Uncaria rhynchophylla Inhibits the Production of Nitric Oxide and Interleukin-1β Through Blocking Nuclear Factor κB,Akt,and Mitogen-Activated Protein Kinase Activation in Macrophages

Abstract

The stems with hook of Uncaria rhynchophylla have been used in traditional medicine as an antipyretic, antihypertensive, and anticonvulsant in China and Korea. In this study, we investigated the mechanism responsible for anti-inflammatory effects of U. rhynchophylla in RAW 264.7 macrophages. The aqueous extract of U. rhynchophylla inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and interleukin (IL)-1β secretion as well as inducible NO synthase (iNOS) expression, without affecting cell viability. Furthermore, U. rhynchophylla suppressed LPS-induced nuclear factor κB (NF-κB) activation, phosphorylation, and degradation of inhibitory protein IκB (IκB)-α, phosphorylation of Akt, extracellular signal-regulated kinase 1/2, p38 kinase, and c-Jun N-terminal kinase. These results suggest that U. rhynchophylla has the inhibitory effects on LPS-induced NO and IL-1β production in macrophages through blockade in the phosphorylation of Akt and mitogen-activated protein kinases, following IκB-α degradation and NF-κB activation.

Introduction

T he dried stems with hook of Uncaria rhynchophylla have been used as an antipyretic, antihypertensive, and anticonvulsant in Oriental medicine. In traditional Korean and Chinese medicine, U. rhynchophylla is classified as a liver-pacifying and wind-extinguishing medicinal and has been used for central nervous system-related symptoms such as tremor, seizure, and epilepsy^1

–5 and also to relieve headache from a cold, eye redness, and skin eruption.⁶ U. rhynchophylla and its constituent alkaloid compounds have been reported to have vasodilative and neuroprotective effects,^{3,5,7

–11} Ca²⁺-blocking activity,¹² phospholipase C inhibitor activity,¹³ and oxygen free radical scavenging activity.^14
–16 Recently, it was reported that U. rhynchophylla relieves atopic dermatitis-like dermatitis¹⁷ and that alkaloid compounds of U. rhynchophylla have anti-inflammatory activity in mouse microglial cells.^18,19 However, the mechanism responsible for its anti-inflammatory effects has not been fully clarified yet.

Macrophages are immune cells usually dispersed throughout the body. They detect pathogenic substances through pattern-recognition receptors and subsequently initiate and regulate inflammatory responses²⁰ using a wide range of soluble pro-inflammatory mediators such as nitric oxide (NO), oxygen free radicals, tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. These inflammatory mediators lead to a variety of responses, including the activation of the T helper 1 cell response, increased expression of adhesion molecules on vascular endothelial cells,²¹ and the induction of acute-phase response proteins by the liver,²² and are generally involved in the development of inflammatory diseases.²³ Thus, the inhibition of excessive production of these inflammatory mediators can be used as criteria to evaluate anti-inflammatory effects of drugs.

Nuclear factor κB (NF-κB) is one of the most important transcription factors and regulates various cellular genes involved in immune and acute-phase inflammatory responses and in cell survival.²⁴ It has been shown to play a major role in expression of genes for inducible NO synthase (iNOS) and cytokines in response to lipopolysaccharide (LPS). NF-κB is a heterodimeric transcription factor composed of p50 and p65 (RelA), but a variety of other Rel-containing dimers are also known to exist.²⁵ In unstimulated cells, NF-κB is present in the cytosol bound to the inhibitory protein IκB (IκB). In response to stimulation such as LPS, IκBs are rapidly ubiquitinated and degraded by 26S proteasome complex. The free NF-κB dimers translocate to the nucleus, bind with high affinity to specific sites in the promoter regions of target genes, and stimulate their transcription. The critical event that triggers the polyubiquitination and degradation of IκBs is their stimulus-dependent phosphorylation at two serine residues (Ser³² and Ser³⁶) that are located within their conserved N-terminal regulatory region.²⁴

Activation of the mitogen-activated protein kinase (MAPK) pathways has been correlated with numerous cellular responses accompanied with NF-κB activation such as proliferation, differentiation, and regulation of specific metabolic pathways in different cell types. Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK)/stress-activated protein kinase, and p38 are activated by two homologous but distinct signaling pathways.²⁶ JNK and p38 are activated in response to inflammatory agents and environmental stress, whereas ERK, the “classic” MAPK, is stimulated primarily by growth factors, cytokines, and tumor promoters; however, activation by LPS also has been demonstrated.²⁷ ERK, JNK, and p38 are rapidly activated by LPS in macrophages and have been implicated in both inflammation and immune response.²⁸

In the present study, we investigated the effects of U. rhynchophylla on LPS-induced inflammatory response (NO and IL-1β release) and further explored the possible mechanisms of this inhibition by U. rhynchophylla in the macrophages. Our results provide a molecular basis for understanding the inhibitory effects of U. rhynchophylla on endotoxin-mediated inflammation.

