JOURNAL OF MEDICINAL FOOD J Med Food 18 (6) 2015, 1–8 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2014.3275
FULL COMMUNICATION
Anti-Inflammatory Effects of 3-(40 -Hydroxyl-30 ,50 -Dimethoxyphenyl)Propionic Acid, an Active Component of Korean Cabbage Kimchi, in Lipopolysaccharide-Stimulated BV2 Microglia Jin-Woo Jeong,1,* Il-Whan Choi,2,* Guk-Heui Jo,1 Gi-Young Kim,3 Jinwoo Kim,4 Hongsuk Suh,4 Chung-Ho Ryu,5 Wun-Jae Kim,6 Kun-Young Park,7 and Yung Hyun Choi1,8 1
Department of Biochemistry, Dongeui University College of Korean Medicine, Busan, Korea. 2 Department of Microbiology, College of Medicine, Inje University, Busan, Korea. 3 Department of Marine Life Sciences, Jeju National University, Jeju, Korea. 4 Department of Chemistry, Pusan National University, Busan, Korea. 5 Division of Applied Life Science, Gyeongsang National University, Jinju, Korea. 6 Department of Urology, Chungbuk National University College of Medicine, Cheongju, Korea. 7 Department of Food and Nutrition, Busan National University, Busan, Korea. 8 Anti-Aging Research Center & Blue-Bio Industry RIC, Dongeui University, Busan, Korea. ABSTRACT We investigated the protective ability of 3-(40 -hydroxyl-30 ,50 -dimethoxyphenyl)propionic acid (HDMPPA), an active principle in Korean cabbage kimchi, against the production of proinflammatory mediators and cytokines, and the mechanisms involved in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. HDMPPA significantly suppressed the production of nitric oxide (NO) and prostaglandin E2, along with the expression of inducible NO synthase and cyclooxygenase-2 in LPS-stimulated BV2 cells, at concentrations with no cytotoxicity. HDMPPA also attenuated the LPS-induced expression and secretion of proinflammatory cytokines, such as tumor necrosis factor-a and interleukin-1b. Furthermore, HDMPPA inhibited LPS-induced nuclear factor-jB (NF-jB) activation, which was associated with the abrogation of IjB-a degradation and phosphorylation, and subsequent decreases in NF-jB p65 levels. Moreover, the phosphorylation of mitogen-activated protein kinases (MAPKs) and Akt, a downstream molecule of phosphatidylinositol-3-kinase (PI3K), in LPS-stimulated BV2 cells was suppressed markedly by HDMPPA. This effect was associated with a significant reduction in the formation of intracellular reactive oxygen species. The findings in this study suggest that HDMPPA may exert anti-inflammatory responses by suppressing LPS-induced expression of proinflammatory mediators and cytokines through blockage of NF-jB, MAPKs, and PI3K/Akt signaling pathways and oxidative stress in microglia.
KEY WORDS: Akt 3-(40 -hydroxyl-30 ,50 -dimethoxyphenyl)propionic acid inflammation MAPKs NF-jB
tion has been implicated in neuronal destruction in various neurodegenerative diseases.3,4 Thus, microglial cells are a key target for therapeutic intervention against neurodegenerative diseases, and it is important to investigate negative regulators of microglial activation and the underlying molecular mechanisms. Kimchi is a traditional fermented Korean side dish made of vegetables with a variety of seasonings and salt. The major ingredients of kimchi are baechu (Korean cabbage), red peppers, garlic, and ginger, which have high levels of dietary fiber, vitamin C, b-carotene, lactic acid bacteria, bsitosterol, minerals, and other health-promoting components.5–8 Several scientific trials to identify the potential health benefits and functional components of kimchi have been conducted. Recently, 3-(40 -hydroxyl-30 ,50 -dimethoxyphenyl)propionic acid (HDMPPA), isolated from a dichloromethane fraction of
INTRODUCTION
M
icroglia function as the first and main form of active immune defense in the central nervous system (CNS). In their activated state, microglia can provide not only resistance to invasive pathogens but also function advantageously in neuron survival, such as clearing damaged cells and toxic cellular debris.1,2 However, microglial cells may become overactivated in abnormal situations and promote neuronal injury through the release of proinflammatory and cytotoxic factors. Moreover, chronic microglial activa*These two authors contributed equally to this work. Manuscript received 29 June 2014. Revision accepted 20 February 2015. Address correspondence to: Yung Hyun Choi, PhD, Department of Biochemistry, Dongeui University College of Korean Medicine, Busanjin-Gu, Busan 614-052, Republic of Korea, E-mail: [email protected]
1
2
JEONG ET AL.
