Anti-inflammatory effects and mechanisms of usnic acid , a compound firstly isolated from lichen Parmelia saxatilis

Usnic acid (UA) was isolated for the first time from Parmelia saxatilis and was confirmed by physical and spectral evidence. Its anti-inflammatory effect and mechanism were explored on lipopolysaccharide (LPS)-stimulated RAW264.7 cell line. The effects of UA on pro-inflammatory cytokines including tumor necrosis factor-alfa (TNF-α), interleukin-6 (IL-6) and interleukin-1 beta (IL-1β), pro-inflammatory mediators such as nitric oxide (NO), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) were studied by sandwich enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (PCR) and Western blot analyses. Similarly, the effect of UA on anti-inflammatory cytokine interleukin-10 (IL-10) and anti-inflammatory mediator heme oxygenase-1 (HO-1) were also studied following the same methods. Furthermore, nuclear factor-κB (NF-κB) was assayed by immunocytochemistry. The results showed that UA has anti-inflammatory effect by down-regulatinng iNOS, COX-2, IL-1β, IL-6 and TNF-α, COX-2 gene expression through the suppression of NF-κB activation and increasing anti-inflammatory cytokine IL-10 and anti-inflammatory mediator HO-1 production.


INTRODUCTION
Lichens have been used for medicinal purpose throughout the ages.As lichens are symbiotic associations between fungus and alga (photobiotic), a number of lichens were screened for antibacterial activity in the 1940s and 1950s following the discovery of penicillin from a fungus (Vartia, 1973) and several compounds were found to be active against mycobacterium species and Gram-positive bacteria.Due to significant antibacterial activity, some extracts of lichens or isolated compounds were used as antibiotics clinically.Parmelia saxatilis belonging to the genus Parmelia was widely used in folk medicine for hundreds of years to treat fever and lumbar pain.However, phytochemical (Ingolfsdotir et al., 1998) and pharmacological reports of P. saxatilis are rare.During the course of searching for anti-inflammatory substances, we found the extract of P. saxatilis having potent antiinflammatory activity.Following bioassay guided investigations on bioactive substances, Usnic acid (UA) was first isolated from P. saxatilis.
of UA have rarely been researched further (Ingolfsdtir, 2002;Li et al., 2007).Hence studying the antiinflammatory mechanism of UA was also undertaken as an objective of the present study.
Inflammation is a multiple process, mediated by activated inflammatory factors or immune cells.During its course, macrophages and monocytes usually play crucial roles in eliciting response cascade in the acute phase of inflammation (Baumann and Gauldie, 1994).After being stimulated, they produce a number of chemokines and enzymes, such as TNF-α, IL-1β (Bertolini et al., 2001;Mongan et al., 2000), IL-6 (Stadnyk et al., 1997) and IL-10 (Hofman, 2004;Inui et al., 2002), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) (Kröncke et al., 1998;Kim et al., 2003), and antiinflammatory mediator (HO-1) (Ogborne et al., 2004), which is macrophage or monocyte-related cytokine, essential for the inflammatory response to pathogenic germs or toxicants.Activation of pro-inflammatory as well as anti-inflammatory cytokines and mediators is the key procedure of inflammatory reaction which subsequently lead to inflammatory impairment and restoration (Naldini and Carraro, 2005).It is well known that nitric oxide (NO) is synthesized by iNOS and involves in diverse physiological processes.An excess NO production is largely thought of causing a variety of inflammatory diseases (Clancy et al., 1998).NF-κB is a nuclear transcription factor regulating the expression of various genes including IL-1β, IL-6, TNF-α and iNOS, which play critical roles in apoptosis, tumor genesis and various autoimmune diseases and inflammation (Lawrence et al., 2001;Riehemann et al., 1999;Renard and Raes, 1999).Thus, suppression of these factors may be an effective therapeutic strategy for preventing inflammatory reaction and diseases (Makarov, 2000).The variation of NF-κB activity could be used for reflecting the anti-inflammatory effect of the inhibitors.
In this study, LPS-stimulated RAW264.7 cell line was used as an inflammation cellular model to explore the anti-inflammatory effect of UA and anti-inflammatory mechanism (Kim et al., 2003).