Materials and Methods

Preparation of extract

The dried stems with hook of U. rhynchophylla were purchased from a local herb store, Kwang Myoung Herb Medicine (Pusan, Republic of Korea), in May 2005. The roots were identified and authenticated based on their microscopic and macroscopic characteristics by Professor Woo Shin Ko, who specializes in traditional Chinese herb medicine at the College of Oriental Medicine, Dongeui University, Pusan. A voucher specimen (number UR-05-05) has been deposited at the Department of Molecular Biology, Pusan National University, Busan, Republic of Korea. The dry roots (300 g) were extracted with distilled water at 100°C for 2 hours. The extract was filtered (pore size, 0.45 μm), freeze-dried (yield, 12 g), and kept at 4°C. The dried extract was dissolved in phosphate-buffered saline and filtered (pore size, 0.22 μm) before use.

Cell culture

Murine macrophage RAW 264.7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with glutamine (1 mM) and 10% fetal bovine serum at 37°C in an atmosphere of 5% CO₂.

Enzyme-linked immunosorbent assay

Cells were incubated with various concentrations of U. rhynchophylla for 1 hour and then treated with LPS (1 μg/mL) for 6 hours. Following a 6-hour incubation, the IL-1β level was quantified in culture medium using an enzyme-linked immunosorbent assay kit (Komabiotech, Seoul, Republic of Korea) according to the manufacturer's instructions.

Measurement of nitrite concentration

NO synthesis in cell cultures was measured by a microplate assay method, as described.²⁹ To measure nitrite, 100-μL aliquots were removed from conditioned medium and incubated with an equal volume of the Griess reagent [1% sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride, and 2.5% H₃PO₄] at room temperature for 10 minutes. Nitrite concentration was determined by measuring the absorbance at 540 nm with a Vmax 96-well microplate spectrophotometer (Molecular Devices, Menlo Park, CA, USA). Sodium nitrite was used as a standard.

Cell viability assay

The cytotoxicity of U. rhynchophylla was assessed using the microculture tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]-based colorimetric assay. The remaining cells after the Griess reaction were used for MTT assay. MTT solution (5 mg/mL) was added to each well (final concentration, 62.5 μg/mL). After incubation for 3 hours at 37°C and 5% CO₂, the supernatant was removed, and the formazan crystals in viable cells were solubilized with 150 μL of dimethyl sulfoxide. The absorbance of each well was then read at 570 nm using the microplate reader.

Western blot analysis

The cells were washed with phosphate-buffered saline three times, scraped off, and lysed with lysis buffer (1% Triton X-100 and 1% deoxycholate). Protein concentration of lysates was determined, equal amounts of protein were separated electrophoretically using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then the gel was transferred to nitrocellulose paper (pore size, 0.45 μm). The blot was incubated with anti-NF-κB p65, IκB-α, α-tubulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-IκB-α, Akt, phospho-Akt, JNK, phospho-JNK, p38, phospho-p38, ERK1/2, phospho-ERK1/2 antibody (Cell Signaling Technology, Danvers, MA, USA), and secondary antibody and then detected by the enhanced chemiluminescence detection system according to the recommended procedure (Amersham, Piscataway, NJ, USA).

Immunofluorescence confocal microscopy

Cells were cultured directly on glass coverslips in a 35-mm-diameter dish. Cells were fixed with 3.5% paraformaldehyde in phosphate-buffered saline for 10 minutes at room temperature and permeabilized with 100% methanol for 10 minutes. To investigate the cellular localization of NF-κB, cells were treated with a polyclonal antibody against NF-κB p65 for 1.5 hours. After extensive washing with phosphate-buffered saline, cells were further incubated with a secondary fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G antibody for 1 hour at room temperature. Nuclei were stained with 1 μg/mL 4′,6-diamidino-2-phenylindole and analyzed by confocal microscopy using a Zeiss (Oberkochen, Germany) LSM 510 Meta microscope.

Statistical analysis

All results were expressed as mean ± SE values. Each experiment was repeated at least three times. Statistical significances were compared between each treated group and analyzed by paired Student's t test. Differences with P < .05 were considered statistically significant.