freeze-dried Korean cabbage kimchi, was identified as an active principle responsible for inhibiting low-density lipoprotein oxidation and 2,2-diphenyl-1-picrylhydrazyl scavenging activity.9 Since then, Kim et al.10 reported that chemically synthesized HDMPPA prevented the development of aortic atherosclerosis in high-cholesterol-fed rabbits and suggested that this antiatherosclerotic effect of HDMPPA may be due to its antioxidative effect at low doses without cholesterol-lowering effects. Furthermore, it was found that HDMPPA decreased atherosclerotic lesions and ameliorated oxidative stress through inhibiting NADPH oxidase activity in the aorta of apolipoprotein E (apoE) knockout (KO) mice.11 Moreover, HDMPPA ameliorated inflammatory response in the aorta of apoE KO mice, probably through inhibiting nuclear factor-jB (NF-jB) expression, suggesting that HDMPPA may exert its vascular protective effects through the preservation of nitric oxide (NO) bioavailability and suppression of the inflammatory response.12 Nevertheless, the specific mechanisms of action have yet to be identified, specifically in neuroinflammatory conditions. Because it has been suggested that lipopolysaccharide (LPS)-activated microglial cells are a good cellular model to assess the efficacy of potential therapeutic compounds for neuroinflammatory disorders,13 in the present study, we sought to examine the anti-inflammatory potential of HDMPPA by investigating its effects on the inflammatory response induced by LPS in murine microglial BV2 cells. To further analyze the underlying mechanisms, the involvement of NF-jB, mitogen-activated protein kinases (MAPKs), and phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathways were also examined. The present study demonstrates that HDMPPA is a candidate with anti-inflammatory actions and suggests a scientific basis for further investigation of HDMPPA against neuroinflammatory conditions. MATERIALS AND METHODS Materials and antibodies HDMPPA was provided by Dr. H.S. Suh, Busan National University (Busan, Korea), dissolved in phosphate-buffered saline (PBS) and diluted with Dulbecco’s modified Eagle’s medium (DMEM) to the desired concentration before use. LPS, Tween-20, bovine serum albumin, and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Rabbit anti-inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1b (IL1b), tumor necrosis factor-a (TNF-a), NF-jB p65, IjB-a, and phosphorylated (p)-IjB-a polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against nucleolin, actin, extracellular signal-regulated kinase (ERK), p-ERK, p38 MAPK, p-p38 MAPK, c-Jun N-terminal kinase ( JNK), p-JNK, Akt, and p-Akt were purchased from Cell Signaling Technology (Danvers, MA, USA). The peroxidase-labeled donkey anti-rabbit immunoglobulin and peroxidase-labeled sheep anti-mouse immunoglobulin were purchased from Amersham Corp. (Arlington Heights, IL, USA). DMEM-
containing l-glutamine (200 mg/L), fetal bovine serum (FBS), penicillin and streptomycin, Triton X-100, and all other chemicals were purchased from Gibco (Grand Island, NY, USA). Cell culture and viability assay BV2 microglia14 were maintained in DMEM supplemented with 10% FBS and 100 U/mL penicillin/streptomycin at 37C with 5% CO2. Cell viability was determined using the MTT reduction assay. Briefly, cell were seeded and treated with different concentrations of HDMPPA in the presence or absence of LPS for 24 h, and 0.5 mg/mL MTT solution was added to each well. After incubation for 3 h at 37C, the resulting dark blue crystals were dissolved with dimethyl sulfoxide. Absorbance values were read at 540 nm on a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Assay of NO production NO production from the cells was determined by measuring the amount of nitrite, a relatively stable oxidation product of NO. Briefly, cells were treated with the indicated concentrations of HDMPPA for 30 min. After LPS stimulation for 24 h, 100 lL of culture medium from each sample was mixed with the same volume of Griess reagent and incubated at room temperature for 10 min in the dark. Absorbance values were read at 540 nm in a microplate reader. NO concentrations were calculated with reference to a standard curve of sodium nitrite generated using known concentrations. Enzyme-linked immunosorbent assay for PGE2, IL-1b, and TNF-a An enzyme-linked immunosorbent assay (ELISA) kit from Cayman Chemical (Ann Arbor, MI, USA) was used for the measurement of PGE2, and kits from R&D Systems (Minneapolis, MN, USA) were used for the measurement of IL-1b and TNF-a in accordance with the manufacturers’ protocols. Briefly, the supernatant of cell culture was collected and microcentrifuged. Samples were applied to each well for the ELISA, and the final measurement was read using a plate reader at 450 nm. The concentration in each sample was calculated according to the standards provided with the kits. RNA extraction and reverse transcription–polymerase chain reaction analysis Total RNA was extracted from BV2 cells using the TRIzol reagent (Invitrogen Co., Carlsbad, CA, USA), according to the manufacturer’s protocol. Equal amounts of RNA (2 lg) were reverse-transcribed to cDNA using oligodT primers and M-MLV reverse transcriptase (Promega Co., Madison, WI, USA) according to the manufacturer’s protocol. Polymerase chain reaction (PCR) was performed using the cDNA as a template, with the following cycle parameters: 40 PCR cycles of denaturing at 94C for 30 sec, annealing at 60C for 30 sec, and extension at 72C for 1 min. The resulting products were separated by
ANTI-INFLAMMATORY EFFECTS OF HDMPPA IN BV2 MICROGLIA
3
electrophoresis on 1% agarose gel, followed by visualization under ultraviolet light after ethidium bromide (0.5 lg/mL) staining. Western blot analysis Cells were washed twice with ice-cold PBS, and the total cellular proteins were extracted with lysis buffer (20 mM sucrose, 1 mM EDTA, 20 lM Tris-HCl, pH 7.2, 1 mM DTT, 10 mM KCl, 1.5 mM MgCl2, and 5 lg/mL aprotinin) for 30 min. In a parallel experiment, nuclear and cytosolic proteins were prepared using nuclear extraction reagents (Pierce, Rockford, IL, USA) according to the manufacturer’s protocol. Equal amounts of cell extracts were separated by electrophoresis using a sodium dodecyl sulfate (SDS)– polyacrylamide gel and then transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA) by electroblotting. After 1 h of blocking with 5% (w/v) nonfat milk in TBST (1.5 M NaCl, 20 mM Tris-Cl, 0.05% [v/v] Tween-20, pH 7.4), the membranes were incubated overnight at 4C with the desired antibodies. The membranes were then washed with TBST prior 1 h incubation at room temperature with horseradish peroxidase-labeled secondary antibodies at room temperature for another 1 h. To reveal the reaction bands, the membrane was reacted with an enhanced chemiluminescence detection system according to the manufacturer’s protocol (Amersham, Piscataway, NJ, USA).
FIG. 1. Effects of HDMPPA on BV2 microglia cell viability. Cells were treated with various concentrations of HDMPPA for 30 min and the cells were then stimulated with 500 ng/mL LPS. After 24 h, cell viability was determined by measuring the absorbance at 540 nm after addition of the MTT reagent, and the results are expressed as the percentage of surviving cells over control cells (no added HDMPPA and LPS). The values shown are means – SDs of three independent experiments. HDMPPA, 3-(40 -hydroxyl-30 ,50 -dimethoxyphenyl)propionic acid; LPS, lipopolysaccharide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; SD, standard deviation.