Plant
The lichen P. saxatilis was purchased from Hubei province herb Co. Ltd.China and identified by Professor Keli Chen (Hubei College of Traditional Chinese Medicine).

Extraction and isolation
The whole air-dried plants (1.0 kg) were extracted at room temperature with 95% ethanol for 2 days.After removal of ethanol in vacuum, the residue was extracted with petroleum ether, then the petroleum ether fraction was condensed in vacuum and the yellow crystal was precipitated.The filtered crystalline precipitate was washed by e4thyl acetate for several times and then recrystallizated with double-distilled water and ethyl acetate.The structure of the crystal was determined using spectroscopic techniques including electron ionization mass spectrometry (EI-MS), 1 H and 13 C NMR, heteronuclear multiple bond correlation (HMBC) spectrometry and X-ray diffraction analysis (Figure 1).

Preparation of cells
The cells were maintained in RPMI 1640 medium supplemented with 100 U/ml of penicillin, 100 μg/ml of streptomycin and 10% fetal bovine serum.Cells were cultured with 5% CO2 in humidified air at 37°C.The crystal of UA was accurately weighed and diluted by 1640 medium to concentrations of 10, 5 and 1 μg/ml, which were then used for treatment in LPS-stimulated RAW 264.7 cell line.Before treatment by LPS, the cells were inoculated into 6, 24 or 96 micro-well plates.24 h later, the cells were observed to be adhering to the bottom of wells; the cell supernatants were disposed from the wells, and then 10 ng/ml LPS with prepared different doses of UA was added to the wells: 10, 5 and 1 μg/ml.There were four kinds of control groups, including positive control group where cells treated with DEXAMETHASONE (DM, 0.5 μg/ml), negative control group, cells treated with Astragalus polysaccharides (APS, 100 μg/ml), blank control group, cells only stimulated by LPS (10 ng/ml) and normal control group where cells incubated by 1640 medium.

MTT assay for the measurement of cell proliferation
Cytotoxic effect of UA was evaluated by conventional MTT assay (Mosmann, 1983).Meanwhile, cell cytopathic effect (CPE) test of the cells was studied using the microplate.Cytotoxicity determinations were conducted by seeding RAW 264.7 cells into microplates at 4 ×10 3 cells per well.After an overnight incubation, compound in MEM containing 10% fetal bovine serum (FBS) was added (contents of the compound were 10, 50 and 100 μg/ml, respectively).
Cells were allowed to grow for an additional 24 h.At 4 h prior to culture termination, 20 μl of the MTT solution (5 mg/ml in phosphate-buffered saline, pH 7.4) was added and the cells were continuously cultured until termination.150 μl dimethyl sulfoxide (DMSO) was added into each well for the solubilization.The optical density (OD) at 490 nm was measured by a Spectramax 250 microplate reader.

Detection of cytokines TNF-α，IL-1β , IL-6 and IL-10 production
After stimulation and treatment on RAW 264.7 cells by LPS with different concentrations of UA for 24 h, cell supernatants of the cell culture were collected and assayed for TNF-α, IL-1β, IL-6 and IL-10 by the sandwich ELISA method with ELISA kits according to the instructions provided by the manufacturer.

Determination of NO production
Levels of NO were determined by the Griess reaction (Green et al., 1982).The samples were assayed in triplicate by a nitrite detection kit according to instructions provided by the manufacturer, and a standard curve was generated in each experiment using NaNO2.

