Protective effect of the methanol extract from Cryptotaenia japonica Hassk. against lipopolysaccharide-induced inflammation in vitro and in vivo
© Kang et al.; licensee BioMed Central Ltd. 2012
Received: 18 May 2012
Accepted: 10 October 2012
Published: 30 October 2012
In folk medicine, the aerial part of Crytotaenia japonica Hassk. (CJ), is applied for treatment of the common cold, cough, urinary problems, pneumonia, and skin rashes. In this paper, the in vitro and in vivo anti-inflammatory activity of CJ methanol extract was tested using lipopolysaccharide (LPS)-induced inflammatory models.
We measured nitric oxide (NO), inducible NO synthase (iNOS), and inflammatory cytokine levels from LPS-stimulated mouse peritoneal macrophages. Also, several cellular signaling molecules which regulate the expressions of these inflammatory markers were examined. Finally, we tested whether oral administration of CJ methanol extract might affect the serum cytokine levels in LPS-injected mice.
CJ methanol extract reduced NO release via iNOS protein inhibition. The extract was also shown to decrease the secretions of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12. Analysis of signaling molecules showed that CJ inhibited the phosphorylation of STAT1, p38, JNK and ERK1/2 as well as IκBα degradation. Finally, CJ decreased the serum levels of TNF-α and IL-6 in LPS-injected mice.
Our results demonstrated the anti-inflammatory activity of CJ methanol extract and its possible underlying mechanisms that involve modulation of IκBα, MAPK, and STAT1 activities.
KeywordsCryptotaenia japonica Hassk. Inflammation Macrophages iNOS Cytokines Signaling
Cryptotaenia japonica Hassk. (CJ) belongs to the Apiaceae family and is a perennial plant distributed in Asia and North America. The aerial part of the plant is used both as a vegetable and a medicinal herb. In folk medicine, CJ is applied for treatment of the common cold, cough, urinary problems, pneumonia, and skin rashes.
Macrophages are professional phagocytes that reside in tissues throughout the body to remove cellular debris and effete cells generated under physiologic conditions. Macrophages also constitute the major cellular components of the inflammatory response. Pathogenic microbes, their byproducts, and host-derived cytokines or other secreted products can stimulate macrophages. Whether the source of insult comes from within or outside the body, the receptors and subsequent signaling molecules employed are similar, resulting in the production of lipid mediators and inflammatory cytokines. However, these responses must be strictly controlled as they may damage healthy tissue and lead to chronic inflammatory disorders such as autoimmune disease, degenerative disease, and cancer.
Signals derived from pathogens or host cells, such as pathogen-associated molecular patterns (PMAP), danger-associated molecular patterns (DAMP) and interferon (IFN)-γ, can activate macrophages[3, 4]. PAMP and DAMP are recognized by various pattern recognition receptors and ultimately cause the activations of mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways, which result in the expressions of many inflammatory genes including inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α and interleukin (IL)-6 and IL-12. IFN-γ, once known as macrophage activation factor, is produced by natural killer (NK) cells early in the immune response and later by type I T helper (Th1) cells. Binding of IFN-γ to its receptor causes the activations of JAK1,2-STAT1, which enhance the expressions of IFN-γ-regulated genes including those required for antigen processing and presentation, antiviral state, and microbicidal functions in macrophages.
Despite the long-lasting use of CJ in folk medicine, scientific evidence for its effectiveness is lacking. A recent study showed that the seed essential oils of CJ have antioxidant and hypolipidemic effects. In this paper, we examined the protective effect of CJ using an lipopolysaccharide (LPS)-induced inflammation model in vitro and in vivo. We also investigated whether this plant modulates cellular signaling molecules which regulate the expressions of inflammatory markers.
Identification of chemical constituents in the methanol extract of the aerial part of Cryptotaenia japonica
Identification of chemical constituents in the methanol extract of the aerial part of Cryptotaenia japonica by GC/MS analysis
Fumaric acid, 2-ethylhexyl undecyl ester
Hexadecanoic acid, methyl ester
7,10,13-Eicosatrienoic acid, methyl ester
Effects of CJ methanol extract on LPS-induced nitric oxide (NO) and inducible NO synthase
Effects of CJ methanol extract on LPS-induced inflammatory cytokines
Effects of CJ methanol extract on IκBα degradation
Effects of CJ methanol extract on MAPK signaling
Effects of CJ methanol extract on STAT1 activation
In vivo effect of CJ methanol extract on the serum cytokines from LPS-injected mice
In this paper, we investigated the in vitro and in vivo anti-inflammatory effects of CJ methanol extract using the LPS-mediated model and found that the extract from this plant was able to suppress the productions of iNOS, TNF-α, IL-6, and IL-12 in activated macrophages. Also, CJ methanol extract inhibited LPS or LPS/IFN-γ-triggered intracellular signaling pathways that end in the activation of such molecules as IκBα, MAPK and STAT1.