Measurement of reactive oxygen species Intracellular reactive oxygen species (ROS) production was monitored using the stable nonpolar dye 20 ,70 dichlorofluorescein diacetate (DCF-DA; Sigma-Aldrich Chemical Co.), which permeates cells readily and is hydrolyzed to the fluorescent dichlorofluorescein (DCF) upon interaction with intracellular ROS. Briefly, cells were incubated with 10 lM DCF-DA for 30 min at 37C in the dark. ROS production in the cells was monitored with a flow cytometer using the Cell-Quest pro software (Becton-Dickinson, Franklin Lakes, NJ, USA). Alternatively, DCF signals in cells were monitored with a fluorescent microscope. Images were prepared using Adobe Photoshop (ver. 8.0). Immunocytochemistry for NF-jB p65 Cells were cultured on glass coverslips in six-well plates for 24 h and pretreated with or without 200 lM HDMPPA for 30 min before treatment with LPS (500 ng/mL) for 30 min. Then, the cells were fixed with 4% paraformaldehyde in PBS for 10 min at 4C and permeabilized with 0.4% Triton X-100 for 20 min at room temperature. The antiNF-jB p65 antibody was applied for 1 h followed by 2 h incubation with FITC-conjugated donkey anti-rabbit IgG ( Jackson ImmunoResearch Lab., West Grove, PA, USA). After washing with PBS, nuclei were counterstained with 4,6diamidino-2-phenyllindile (DAPI) solution (Sigma-Aldrich Chemical Co.) for 15 min in dark, and fluorescence was visualized using a fluorescence microscope (Carl Zeiss, Jena, Germany).
FIG. 2. Inhibition of NO and PGE2 production by HDMPPA in LPSstimulated BV2 microglia. BV2 microglial cells were pretreated with various concentrations of HDMPPA for 30 min before incubation with LPS (500 ng/mL) for 24 h. (A) Nitrite content was measured using the Griess reaction, and (B) PGE2 concentration was measured in the culture medium using the commercial ELISA kit. Each value indicates the mean – SD and is representative of results obtained from three independent experiments. *P < .05, significantly different from the values obtained for cells treated with LPS in the absence of HDMPPA. ELISA, enzyme-linked immunosorbent assay; NO, nitric oxide.
4
JEONG ET AL.
Statistical analyses Data are expressed as means – standard deviations of three separate experiments and were analyzed for statistical significance using analysis of variance, followed by Scheffe’s test for multiple comparisons. Values of P < .05 were considered to indicate statistical significance. We used the Sigma Stat software. RESULTS Effect of HDMPPA on BV2 cell viability The effect of HDMPPA on BV2 cell viability was measured in the concentration range of 50–200 lM using an MTT-based viability assay after incubating the cells for 24 h in the absence or presence of LPS. As shown in Figure 1, cell viability was not significantly affected by HDMPPA treatment compared with the control. Cotreatments with HDMPPA and LPS were also not cytotoxic at any of the concentrations used. Thus, concentrations of HDMPPA from 50 to 200 lM were used in subsequent experiments.
FIG. 3. Downregulation of LPS-induced iNOS and COX-2 mRNA and protein expression by HDMPPA pretreatment in BV2 microglia. (A) Cells were pretreated with different concentrations of HDMPPA for 30 min, then with LPS (500 ng/mL), and incubated for 24 h. Total RNA was prepared for RT-PCR analysis of COX-2 and iNOS gene expression. The amplified PCR products were run on a 1% agarose gel and visualized by ethidium bromide staining. (B) Total cellular proteins (30 lg) were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and detected with anti-COX-2 and iNOS antibodies, and an ECL detection system. GAPDH and actin were used as internal controls for the RT-PCR and Western blot assays, respectively. COX-2, cyclooxygenase-2; ECL, enhanced chemiluminescence; GAPDH, glyceraldehyde 3-phophate dehydrogenase; iNOS, inducible NO synthase; RT-PCR, reverse transcription–polymerase chain reaction; SDS, sodium dodecyl sulfate.
HDMPPA reduces LPS-induced NO production and iNOS expression To characterize the nature of the anti-inflammatory actions of HDMPPA, we initially examined its effects on the production of NO. As shown in Figure 2A, LPS alone was able to induce NO production markedly from BV2 cells; however, HDMPPA decreased LPS-induced NO production in a dose-dependent manner. To examine whether the inhibitory effect of HDMPPA on NO production was associated with decreased iNOS expression, we investigated the levels of iNOS mRNA by reverse transcription–polymerase chain reaction (RT-PCR). HDMPPA inhibited the levels of iNOS mRNA induced by LPS stimulation in a dosedependent manner (Fig. 3A). Consistent with these results, Western blot analysis showed that iNOS protein expression also decreased following HDMPPA pretreatment (Fig. 3B). HDMPPA inhibits LPS-induced PGE2 production and COX-2 expression To investigate the effects of HDMPPA on the production of PGE2, the amount of PGE2 released by BV2 cells was measured using ELISA. The results showed that exposure of BV2 cells to LPS increased the production of PGE2;
FIG. 4. Inhibition of LPS-induced IL-1b and TNF-a production by HDMPPA in BV2 microglia. BV2 cells were pretreated with various concentrations of HDMPPA for 30 min before exposure to LPS (500 ng/mL). Following incubation for 24 h, the levels of IL-1b (A) and TNF-a (B) present in the supernatants were measured. Each value indicates the mean – SD and is representative of results obtained from three independent experiments. *P < .05, significantly different from the value obtained with cells treated with LPS in the absence of HDMPPA. IL-1b, interleukin-1b; TNF-a, tumor necrosis factor-a.
ANTI-INFLAMMATORY EFFECTS OF HDMPPA IN BV2 MICROGLIA
however, HDMPPA decreased the levels of PGE2 production significantly from LPS-stimulated BV2 cells in a dosedependent manner (Fig. 2B). HDMPPA also suppressed mRNA expression and protein induction of COX-2 in LPS-stimulated BV2 cells in a concentration-dependent manner (Fig. 3). HDMPPA attenuates LPS-induced proinflammatory cytokines production and expression Next step, we investigated HDMPPA’s effect on the production of proinflammatory cytokines, such as IL-1b and TNF-a, by ELISA. Unstimulated BV2 cells produced low amounts of these cytokines. However, in LPS-stimulated cells, a significant increase in the secretion of IL-1b and TNF-a was observed and it was significantly inhibited, in a concentration-dependent manner, by HDMPPA pretreatment (Fig. 4). Similar to the ELISA result, stimulation with LPS increased the mRNA and protein expression of IL-1b and TNF-a. Meanwhile, pretreatment with HDMPPA markedly attenuated their expression (Fig. 5).