Quantitative real-time PCR for detecting mRNA of TNF-α, COX-2, iNOS and HO-1
TRIzol Reagent (Gibco BRL) was added according to manufacturer's protocol and stored at −80°C before use.The total RNA for detection of pro-inflammatory factors TNF-α, iNOS and COX-2 were extracted at 4 h after the cells were stimulated with LPS and treated by UA; 18 h after the cells stimulation and treatment was for extracting the RNA of HO-1.Quantitative realtime polymerase chain reaction (PCR) was performed in a LightCycler instrument (Roche Diagnostics, Mannheim, Germany) with the FastStart DNA Master SYBR Green I kit (also from Roche), and the results were analyzed with LDCA software supplied with the machine (Livak and Schmittgen, 2000) [23].The primers used were detailed in Table 1 (Zhao et al., 2007).

Western blot analysis of COX-2 and HO-1
RAW 264.7 cells were incubated with or without LPS in the presence or absence of UA for 24 h.The cells were harvested, washed twice with ice-cold phosphate-buffered saline (PBS), and resuspended in PBS containing 0.1 mM phenylmethylsulfonyl fluoride.They were laid by three cycles of freezing and thawing in liquid nitrogen.The cytokine fractions were obtained from each supernatant after 12,000 g centrifugation at 4°C for 20 min.Samples (30 μg protein) were separated on 8% SDS-PAGE and transferred to nitrocellulose membranes.The membranes were blocked with 5% nonfat milk in TBST (0.1%) for 1 h and then incubated with polyclonal antibody for goat HO-1 (1:6000 dilutions) or polyclonal antibody for COX-2 (1:6000 dilutions) in TBST containing 1% nonfat milk for 1 h.The membranes were then hybridized with secondary antibody conjugated with horseradish peroxidase (anti-rabbit and anti-mouse IgG-HRP, 1:2000 dilutions, Santa Cruz Biotechnology, USA) for 1 h and washed five times with TBST.The membranes were immediately incubated with ECL detection kits (Pierce Biotechnology Co., Ltd, USA) for 2 min and later exposed to X-ray film.

Immunocytochemical assay on NF-κB
Covers slips were placed in the wells to allow the cells crawl on them.After treatment with LPS and UA, the cells were washed, fixed and blocked.The first antibody (anti-mouse NF-κB IgG) (diluted to 1:100) was added then.12 h later, the cells were mixed with the horseradish peroxidase-labelled second antibody (biotinylated anti-IgG) (diluted to 1:75) at room temperature for 30 min.Then the colorization with diaminobenzidine, counterstaining with hematoxylin, ethanol dehydration (orderly by 75, 95 and 100%), dimethyl benzene clearance and mounting observation were performed in sequence.Each sample was randomly chosen at 5 visual fields in microscope and positive cells were counted.NF-κB was detected 24 h after treatment with UA.

Statistical analysis
Student's t-test was used in determining the statistical significance of differences between the values for the various experimental and control groups.Data were expressed as means ± standard deviation (SD) and p-values of 0.05 or less were considered to be statistically significance.

RESULTS AND DISCUSSION
From petroleum ether fraction of ethanol extract of P. saxatilis, a crytalline compound was isolated which was confirmed as usnic acid by comparing its physical and spectral data with authentic literature (Kumar et al., 1996).It is the first time that usnic acid was found occuring in P. saxatilis.

In vitro cytotoxicity of UA from P. saxatilis
Pre-treatment of unstimulated RAW264.7 cells with UA of no more than 10 μg/ml for 24 h did not significantly affect cell viability.The cytopathic effect (CPE) test also gave the same result (the content of the compound was no more than 10 μg/ml) (Figure 2).The result suggested that UA have no cytotoxicity to RAW264.7 cell line.

Effects of UA on the pro-inflammatory cytokines and mediators produced by LPS stimulation
After the cells were stimulated by LPS and treated with UA for various times, it was found that UA significantly decreased the secretion of TNF-α, IL-1β, IL-6 and NO compared to single LPS stimulation (p < 0.01 or p < 0.05) (Figure 3a to d).Furthermore, the higher the dose of UA, the greater the influence on antagonizing proinflammatory cytokines, such results showed a dosedependent relation of UA on anti-inflammatory effect.Similarily, UA strikingly decreased the levels of COX-2 protein, TNF-α mRNA and COX-2 mRNA compared to blank control (p < 0.01 or p < 0.05) (Figure 3e to g), which suggested that UA might control pro-inflammatory factors production not only at levels of proteins but also at transcriptional and translational level.Furthermore, the effect of UA on iNOS mRNA (Figure 3h) was coincidental with that on NO (Figure 3d).