NO is a signaling molecule; it diffuses into the cytosol of neighboring cells and binds to the iron cofactor of guanylate cyclase, triggering activation of the enzyme and elevating intracellular cGMP concentrations. However, NO is also a free radical; it reacts with reactive oxygen species to produce peroxynitrite, a potent oxidant that inactivates target proteins by direct nitrosylation. The main control of NO production is determined by iNOS, and NF-κB, STAT1, and AP-1 are among the known transcription factors involved in the regulation of iNOS expression. In particular, NF-κB is a target modulated by many iNOS inhibitors such as glucocorticoids and antioxidants. IκBα degradation is critical for the regulation of NF-κB. IκBα is the prototypical protein of the IκB protein family (IκBα, IκBβ, IκBγ, IκBε, Bcl-3, p100, and p105). Phospho-IκBα is subject to polyubiquitination by E2 UbcH5 and E3 SCFβTrCP and is then degraded by the 20S proteasome. Our results indicate that the action of CJ methanol extract occurred in the pathways linking LPS to IKK.
TNF-α and IL-6 play major roles in vascular permeability, neutrophil recruitment, blood clotting, and acute phase protein synthesis: all of which are characteristics of acute inflammation. IL-12 activates NK cells and promotes the differentiation of T-helper cells into IFN-γ-secreting Th1 cells, which enhance macrophage activity. The MAPK signaling pathway mediates the LPS-triggered expressions of TNF-α, IL-6, and IL-12[4, 12]. The inhibitions of p38, JNK and ERK1/2 by CJ methanol extract may explain part of the mechanism that underlies the suppression of these pro-inflammatory cytokines.
IFN-γ upregulates the receptors for PAMP and DAMP, resulting in enhanced macrophage function. IFN-γ-dependent biological responses were impaired in STAT1-deficient mice. STAT1 has two phosphorylation sites, one at tyrosine 701 and the other at serine 727. Phosphorylation at tyrosine 701 is a direct result of IFN-γ exposure while phosphorylation of serine 727 requires a separate signaling pathway. LPS is able to induce phosphorylation at tyrosine 701 in a delayed manner, but uses the same IFN-γ receptor-mediated pathway. Our experimental model utilized both IFN-γ and LPS to fully activate STAT1. Inhibition of STAT1 phosphorylation at tyrosine 701 by CJ methanol extract may contribute to the downregulation of macrophage activity.
Many medicinal and food plants that belong to the Apiaceae family contain bioactive polyacetylenes. Falcarinol type polyacetylenes have been demonstrated to inhibit the release of NO and inflammatory cytokines in LPS-activated macrophages[16, 17]. Catechol, a polyphenol found in CJ, has been reported to be a potent inhibitor of iNOS expression and NF-κB activation. Presumably, part of the anti-inflammatory activity of CJ may be due to the presence of polyacetylene compound and catechol.
Taken together, the aerial part of CJ methanol extract was effective in suppressing the production of iNOS, TNF-α, IL-6, and IL-12 in LPS-stimulated macrophages in vitro and in vivo. The anti-inflammatory action of this plant includes modulation of STAT1 and MAPK activation as well as IκBα degration. Future study is required to characterize the active compounds of CJ extract.
The aerial parts of Crytotaenia japonica Hassk. were collected in the Medicinal Herb Garden of Kyung Hee Univeristy (Yongin) in May 2009. A voucher sample specimen (CJ-01) was deposited in the laboratory of Herbology, College of Oriental Medicine, Kyung Hee University. The dried plant was boiled three times in 100% methanol for 2 h. The extract was filtered, concentrated in vacuo, and dried with a lyophilizer. The yield of the extract was approximately 24.7%. The powdered extract was dissolved in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA) and sterilized by passing through a 0.22 μm syringe filter. A maximum of DMSO used for in vitro studies was 0.1%.
Gas chromatography / mass spectrometery
One mg of CJ methanol extract dissolved in 0.01 ml of DMSO was examined by gas chromatography coupled with mass spectrometer (Perkin Elmer Clarus 600T). A DB-5MS capillary column (30m x 0.25mm, film thickness 0.25μm) was used for the separation of constituents. The column temperatures were programmed from 50°C hold in initial 3 min to 140°C hold in 8.5 min, and then 310°C hold in 35 min. A constant flow rate of 1.0 ml/min was applied by using helium as the carrier gas. The electron energy for the mass selective detector was 70 eV. The temperature of the ion source was set at 255°C. Mass selective detector was used in SCAN mode over a mass scan range at m/z 50 to 600.
