatically compared with control group (Fig. 4) . However, pretreatment with Prime-O-glucosylcimifugin (2.5, 5 or 10 mg/kg) significantly down-regulated the levels of TNF-α, IL-1β and IL-6 in a dose-dependent manner ( ⁎ P b 0.05, ⁎⁎ P b 0.01).

Seven hours after LPS administration, the BALF was collected to evaluate the total cell counts and the number of inflammatory cells in BALF, such as macrophages and neutrophils. As shown in Fig. 5 , LPS challenge markedly increased the number of total cells, neutrophils, and macrophages compared to the control group (P b 0.01). In addition, pretreatment with Prime-O-glucosylcimifugin was found to significantly decrease the number of total cells, neutrophils and macrophages ( ⁎ P b 0.05, ⁎⁎ P b 0.01).

To evaluate LPS-induced changes in pulmonary vascular permeability to water, we evaluated the wet weight to dry weight ratio of the lungs. The lung W/D ratio was evidently higher at 7 h after LPS administration compared with the control mice as illustrated ( ⁎⁎ P b 0.01). Pretreatment of mice with Prime-O-glucosylcimifugin significantly reduced the water gain (Fig. 6) ( ⁎ P b 0.05, ⁎⁎ P b 0.01) . BALF was collected at 7 h following LPS challenge to analyze the inflammatory cytokines TNF-α (Fig. 4A) , IL-1β (Fig. 4B ) and IL-6 (Fig. 4C) . The values presented are the mean ± SEM (n = 6 in each group). ## P b 0.01 vs. control group, ⁎ P b 0.05, ⁎⁎ P b 0.01 vs. LPS group.

The MPO activity (Fig. 7) was determined to assess the effects of Prime-O-glucosylcimifugin on neutrophil accumulation within pulmonary tissues. LPS challenge resulted in significantly increased lung MPO activity compared with the control group ( ⁎⁎ P b 0.01). However, this increase was reduced by Prime-O-glucosylcimifugin ( ⁎ P b 0.05, ⁎⁎ P b 0.01).

To evaluate the effect of Prime-O-glucosylcimifugin on ALI, we observed histological changes after Prime-O-glucosylcimifugin treatment in LPS-treated mice. In the LPS group, the lungs were significantly damaged with inflammatory cell infiltration, alveolar wall thickening and interstitial edema. In contrast, Prime-O-glucosylcimifugin (2.5, 5 or 10 mg/kg) was found to decrease these histopathological changes (Fig. 8) .

The results of this study indicate that Prime-O-glucosylcimifugin had a promising anti-inflammatory activity. Treatment with Prime-O-glucosylcimifugin before LPS challenge can attenuate LPS-induced inflammatory responses in RAW 264.7 cells and significantly protect mice against LPS-induced ALI.

The study primarily focused on anti-inflammatory effects of Prime-O-glucosylcimifugin on LPS-stimulated RAW 264.7 cells. LPS, a glycolipid that constitutes the major portion of the outermost membrane of Gram-negative bacteria, can bind to the cell membrane receptor of the monocytes/macrophages and endothelial cells, then activate the signal-transduction system, thus resulting in the synthesis and release of cytokines and inflammatory mediators [22] . Excessive production of pro-inflammatory cytokines not only enhances immune responses by fighting invading pathogens, but also has deleterious effects like perturbing regular hemodynamic and metabolic balances [23] . Thus, it is an important target in the treatment of inflammatory diseases to inhibit the pro-inflammatory mediator. As shown in Fig. 2 , the levels of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 were down-regulated while the level of IL-10 was up-regulated in the Prime-O-glucosylcimifugin group. IL-10, an anti-inflammatory, is well known to down-regulate the production of TNF-α, IL-1β, IL-6, IL-12 and NO [24] [25] [26] . A number of cytokines in combination with endotoxin can cause expression of inducible nitric oxide synthase (iNOS) in macrophages [27] . Inducible NOS is an important pharmacological target in inflammatory and mutagenesis research. It has been proved that Prime-O-glucosylcimifugin had inhibitory effect on the synthesis of NO induced by LPS in RAW 264.7 cells [28, 29] . Combined with the finding of our study, it seems like Prime-O-glucosylcimifugin inhibits the synthesis of NO by producing IL-10 in RAW 264.7 cells. The anti-inflammatory actions of IL-10 can interfere with the production of pro-inflammatory cytokines through the suppression of NF-κB activation by preserving the expression of IκB protein. NF-kB is a key transcriptional factor involved in regulating the expression of proinflammatory mediators, including cytokines, chemokines, and adhesion molecules, thereby playing a critical role in mediating inflammatory responses [30, 31] . Under resting conditions, NF-κB is held inactive by IκB. However, NF-κB can be activated by some stimulation of various receptors including TNF receptor, Toll-like receptors (TLRs) and T-cell receptor (TCR). Persistent activation of NF-κB is central to the pathogenesis of many inflammatory lung disorders including chronic obstructive pulmonary, asthma, pneumonia, and acute lung injury [32] . The activation of NF-κB is implicated in the MAPK signaling pathway. Inhibition of MAPK family pathway, such as ERK, p38, and JNK, alleviates the production of pro-inflammatory cytokines [33] . Therefore, we investigate the possibility that Prime-O-glucosylcimifugin inhibits the production of TNF-α, IL-1β, and IL-6 via interfering with the activation of MAPK and NF-κB. The results showed that Prime-Oglucosylcimifugin obviously not only inhibited NF-κB activation, but also inhibited LPS-induced phosphorylation of ERK1/2, JNK and p38 in RAW 264.7 cells (Fig. 3) . Based on the above observations, our results suggest that Prime-O-glucosylcimifugin had antiinflammatory ability by suppressing the expression of pro-inflammatory cytokines through blocking the activation of MAPK and NF-κB pathways in vitro. Acute lung injury is characterized by systemic airway inflammatory response including cytokines (e.g., TNF-α, IL-6, IL-8), chemokines, pro-inflammatory mediators and a variety of cells, which regulate the migration and pulmonary infiltration of neutrophils into the interstitial tissue [34] . Neutrophils are an important component of the inflammatory response that characterizes acute lung injury (ALI) [35] . Once an inflammatory response is initiated, neutrophils are the first cells to be recruited to sites of infection or injury. Although neutrophils have beneficial actions in eradicating microbial infections, excessive neutrophil activation, with resultant release of cytokines and other pro-inflammatory mediators, results in tissue injury and contributes to the development of organ dysfunction, such as ALI [9] .