Effects of UA on anti-inflammatory cytokines and mediators produced by LPS stimulation
The IL-10 levels of cells treated with UA were similar to that of DM treatment, which showed UA and DM had a similar inhibitory effect on anti-inflammatory cytokine (Figure 4a).The levels of HO-1 mRNA and protein in UA treatment and DM and APS treatment cells were significantly higher than those in single LPS stimulation and normal group cells (p < 0.05) (Figure 4b and c).Furthermore, the higher the dose of UA, the greater the effect on expression of HO-1 mRNA and protein, which suggested that UA might promote regression of inflammation and control anti-inflammatory mediators production at levels of proteins and by transcriptional and translational in a dose-dependent manner.

Effect of UA on NF-κB of usnic acid
NF-κB activation was significantly blocked by UA (Figure 5), which suggested that the suppression of IL-1β, IL-6, TNF-α, iNOS and COX-2 gene expression by UA might be due to the attenuation of NF-κB activation.Though UA is commonly occuring in Lichens, this is the first time that the compound is isolated from P. saxatilis.We firstly found that UA could inhibit TNF-α, IL-1β and IL-6 in LPSstimulated RAW 264.7 cells; these pro-inflammatory cytokines and mediators possess a multitude of biological activities linked to the immunopathology of acute or chronic inflammatory diseases (Bertolini et al., 2001;Plutzky, 2001).These results suggested that the observed changes of proinflammatory cytokines productions might be associated with anti-inflammatory effect of UA.Furthermore, we studied the effect of UA on anti-inflammatory cytokines and mediators, such as IL-10 and HO-1 (Zhao et al., 2007), and found that it could increase the production of IL-10 and HO-1 protein (and gene levels, which illustrates the dual effects of UA on antagonizing antagonizing pro-inflammatory and augmenting antiinflammatory mediators. The results also showed that UA significantly inhibited the gene expression of iNOS and COX-2, which resulted in the suppression of NO production in LPS-stimulated RAW264.7 cells.Meanwhile, NF-κB is a nuclear transcription factor that regulates the expression of various genes, including IL-1β, TNF-α, iNOS and COX-2 (Figures 6 to 14) during inflammation process.We also investigated whether UA could inhibit NF-κB production or not.The

Conclusion
The molecular mechanisms on anti-inflammation of UA firstly isolated from P. saxatilis had been addressed in this study.The strong anti-inflammatory activity was down-regulating pro-inflammatory cytokines and mediators via suppressing NF-kB and up-regulating antiinflammatory factors of IL-10 and HO-1.A further evaluation of UA on anti-inflammation is undergoing.

Figure 2 .Figure 3 .
Figure 2. Results of CPE of usnic acid (UA).(a) The cell before treatment by UA, (b) The cell after treatment by UA (1 × 40 times in microscope).

Figure 4 .Figure 5 .
Figure 4. Effects of UA on IL-1β production.RAW 264.7 cells were treated with LPS (10 ng/ml) in the presence of various concentrations of UA.IL-1β was measured in the cell culture media by sandwich ELISA.Data shown were the mean ± SD (n = 3).*p < 0.05; **p < 0.01 versus LPS alone, # p < 0.05 or ## p < 0.01 compared to normal cell.

Figure 6 .Figure 7 .
Figure 6.Effects of UA on on NO production.RAW264.7 cells were treated with LPS (10 ng/ml) in the presence of various concentrations of UA.NO levels were measured in the cell culture media by ELISA.Data shown were the mean ±SD (n = 3).*p < 0.05; **p < 0.01 versus LPS alone, # p < 0.05 or ## p < 0.01 compared to normal cell.