Results

Effect of U. rhynchophylla on NO synthesis and iNOS expression in macrophages

To investigate the anti-inflammatory effect of U. rhynchophylla, we examined the effect of U. rhynchophylla on NO synthesis. RAW 264.7 macrophage cells were incubated with U. rhynchophylla for 1 hour and stimulated with LPS (1 μg/mL) for 24 hours. The amount of NO released into culture medium was measured by the Griess method. Whereas LPS-treated cells produced a large amount of NO, U. rhynchophylla suppressed NO release into culture supernatant in a dose-dependent manner (Fig. 1A). When macrophages were treated with 2 mg/mL U. rhynchophylla, the level of NO released was decreased to the basal level. Because NO is produced by iNOS in macrophages, we analyzed the effect of U. rhynchophylla on the expression of iNOS by western blot analysis. RAW 264.7 cells were treated with U. rhynchophylla as mentioned above. The levels of iNOS were dramatically reduced by U. rhynchophylla in a dose-dependent manner (Fig. 1B). Next, we examined the effect of U. rhynchophylla on pro-inflammatory cytokine production. RAW 264.7 cells were challenged with LPS in the presence or absence of U. rhynchophylla, and the levels of IL-1β in the medium were measured. Whereas LPS-treated cells produced a large amount of IL-1β, U. rhynchophylla suppressed the release of IL-1β into culture supernatant in a dose-dependent manner (Fig. 1C). On the other hand, cell viability was measured by MTT assay to examine whether the effect of U. rhynchophylla is due to its cytotoxicity. As shown in Figure 2, U. rhynchophylla did not decrease cell viability but rather protected the toxicity of LPS. This result suggests that U. rhynchophylla inhibits NO and IL-1β release without affecting cell viability.

FIG. 1.

Effect of U. rhynchophylla (UR) on nitric oxide and interleukin (IL)-1β production in macrophages. (A) RAW 264.7 macrophages were incubated with various concentrations of UR for 1 hour and then stimulated with 1 μg/mL lipopolysaccharide (LPS) for 24 hours at 37°C. The amount of nitrite released was measured by the Griess method. (B) Equal amounts of cytosolic extracts were analyzed by western blotting with anti-inducible nitric oxide synthase (iNOS) antibody. Western blot detection of α-tubulin was estimated with a protein-loading control for each lane. (C) RAW 264.7 macrophages were incubated with various concentrations of UR for 1 hour and then treated with LPS (1 μg/mL) for 6 hours. IL-1β in the cultured supernatant was measured by enzyme-linked immunosorbent assay kit. Data are mean ± SE values of three independent experiments. ^* P < .05 versus LPS-treated group.

FIG. 2.

Effect of UR on cell viability. RAW 264.7 macrophages were incubated with various concentrations of UR in the presence of 1 μg/mL LPS for 24 hours. Then cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described in Materials and Methods. Data represent the relative viability relative to the control group and are mean ± SE values of three independent experiments.

Effect of U. rhynchophylla on NF-κB activity in macrophages

Because NF-κB plays a critical role in LPS-induced iNOS^30,31 and cytokine expression,²⁴ we investigated whether U. rhynchophylla suppresses iNOS and cytokine expression through the regulation of NF-κB activity. Like other members of the NF-κB family, p65 resides in the cytoplasm in an inactive form bound to inhibitory IκB proteins. Cellular activation results in the nuclear translocation of NF-κB for initiating gene transcription. We therefore assessed the nuclear translocation of NF-κB p65 subunit by immunofluorescence. As shown in Figure 3, NF-κB p65 immunoreactivity was almost absent from the nucleus of untreated cells, whereas it was evenly distributed throughout the cytoplasm. LPS-stimulated cells showed co-staining of p65 and 4′,6-diamidino-2-phenylindole indicating a shift of NF-κB p65 towards the nucleus. However, the nuclear translocation of NF-κB p65 was inhibited by U. rhynchophylla in a dose-dependent manner. The level of NF-κB p65 of the U. rhynchophylla-alone group was similar to that of the control group (data not shown).

FIG. 3.

Inhibitory effect of UR on nuclear translocation of nuclear factor κB (NF-κB). RAW 264.7 macrophages were incubated with various concentration of UR for 1 hour and then stimulated with LPS (1 μg/mL) for 30 minutes. Fixed cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) or anti-NF-κB p65 antibody and fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G antibody. Images were taken by confocal microscopy. Bar = 20 μm.