High levels of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6, perform a central role in the initiation and propagation of the inflammatory cascade in LPS-induced ALI [36] . Cytokines, such as TNF-α, IL-1β, IL-6, IL-8, and IL-10, that are secreted by alveolar macrophages stimulate more chemotaxis and attract more neutrophils to injured lungs [37] [38] [39] . In our study, the levels of TNF-α, IL-1β and IL-6 in BALF were lower in the Prime-O-glucosylcimifugin group than in the LPS group. These reductions may have contributed to the decreased neutrophil count in BALF in the LPS-induced ALI model treated with Prime-O-glucosylcimifugin.

Edema is a typical symptom of inflammation both in systemic inflammation and in local inflammation [40] . Permeability edema which accompanies acute lung injury, severe pneumonia and acute respiratory distress syndrome (ARDS) is associated with alveolar fluid clearance capacity reduction, increase of capillary endothelial permeability, and alveolar epithelial barrier disruption [41] . To quantify the magnitude of pulmonary edema, we determined the W/D ratio of the lung tissue and Prime-O-glucosylcimifugin was shown to inhibit this ratio. On the other hand, MPO is an enzyme located mainly in the primary granules of neutrophils, thus MPO activity in the parenchyma reflects the adhesion and margination of neutrophils in the lung [42] . In the LPS-induced ALI model, a large amount of PMN is recruited from peripheral blood into the lung, producing a substantial amount of MPO and reactive oxygen derivatives, and finally resulting in a cascade-like response and tissue damage [12, 43] . By contrast, administration of Prime-O-glucosylcimifugin markedly suppressed LPSinduced BALF neutrophilia (Fig. 6 ) and MPO activity (Fig. 7) , as well as ameliorated the histopathologic changes in lung tissue produced by LPS challenge (Fig. 8) . Recent studies that have been conducted on pravastatin [44] , ceftiofur [45] , and pinocembrin [46] showed the same regulatory effects as Prime-O-glucosylcimifugin, suggesting that this agent may be an important regulator of inflammatory responses. Taken together, these results indicate that the protective effects of Prime-Oglucosylcimifugin on acute lung injury in a mouse model induced by LPS may be due to its inhibition of inflammatory mediators and limitation of inflammatory response in the lung.

In conclusion, the findings of this study showed that Prime-Oglucosylcimifugin has a promising anti-inflammatory effect and a protective effect against LPS-induced ALI. Pretreatment with Prime-O-glucosylcimifugin inhibited the release of in vitro and in vivo inflammatory responses by counteracting MAPK and NF-κB activation. These findings strongly suggest that Prime-O-glucosylcimifugin has a potent anti-inflammatory activity and may represent a novel strategy for the modulation of inflammatory responses. Further studies are warranted to investigate the clinical usefulness of Prime-Oglucosylcimifugin.

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This work was supported by grants from the National Nature Science Foundation of China (No. 30972212) and the China Postdoctoral Science Foundation (No. 20090461034) .