BALB/c mice (male, 8 weeks of age) were purchased from the Korean branch of Taconic, Samtaco (Osan, Korea), kept in a temperature-and humidity-controlled, pathogen-free animal facility at Kyung Hee University and provided standard mouse chow and water ad libitum. The mice were maintained in accordance with the Guide for the Care and Use of Laboratory Animals issued by the United States National Research Council (1996), and the protocol was approved by the Kyung Hee University Institutional Animal Care and Use Committee.
Isolation of peritoneal macrophages
Mice were injected intraperitoneally with 2 ml of sterile thioglycollate medium (BD, Sparks, MD, USA). Three days later, macrophages were collected by peritoneal lavage with cold Dulbecco’s modified Eagle’s medium (DMEM). Cells were resuspended in DMEM with 10% fetal bovine serum and incubated for 2 h in a humidified atmosphere of 5% CO2 at 37°C. Non-adherent cells were removed and the resulting adherent cell population consisted of 95% macrophages, as determined by morphology and non-specific esterase staining.
Cells were seeded at 4x104/ 0.1 ml in 96-well plates and stimulated for 24 h at increasing concentrations of CJ methanol extract. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopnehyl)-2H-tetrazolium (MTS) (Promega, Madison, WI, USA). Optical density was read at 490 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Measurement of nitrites
Cells were seeded at 2x106/ 2.0 ml in 6-well plates and primed for 2 h with 0.5 ng/ml of IFN-γ (BD Pharmingen, San Diego, CA, USA) before addition of LPS and CJ methanol extract. At 18 h after LPS stimulation, supernatant and cell pellets were used for subsequent assays. 50 μl medium was incubated with an equal volume of Griess reagent (Sigma) for 15 min at room temperature. The absorbance at 550 nm was measured with the microplate reader.
Supernatants or sera were appropriately diluted and the levels of cytokines were measured by ELISA according to the manufacturer’s protocol (BD Pharmingen).
Analysis of signaling molecules
Cells were seeded at 3x106/ 2.0 ml in 6-well plates and pre-treated for 1 h with CJ methanol extract and then stimulated with LPS for additional 15 min or 3 h. For the measurement of phospho-STAT1, cells were primed with IFN-γ.
Total proteins were prepared by resuspending the cells in lysis buffer (50 mM Tris–HCl, pH 7.5; 150 mM NaCl; 1mM EDTA; 20mM NaF; 0.5% NP-40; and 1% Triton X-100) containing a phosphatase inhibitor (Sigma) and a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Protein concentration was determined using the Bradford assay. Cell extracts were run on an 8% or 10% sodiumdodecyl sulfate-polyacrylamide gel and were transferred to polyvinylidene fluoride. The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h and then incubated overnight at 4°C incubated with IκBα, β-tubulin (Santa Cruz Biotechnology, CA, USA), iNOS (BD Pharmingen), phospho-IκBα, phospho-JNK, JNK, phospho-p38, p38, phospho-ERK1/2, ERK1/2, phospho-STAT1, or STAT1 (Cell Signaling Technology, CA, USA) diluted 1/1000 in 5% skim milk in TBST. The blots were washed with TBST and incubated for 1 h with anti-rabbit or anti-mouse HRP-conjugated antibody (diluted 1:5000 in 5% skim milk in TBST). Immunoreactive bands were developed using an enhanced chemiluminescence system (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
In vivo experiment
CJ methanol extract dissolved in water ( 25, 100, and 400 mg/kg) was orally given for 1 week. On day 7, intraperitoneal injection of LPS (1.3 mg/kg) was performed and 1 h later mice were anesthetized with ether and blood was obtained by cardiac puncture.
Statistical differences among the means of multiple groups were determined by using one way ANOVA followed by Dunnet’s post hoc test. The difference between the two means was assessed using non-paired student’s t test. Calculations were carried out using SPSS version 12. P values less than 0.05 were considered significant.
Cryptotaenia japonica Hassk
Pathogen-associated molecular pattern
Danger-associated molecular pattern
- NK cells:
Natural killer cells
Inducible nitric oxide synthase
Tumor necrosis factor-α
Inhibitor of κB
Mitogen-activated protein kinase
Signal transducers and activators of transcription
Extracellular signal-related kinase
C-Jun N-terminal kinase
Enzyme linked immunosorbent assay.
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