Suppression of IκB-α phosphorylation and degradation by U. rhynchophylla

Next, we investigated the effects of U. rhynchophylla on the upstream signaling pathway of NF-κB, that is, IκB-α degradation. It has been known that the degradation of IκB-α is provoked by their stimulus-dependent phosphorylation at two serine residues (Ser³² and Ser³⁶) located within their conserved N-terminal regulatory region.²⁴ RAW 264.7 cells were pretreated with various concentrations of U. rhynchophylla for 1 hour and stimulated with LPS (1 μg/mL) for 15 minutes, and then the levels of IκB-α or phospho-IκB-α protein were examined by western blotting. As shown in Figure 4, IκB-α was degraded in response to LPS, but U. rhynchophylla suppressed the degradation of IκB-α in a dose-dependent manner. Incubation of macrophages with LPS for 15 minutes also caused significant phosphorylation of IκB-α, but U. rhynchophylla markedly inhibited the phosphorylation of IκB-α.

FIG. 4.

Inhibitory effect of UR on LPS-stimulated phosphorylation and degradation of inhibitory protein IκB (IκB)-α. RAW 264.7 macrophages were incubated with various concentrations of UR for 1 hour and then stimulated with 1 μg/mL LPS for 15 minutes. Cells were harvested, and equal amounts of cytosolic extracts were analyzed by western blotting with anti-IκB-α or anti-phospho-IκB-α (p-IκB-α) antibody. Western blot detection of α-tubulin was estimated as the protein-loading control for each lane.

Inhibitory effect of U. rhynchophylla on LPS-induced Akt and MAPK activities

To elucidate the molecular target of U. rhynchophylla in the further upstream signaling pathway, we examined the effect of U. rhynchophylla on Akt and MAPK activities that regulate the function of NF-κB.^32

–38 Because these kinases have been known to be phosphorylated and activated in response to LPS, Akt, ERK1/2, p38, and JNK activities were monitored by its phosphorylation. RAW 264.7 cells were treated with indicated concentrations of U. rhynchophylla for 1 hour and then stimulated with 1 μg/mL LPS for 15 minutes. U. rhynchophylla suppressed LPS-induced activation of Akt, ERK1/2, p38, and JNK in a dose-dependent manner (Fig. 5), whereas the amount of nonphosphorylated kinases was unaffected by either LPS or U. rhynchophylla treatment. U. rhynchophylla alone did not affect the level of phosphorylated kinases (data not shown).

FIG. 5.

Effect of UR on Akt, extracellular signal-regulated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase (JNK) activity in LPS-stimulated macrophages. RAW 264.7 macrophages were treated with indicated concentrations of UR for 1 hour and stimulated with 1 μg/mL LPS for 15 minutes. Equal amounts of cell extracts were analyzed by western blotting with anti-phosphokinase (p-) antibodies, respectively. Western blot detection of nonphosphorylated kinases was estimated with the protein-loading control for each lane.

Discussion

In this study, we investigated anti-inflammatory effect of U. rhynchophylla in LPS-stimulated macrophages. U. rhynchophylla significantly inhibited LPS-induced NO production and iNOS expression in macrophages without appreciable cytotoxic effects (Figs. 1 and 2). The expression of iNOS and the release of large amounts of NO by macrophages are considered to play a significant role in the pathogenesis of various inflammatory disorders. Inhibition of iNOS activity in macrophages may thus represent an interesting target to treat various diseases associated with increased NO production. In fact, administration of selective iNOS inhibitors has been reported to attenuate osteoarthritis,³⁹ periodontitis,⁴⁰ experimental autoimmune myocarditis,⁴¹ multiple sclerosis,⁴² and shock.⁴³ We also found that U. rhynchophylla impaired LPS-induced secretion of pro-inflammatory cytokines (IL-1β) in macrophages. Several pro-inflammatory cytokines such as tumor necrosis factor-α, IL-1β, and IL-6 are known to play key roles in the induction and perpetuation of inflammation in macrophages. Thus these results suggest that U. rhynchophylla exhibits an anti-inflammatory effect via inhibition of NO and pro-inflammatory cytokine synthesis in macrophages. It is also supposed that U. rhynchophylla can be used as a drugfor other inflammatory diseases as well as neuro-inflammation.^10,18,19