5
stress under our experimental conditions. As shown in Figure 6A, the high fluorescence intensity of the DCF product generated by LPS-induced intracellular ROS was decreased significantly by pretreatment with 200 lM HDMPPA, but HDMPPA itself had no effect on ROS production, relative to untreated control cells. These results were confirmed by flow cytometry, which showed a reduction in DCF fluorescence in HDMPPA-treated and LPS-stimulated cells compared with LPS-stimulated cells (Fig. 6B).
HDMPPA reduces LPS-induced ROS generation To further understand the possible underlying mechanisms involved in the anti-inflammatory effects of HDMPPA, we measured the formation of ROS in cells with the DCF-DA assay to determine whether HDMPPA reduced oxidative
FIG. 5. Downregulation of LPS-induced IL-1b and TNF-a mRNA and protein expression by HDMPPA pretreatment in BV2 microglia. (A) Cells were pretreated with different concentrations of HDMPPA for 30 min, then with LPS (500 ng/mL), and incubated for 24 h. Total RNA was prepared for RT-PCR analysis of COX-2 and iNOS gene expression. The amplified PCR products were run on a 1% agarose gel and visualized by ethidium bromide staining. (B) Total cellular proteins (30 lg) were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and detected with anti-COX-2 and iNOS antibodies, and an ECL detection system. GAPDH and actin were used as internal controls for the RT-PCR and Western blot assays, respectively.
FIG. 6. Scavenging effects of HDMPPA on intracellular ROS generation in LPS-stimulated BV2 microglia. (A) Cells were pretreated with or without 200 lM HDMPPA for 30 min before treatment with LPS (500 ng/mL) for 30 min, and then ROS generation was measured using a flow cytometer. The results are presented as the mean of two independent experiments. (B) Representative fluorescent images illustrate that the fluorescence intensity of DCF produced from DCF-DA by ROS was elevated in LPS-treated BV2 cells compared with the control untreated cells. HDMPPA had no effect on its own but lowered the fluorescence intensity of DCF in LPS-treated cells. DCF-DA, 20 ,70 dichlorofluorescein diacetate; ROS, reactive oxygen species. Color images available online at www.liebertpub.com/jmf
6
JEONG ET AL.
HDMPPA blocks LPS-induced NF-jB activation
HDMPPA inhibits LPS-induced Akt and MAPKs activation
To understand whether HDMPPA blocked the NF-jB pathway, implicated in the transcriptional regulation of inflammatory mediators and cytokines in LPS-activated BV2 microglia, cytosolic and nuclear extracts prepared from cells were assayed for the degradation and phosphorylation of IjB-a and subsequent translocation of NF-jB to the nucleus. As shown in Figure 7A, the amount of NF-jB p65 in the nucleus was increased markedly on exposure to LPS alone. However, the expression levels of NF-jB p65 protein in the nuclear fractions were significantly attenuated by pretreatment with HDMPPA. Because the degradation of IjB-a is a key step in NF-jB activation with LPS, we examined the effect of HDMPPA on LPS-induced degradation and phosphorylation of IjB-a protein. Our results indicated that IjB-a proteins were degraded and phosphorylated in BV2 cells by a 30-min treatment with LPS, and the degradation and phosphorylation were noticeably prevented by HDMPPA pretreatment, indicating that HDMPPA decreases the NF-jB activity in LPSstimulated BV2 cells by suppressing the phosphorylation or degradation of IjB-a. Additionally, an immunocytochemical analysis revealed that the NF-jB p65 protein was located primarily in the cytosol in the untreated condition (Fig. 7B). When BV2 cells were exposed to LPS, the NF-jB p65 protein appeared in nuclei within 1 h; however, HDMPPA pretreatment reduced NF-jB p65 nuclear immunoreactivity, suggesting that HDMPPA inhibited the translocation of NF-jB p65 protein from the cytosol to the nucleus.
Because there is accumulating evidence indicating roles for PI3K/Akt and MAPKs signaling pathways in mediating inflammatory responses, we investigated whether the antiinflammatory potentials of HDMPPA were mediated through these pathways. As indicated in Figure 8, LPS alone treatment strongly activated Akt and MAPKs, including ERK, JNK, and p38 MAPK (their phosphorylated forms are shown), within 1 h. However, pretreatment of the cells with HDMPPA suppressed the LPS-induced phosphorylation of the Akt and MAPKs markedly, while their unphosphorylated forms remained the same, indicating that signal transduction by Akt and MAPKs may effectively be blocked by HDMPPA. DISCUSSION It is generally accepted that excess production and accumulation of NO, PGE2, and ROS by activated microglial cells can contribute to neurodegeneration. After LPS stimulation, microglia show an increase in iNOS and COX-2, key enzymes for the production of NO and PGE2, respectively, during inflammation, and contributors to the development of chronic neurological disorders.15,16 Thus, agents with the ability to inhibit iNOS and COX-2 expression may be beneficial in the treatment of conditions associated with NO and PGE2 overproduction, including inflammatory diseases. Accumulating evidence also indicates that oxidative components contribute to LPS-induced iNOS and COX-2
FIG. 7. Inhibition of LPS-induced nuclear accumulation of NF-jB p65 and degradation of IjB-a by HDMPPA pretreatment in BV2 microglia. Cells were pretreated with or without 200 lM HDMPPA for 30 min before treatment with LPS (500 ng/mL) for 30 min. (A) Nuclear and cytosolic proteins were separated on 10% SDS-polyacrylamide gels, followed by Western blotting using anti-NF-jB p65, anti-IjB-a, and anti-p-IjB-a antibodies. Nucleolin and actin were used as internal controls for the nuclear and cytosolic fractions, respectively. (B) Cells were pretreated with 200 lM HDMPPA for 30 min, LPS (500 ng/mL) was then added, and cells were incubated for 30 min. Localization of NF-jB p65 was visualized with fluorescence microscopy after immunofluorescence staining with NF-jB p65 antibody (red). Cells were stained with DAPI for visualization of the nuclei (blue). A representative example of three independent experiments is shown. DAPI, 4,6-diamidino-2-phenyllindile; NF-jB, nuclear factor-jB. Color images available online at www.liebertpub.com/jmf
ANTI-INFLAMMATORY EFFECTS OF HDMPPA IN BV2 MICROGLIA
FIG. 8. Effect of HDMPPA on LPS-induced MAPKs and Akt activation in BV2 microglia. BV2 cells were pretreated with 200 lM HDMPPA for 30 min before exposure to LPS (500 ng/mL), and total proteins were isolated at the times indicated following exposure to LPS. The proteins were separated on SDS-polyacrylamide gels followed by Western blot analysis using the indicated antibodies and an ECL detection system. Actin was used as an internal control for Western blot assays. MAPKs, mitogen-activated protein kinases.
expression.17,18 Thus, the management of cellular ROS levels could be regarded as key to the regulation of NO and PGE2 production. In this study, HDMPPA significantly inhibited LPS-induced NO and PGE2 productions by suppressing iNOS and COX-2 expression in BV2 cells in a concentration-dependent manner, which was associated with HDMPPA’s ROS scavenging activity. These data indicate that HDMPPA acts by regulating NO, PGE2, and ROS generation and that it could be a suppressor of microglial activation. Additionally, IL-1b and TNF-a are major proinflammatory cytokines that are produced by activated microglia in CNS inflammation, and their excessive production due to several stimuli, such as LPS, b-amyloid, and traumatic brain injury, has linked with many neurodegenerative disorders in the CNS.4,19,20 In this study, we investigated whether HDMPPA inhibited LPS-induced production of these cytokines. The results indicated that HDMPPA inhibits the LPSinduced release of proinflammatory cytokines, IL-1b and TNF-a in BV2 cells. We also found that the inhibitory effects of HDMPPA on the LPS-induced release of proinflammatory cytokines correlated with their ability to suppress the expression of their genes, suggesting that HDMPPA may modulate gene expression levels of cytokines; these levels then control their release.
7
Various intracellular signaling pathways are involved in inflammatory mediator expression. Several inflammatory stimuli, including LPS, commonly activate the NF-jB, MAPK, and PI3K/Akt signaling pathways in microglia. Among them, NF-jB is known to play a critical role in the expression of proinflammatory enzymes and cytokines related to the inflammatory process. It is known that blockade of NF-jB transcriptional activity in microglia can also suppress the expression of iNOS, COX-2, and proinflammatory cytokines, including IL-1b and TNF-a.21,22 NF-jB is present normally in the cytoplasm as a heterotrimer complex consisting of p50, p65, and IjB subunits. Upon activation of the complex, phosphorylation and degradation of IjB exposes nuclear localization signals on the p50/p65 complex, allowing NF-jB translocation to the nucleus and binding to specific regulatory sequences in the genomic DNA, thus controlling gene transcription.23,24 The MAPK family proteins—ERK, JNK, and p38 MAPK—also appear to play key roles in inflammatory processes. Previous studies have shown that the activation of MAPKs has a significant effect on the regulation of COX-2, iNOS, and proinflammatory cytokine gene expression by controlling the activation of NF-jB in microglia.25,26 Akt also can modulate the NF-jB activation through IjB phosphorylation and degradation,27,28 which has been implicated in the inflammatory pathway in LPS-stimulated BV2 microglial cells.29–31 Moreover, excessive accumulation of intracellular ROS triggers the release of inflammatory factors in microglia through the activation of downstream signaling molecules, such as NF-jB and MAPKs.8,32–34 Thus, it is possible that neuroprotective mechanisms are associated with the inhibition of NF-jB, PI3K/Akt, and MAPKs signaling pathways in activated microglia. In the present study, we found that HDMPPA effectively attenuated LPS-induced IjB-a degradation and phosphorylation as well as the nuclear translocation of NF-jB p65. We also demonstrated that HDMPPA inhibited LPS-induced phosphorylation of MAPKs and Akt in LPS-activated BV2 microglia associated with the inhibition of intracellular ROS accumulation. This indicates that the transcriptional downregulation of inflammatory mediators and cytokines by HDMPPA in activated microglia is, at least part, due to the blocking of the NF-jB, MAPKs, and PI3K/ Akt pathways by inhibiting the generation of ROS in LPSactivated BV2 microglia. In conclusion, the present observations identify a potential anti-inflammatory role for HDMPPA against neuroinflammatory toxicity resulting from the presence of inflammatory mediators and cytokines, through the inhibition of NF-jB, MAPKs, and PI3K/Akt activation. Although the neuroprotective actions of HDMPPA need to be examined further using in vivo models, our results indicate that HDMPPA is a novel compound with potential for the treatment of neurodegenerative diseases that involve microglial activation.
ACKNOWLEDGMENTS This research was supported by Grants from the Globalization of Korean Foods R&D Program (912001-1), funded by
8
JEONG ET AL.