A major transcriptional regulator of iNOS and cytokine genes is NF-κB, which is also a key regulator of a variety of genes involved in immune and inflammatory responses.²⁴ Inappropriate regulation of NF-κB is directly involved in a wide range of human disorders, including a variety of cancers, neurodegenerative diseases, arthritis, asthma, inflammatory bowel disease, sepsis, and numerous other inflammatory conditions.^44

–47 Therefore, the development of a drug that controls NF-κB is a promising strategy for the treatment of inflammatory disease.⁴⁶ Our study showed that U. rhynchophylla significantly inhibited NF-κB translocation into the nucleus. In addition, U. rhynchophylla suppressed not only the LPS-stimulated degradation but also phosphorylation of IκB-α. Therefore these results suggest that U. rhynchophylla inhibits NF-κB activity through the suppression of phosphorylation and degradation of IκB-α and that U. rhynchophylla might target upstream kinases that phosphorylate IκB-α.

MAPKs and Akt have been shown to be involved in the LPS-mediated induction of many inflammatory genes, including the iNOS gene, and are important for the activation of NF-κB.^27,48 MAPKs are a family of serine/threonine protein kinases that are an important part of intracellular signaling pathways, connecting extracellular signals to intracellular regulatory proteins. MAPK family members ERK1/2 and p38 MAPK are known to participate in the regulation of LPS-induced pro-inflammatory cytokine production.^28,32,33 The third signal transduction pathway of the MAPK family is the JNK pathway, which is also activated primarily by cellular stress and cytokines, and its downstream targets include transcription factors important in cytokine expression.^49,50 In addition, Akt/protein kinase B is a serine/threonine kinase that is best known for its ability to inhibit cell death pathways.^51
–53 Activation of Akt by growth factors and cytokines generally occurs via the phosphoinositide-3-kinase pathway. Upon stimulation, phosphoinositide-3-kinase phosphorylates specific phosphoinositide lipids, which accumulate in the plasma membrane, creating docking sites for Akt. At the plasma membrane Akt undergoes phosphorylation at two residues, leading to its activation. Although NF-κB and Akt were initially thought to be components of distinct signaling pathways, several studies have demonstrated convergence of the NF-κB and Akt signaling pathways.^37,38 Indeed, IκB kinase is a substrate of Akt; thus, activation of Akt stimulates NF-κB activity. Thus, the activations of ERK1/2, p38, JNK, and Akt are used as a hallmark of LPS-induced signal transduction in macrophages. Therefore, to further confirm the inhibitory mechanism of NF-κB activation by U. rhynchophylla, we investigated the effects of U. rhynchophylla on phosphorylation of Akt and MAPKs in macrophages stimulated with LPS, and it was found that Akt, ERK1/2, p38, and JNK phosphorylations were suppressed by U. rhynchophylla in a dose-dependent manner. U. rhynchophylla greatly inhibited the LPS-induced ERK1/2, p38, and JNK activation at the same range of doses necessary to prevent the NF-κB activation and iNOS expression after LPS application. These results suggest that U. rhynchophylla inhibits LPS-induced NF-κB activation by down-regulating the phosphorylations of Akt, ERK1/2, p38, and JNK. Inhibition of Akt, ERK1/2, p38, and JNK activities might suppress NF-κB directly and/or indirectly through the inhibition of IκB kinase activity.

We examined the effects of crude extract of U. rhynchophylla in this study. Because rhynchophylline and isorhynchophylline are known to be the major components of U. rhynchophylla,^7,9,18,19 rhynchophylline and/or isorhynchophylline might exhibit these anti-inflammatory effects in response to LPS. Further study should include extraction and identification of the responsible compound.

In conclusion, we demonstrated that U. rhynchophylla inhibited NO release, iNOS expression, and IL-1β production in LPS-stimulated macrophages and that these effects are mediated by inhibition of the activity of IκB/NF-κB and the phosphorylation of Akt, ERK1/2, p38, and JNK. Our results provide a molecular basis for understanding the inhibitory effects of U. rhynchophylla stem and hook on endotoxin-mediated inflammation. U. rhynchophylla could be developed into potent anti-inflammatory drugs and could perhaps be used in pathological processes, including septic shock, arthritis, and inflammatory bowel disease as well as neurodegenerative diseases.

Footnotes

Acknowledgment

This study was supported by the Research Fund Program of Research Institute for Basic Sciences, Pusan National University, Busan, Republic of Korea, 2009, project number RIBS-PNU-2009-0016000.

Author Disclosure Statement

No competing financial interests exist. All the authors have substantially participated in the investigation, data analysis, and the preparation of the manuscript and accept full responsibility for its content.

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