the Ministry of Food, Agriculture, Forestry and Fisheries and the National Research Foundation of Korea (NRF) Grant funded by the Korea government (NRF-2014R1A2A1A09006983 and 2012046358), Republic of Korea. AUTHOR DISCLOSURE STATEMENT No competing financial interests exist. REFERENCES 1. Block ML, Hong JS: Microglia and inflammation-mediated neurodegeneration: Multiple triggers with a common mechanism. Prog Neurobiol 2005;76:77–98. 2. Glezer I, Simard AR, Rivest S: Neuroprotective role of the innate immune system by microglia. Neuroscience 2007;147:867–883. 3. Dheen ST, Kaur C, Ling EA: Microglial activation and its implications in the brain diseases. Curr Med Chem 2007;14:1189–1197. 4. Smith JA, Das A, Ray SK, Banik NL: Role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases. Brain Res Bull 2012;87:10–20. 5. Cheigh HS, Park KY: Biochemical, microbiological, and nutritional aspects of kimchi (Korean fermented vegetable products). Crit Rev Food Sci Nutr 1994;34:175–203. 6. Park H, Kim HS: Korean traditional natural herbs and plants as immune enhancing, antidiabetic, chemopreventive, and antioxidative agents: A narrative review and perspective. J Med Food 2014;17: 21–27. 7. Choi IH, Noh JS, Han JS, Kim HJ, Han ES, Song YO: Kimchi, a fermented vegetable, improves serum lipid profiles in healthy young adults: Randomized clinical trial. J Med Food 2013;16: 223–229. 8. Park KY, Jeong JK, Lee YE, Daily JW 3rd: Health benefits of kimchi (Korean fermented vegetables) as a probiotic food. J Med Food 2014;17:6–20. 9. Lee YM, Kwon MJ, Kim JK, Suh HS, Choi JS, Song YO: Isolation and identification of active principle in Chinese cabbage kimchi responsible for antioxidant effect. Korean J Food Sci Technol 2004;36:129–133. 10. Kim HJ, Lee JS, Chung HY, et al.: 3-(40 -hydroxyl-30 ,50 dimethoxyphenyl)propionic acid, an active principle of kimchi, inhibits development of atherosclerosis in rabbits. J Agric Food Chem 2007;55:10486–10492. 11. Noh JS, Kim HJ, Kwon MJ, Song YO: Active principle of kimchi, 3-(40 -hydroxyl-30 ,50 -dimethoxyphenyl)propionic acid, retards fatty streak formation at aortic sinus of apolipoprotein E knockout mice. J Med Food 2009;12:1206–1212. 12. Noh JS, Choi YH, Song YO: Beneficial effects of the active principle component of Korean cabbage kimchi via increasing nitric oxide production and suppressing inflammation in the aorta of apoE knockout mice. Br J Nutr 2013;109:17–24. 13. Chao CC, Gekker G, Hu S, Peterson PK: Human microglial cell defense against Toxoplasma gondii. The role of cytokines. J Immunol 1994;152:1246–1252. 14. Blasi E, Barluzzi R, Bocchini V, Mazzolla R, Bistoni F: Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus. J Neuroimmunol 1990;27:229–237. 15. Minghetti L, Pocchiari M: Cyclooxygenase-2, prostaglandin E2, and microglial activation in prion diseases. Int Rev Neurobiol 2007;82:265–275.
16. Kanwar JR, Kanwar RK, Burrow H, Baratchi S: Recent advances on the roles of NO in cancer and chronic inflammatory disorders. Curr Med Chem 2009;16:2373–2394. 17. Pawate S, Shen Q, Fan F, Bhat NR: Redox regulation of glial inflammatory response to lipopolysaccharide and interferongamma. J Neurosci Res 2004;77:540–551. 18. Munhoz CD, Garcı´a-Bueno B, Madrigal JL, Lepsch LB, Scavone C, Leza JC: Stress-induced neuroinflammation: Mechanisms and new pharmacological targets. Braz J Med Biol Res 2008;41: 1037–1046. 19. Whitney NP, Eidem TM, Peng H, Huang Y, Zheng JC: Inflammation mediates varying effects in neurogenesis: Relevance to the pathogenesis of brain injury and neurodegenerative disorders. J Neurochem 2009;108:1343–1359. 20. Lee CY, Landreth GE: The role of microglia in amyloid clearance from the AD brain. J Neural Transm 2010;117:949–960. 21. Mattson MP, Camandola S: NF-kappaB in neuronal plasticity and neurodegenerative disorders. J Clin Invest 2001;107: 247–254. 22. Zheng Z, Yenari MA: Post-ischemic inflammation: Molecular mechanisms and therapeutic implications. Neurol Res 2004;26: 884–992. 23. Guha M, Mackman N: LPS induction of gene expression in human monocytes. Cell Signal 2001;13:85–94. 24. Li Q, Verma IM: NF-kappaB regulation in the immune system. Nat Rev Immunol 2002;2:725–734. 25. Gosselin RD, Suter MR, Ji RR, Decosterd I: Glial cells and chronic pain. Neuroscientist 2010;16:519–531. 26. Ji RR, Gereau RW 4th, Malcangio M, Strichartz GR: MAP kinase and pain. Brain Res Rev 2009;60:135–148. 27. Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB: NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 1999;401:82–85. 28. Chan CB, Ye K: Phosphoinositide 3-kinase enhancer (PIKE) in the brain: Is it simply a phosphoinositide 3-kinase/Akt enhancer? Rev Neurosci 2012;23:153–161. 29. Lee SH, Lee JH, Oh EY, et al.: Ethanol extract of Cnidium officinale exhibits anti-inflammatory effects in BV2 microglial cells by suppressing NF-jB nuclear translocation and the activation of the PI3K/Akt signaling pathway. Int J Mol Med 2013; 32:876–882. 30. Park HY, Han MH, Park C, et al.: Anti-inflammatory effects of fucoidan through inhibition of NF-jB, MAPK and Akt activation in lipopolysaccharide-induced BV2 microglia cells. Food Chem Toxicol 2011;49:1745–1752. 31. Jeong JW, Lee WS, Shin SC, et al.: Anthocyanins downregulate lipopolysaccharide-induced inflammatory rsponses in BV2 microglial cells by suppressing the NF-jB and Akt/MAPKs signaling pathways. Int J Mol Sci 2013;14:1502–1515. 32. Rosenberger J, Petrovics G, Buzas B: Oxidative stress induces proorphanin FQ and proenkephalin gene expression in astrocytes through p38- and ERK-MAP kinases and NF-kappaB. J Neurochem 2001;79:35–44. 33. Kim BH, Oh I, Kim JH, et al.: Anti-inflammatory activity of compounds isolated from Astragalus sinicus L. in cytokineinduced keratinocytes and skin. Exp Mol Med 2014;46:e87. 34. Kim M, Li YX, Dewapriya P, Ryu B, Kim SK: Floridoside suppresses pro-inflammatory responses by blocking MAPK signaling in activated microglia. BMB Rep 2013;46:398–403.
FULL COMMUNICATION
Anti-Inflammatory Effects of 3-(40 -Hydroxyl-30 ,50 -Dimethoxyphenyl)Propionic Acid, an Active Component of Korean Cabbage Kimchi, in Lipopolysaccharide-Stimulated BV2 Microglia Jin-Woo Jeong,1,* Il-Whan Choi,2,* Guk-Heui Jo,1 Gi-Young Kim,3 Jinwoo Kim,4 Hongsuk Suh,4 Chung-Ho Ryu,5 Wun-Jae Kim,6 Kun-Young Park,7 and Yung Hyun Choi1,8 1
Department of Biochemistry, Dongeui University College of Korean Medicine, Busan, Korea. 2 Department of Microbiology, College of Medicine, Inje University, Busan, Korea. 3 Department of Marine Life Sciences, Jeju National University, Jeju, Korea. 4 Department of Chemistry, Pusan National University, Busan, Korea. 5 Division of Applied Life Science, Gyeongsang National University, Jinju, Korea. 6 Department of Urology, Chungbuk National University College of Medicine, Cheongju, Korea. 7 Department of Food and Nutrition, Busan National University, Busan, Korea. 8 Anti-Aging Research Center & Blue-Bio Industry RIC, Dongeui University, Busan, Korea. ABSTRACT We investigated the protective ability of 3-(40 -hydroxyl-30 ,50 -dimethoxyphenyl)propionic acid (HDMPPA), an active principle in Korean cabbage kimchi, against the production of proinflammatory mediators and cytokines, and the mechanisms involved in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. HDMPPA significantly suppressed the production of nitric oxide (NO) and prostaglandin E2, along with the expression of inducible NO synthase and cyclooxygenase-2 in LPS-stimulated BV2 cells, at concentrations with no cytotoxicity. HDMPPA also attenuated the LPS-induced expression and secretion of proinflammatory cytokines, such as tumor necrosis factor-a and interleukin-1b. Furthermore, HDMPPA inhibited LPS-induced nuclear factor-jB (NF-jB) activation, which was associated with the abrogation of IjB-a degradation and phosphorylation, and subsequent decreases in NF-jB p65 levels. Moreover, the phosphorylation of mitogen-activated protein kinases (MAPKs) and Akt, a downstream molecule of phosphatidylinositol-3-kinase (PI3K), in LPS-stimulated BV2 cells was suppressed markedly by HDMPPA. This effect was associated with a significant reduction in the formation of intracellular reactive oxygen species. The findings in this study suggest that HDMPPA may exert anti-inflammatory responses by suppressing LPS-induced expression of proinflammatory mediators and cytokines through blockage of NF-jB, MAPKs, and PI3K/Akt signaling pathways and oxidative stress in microglia.
KEY WORDS: Akt 3-(40 -hydroxyl-30 ,50 -dimethoxyphenyl)propionic acid inflammation MAPKs NF-jB
tion has been implicated in neuronal destruction in various neurodegenerative diseases.3,4 Thus, microglial cells are a key target for therapeutic intervention against neurodegenerative diseases, and it is important to investigate negative regulators of microglial activation and the underlying molecular mechanisms. Kimchi is a traditional fermented Korean side dish made of vegetables with a variety of seasonings and salt. The major ingredients of kimchi are baechu (Korean cabbage), red peppers, garlic, and ginger, which have high levels of dietary fiber, vitamin C, b-carotene, lactic acid bacteria, bsitosterol, minerals, and other health-promoting components.5–8 Several scientific trials to identify the potential health benefits and functional components of kimchi have been conducted. Recently, 3-(40 -hydroxyl-30 ,50 -dimethoxyphenyl)propionic acid (HDMPPA), isolated from a dichloromethane fraction of
INTRODUCTION
M
icroglia function as the first and main form of active immune defense in the central nervous system (CNS). In their activated state, microglia can provide not only resistance to invasive pathogens but also function advantageously in neuron survival, such as clearing damaged cells and toxic cellular debris.1,2 However, microglial cells may become overactivated in abnormal situations and promote neuronal injury through the release of proinflammatory and cytotoxic factors. Moreover, chronic microglial activa*These two authors contributed equally to this work. Manuscript received 29 June 2014. Revision accepted 20 February 2015. Address correspondence to: Yung Hyun Choi, PhD, Department of Biochemistry, Dongeui University College of Korean Medicine, Busanjin-Gu, Busan 614-052, Republic of Korea, E-mail: [email protected]
1
2
JEONG ET AL.
freeze-dried Korean cabbage kimchi, was identified as an active principle responsible for inhibiting low-density lipoprotein oxidation and 2,2-diphenyl-1-picrylhydrazyl scavenging activity.9 Since then, Kim et al.10 reported that chemically synthesized HDMPPA prevented the development of aortic atherosclerosis in high-cholesterol-fed rabbits and suggested that this antiatherosclerotic effect of HDMPPA may be due to its antioxidative effect at low doses without cholesterol-lowering effects. Furthermore, it was found that HDMPPA decreased atherosclerotic lesions and ameliorated oxidative stress through inhibiting NADPH oxidase activity in the aorta of apolipoprotein E (apoE) knockout (KO) mice.11 Moreover, HDMPPA ameliorated inflammatory response in the aorta of apoE KO mice, probably through inhibiting nuclear factor-jB (NF-jB) expression, suggesting that HDMPPA may exert its vascular protective effects through the preservation of nitric oxide (NO) bioavailability and suppression of the inflammatory response.12 Nevertheless, the specific mechanisms of action have yet to be identified, specifically in neuroinflammatory conditions. Because it has been suggested that lipopolysaccharide (LPS)-activated microglial cells are a good cellular model to assess the efficacy of potential therapeutic compounds for neuroinflammatory disorders,13 in the present study, we sought to examine the anti-inflammatory potential of HDMPPA by investigating its effects on the inflammatory response induced by LPS in murine microglial BV2 cells. To further analyze the underlying mechanisms, the involvement of NF-jB, mitogen-activated protein kinases (MAPKs), and phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathways were also examined. The present study demonstrates that HDMPPA is a candidate with anti-inflammatory actions and suggests a scientific basis for further investigation of HDMPPA against neuroinflammatory conditions. MATERIALS AND METHODS Materials and antibodies HDMPPA was provided by Dr. H.S. Suh, Busan National University (Busan, Korea), dissolved in phosphate-buffered saline (PBS) and diluted with Dulbecco’s modified Eagle’s medium (DMEM) to the desired concentration before use. LPS, Tween-20, bovine serum albumin, and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Rabbit anti-inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1b (IL1b), tumor necrosis factor-a (TNF-a), NF-jB p65, IjB-a, and phosphorylated (p)-IjB-a polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against nucleolin, actin, extracellular signal-regulated kinase (ERK), p-ERK, p38 MAPK, p-p38 MAPK, c-Jun N-terminal kinase ( JNK), p-JNK, Akt, and p-Akt were purchased from Cell Signaling Technology (Danvers, MA, USA). The peroxidase-labeled donkey anti-rabbit immunoglobulin and peroxidase-labeled sheep anti-mouse immunoglobulin were purchased from Amersham Corp. (Arlington Heights, IL, USA). DMEM-
containing l-glutamine (200 mg/L), fetal bovine serum (FBS), penicillin and streptomycin, Triton X-100, and all other chemicals were purchased from Gibco (Grand Island, NY, USA). Cell culture and viability assay BV2 microglia14 were maintained in DMEM supplemented with 10% FBS and 100 U/mL penicillin/streptomycin at 37C with 5% CO2. Cell viability was determined using the MTT reduction assay. Briefly, cell were seeded and treated with different concentrations of HDMPPA in the presence or absence of LPS for 24 h, and 0.5 mg/mL MTT solution was added to each well. After incubation for 3 h at 37C, the resulting dark blue crystals were dissolved with dimethyl sulfoxide. Absorbance values were read at 540 nm on a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Assay of NO production NO production from the cells was determined by measuring the amount of nitrite, a relatively stable oxidation product of NO. Briefly, cells were treated with the indicated concentrations of HDMPPA for 30 min. After LPS stimulation for 24 h, 100 lL of culture medium from each sample was mixed with the same volume of Griess reagent and incubated at room temperature for 10 min in the dark. Absorbance values were read at 540 nm in a microplate reader. NO concentrations were calculated with reference to a standard curve of sodium nitrite generated using known concentrations. Enzyme-linked immunosorbent assay for PGE2, IL-1b, and TNF-a An enzyme-linked immunosorbent assay (ELISA) kit from Cayman Chemical (Ann Arbor, MI, USA) was used for the measurement of PGE2, and kits from R&D Systems (Minneapolis, MN, USA) were used for the measurement of IL-1b and TNF-a in accordance with the manufacturers’ protocols. Briefly, the supernatant of cell culture was collected and microcentrifuged. Samples were applied to each well for the ELISA, and the final measurement was read using a plate reader at 450 nm. The concentration in each sample was calculated according to the standards provided with the kits. RNA extraction and reverse transcription–polymerase chain reaction analysis Total RNA was extracted from BV2 cells using the TRIzol reagent (Invitrogen Co., Carlsbad, CA, USA), according to the manufacturer’s protocol. Equal amounts of RNA (2 lg) were reverse-transcribed to cDNA using oligodT primers and M-MLV reverse transcriptase (Promega Co., Madison, WI, USA) according to the manufacturer’s protocol. Polymerase chain reaction (PCR) was performed using the cDNA as a template, with the following cycle parameters: 40 PCR cycles of denaturing at 94C for 30 sec, annealing at 60C for 30 sec, and extension at 72C for 1 min. The resulting products were separated by
ANTI-INFLAMMATORY EFFECTS OF HDMPPA IN BV2 MICROGLIA
3
electrophoresis on 1% agarose gel, followed by visualization under ultraviolet light after ethidium bromide (0.5 lg/mL) staining. Western blot analysis Cells were washed twice with ice-cold PBS, and the total cellular proteins were extracted with lysis buffer (20 mM sucrose, 1 mM EDTA, 20 lM Tris-HCl, pH 7.2, 1 mM DTT, 10 mM KCl, 1.5 mM MgCl2, and 5 lg/mL aprotinin) for 30 min. In a parallel experiment, nuclear and cytosolic proteins were prepared using nuclear extraction reagents (Pierce, Rockford, IL, USA) according to the manufacturer’s protocol. Equal amounts of cell extracts were separated by electrophoresis using a sodium dodecyl sulfate (SDS)– polyacrylamide gel and then transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA) by electroblotting. After 1 h of blocking with 5% (w/v) nonfat milk in TBST (1.5 M NaCl, 20 mM Tris-Cl, 0.05% [v/v] Tween-20, pH 7.4), the membranes were incubated overnight at 4C with the desired antibodies. The membranes were then washed with TBST prior 1 h incubation at room temperature with horseradish peroxidase-labeled secondary antibodies at room temperature for another 1 h. To reveal the reaction bands, the membrane was reacted with an enhanced chemiluminescence detection system according to the manufacturer’s protocol (Amersham, Piscataway, NJ, USA).
FIG. 1. Effects of HDMPPA on BV2 microglia cell viability. Cells were treated with various concentrations of HDMPPA for 30 min and the cells were then stimulated with 500 ng/mL LPS. After 24 h, cell viability was determined by measuring the absorbance at 540 nm after addition of the MTT reagent, and the results are expressed as the percentage of surviving cells over control cells (no added HDMPPA and LPS). The values shown are means – SDs of three independent experiments. HDMPPA, 3-(40 -hydroxyl-30 ,50 -dimethoxyphenyl)propionic acid; LPS, lipopolysaccharide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; SD, standard deviation.
Measurement of reactive oxygen species Intracellular reactive oxygen species (ROS) production was monitored using the stable nonpolar dye 20 ,70 dichlorofluorescein diacetate (DCF-DA; Sigma-Aldrich Chemical Co.), which permeates cells readily and is hydrolyzed to the fluorescent dichlorofluorescein (DCF) upon interaction with intracellular ROS. Briefly, cells were incubated with 10 lM DCF-DA for 30 min at 37C in the dark. ROS production in the cells was monitored with a flow cytometer using the Cell-Quest pro software (Becton-Dickinson, Franklin Lakes, NJ, USA). Alternatively, DCF signals in cells were monitored with a fluorescent microscope. Images were prepared using Adobe Photoshop (ver. 8.0). Immunocytochemistry for NF-jB p65 Cells were cultured on glass coverslips in six-well plates for 24 h and pretreated with or without 200 lM HDMPPA for 30 min before treatment with LPS (500 ng/mL) for 30 min. Then, the cells were fixed with 4% paraformaldehyde in PBS for 10 min at 4C and permeabilized with 0.4% Triton X-100 for 20 min at room temperature. The antiNF-jB p65 antibody was applied for 1 h followed by 2 h incubation with FITC-conjugated donkey anti-rabbit IgG ( Jackson ImmunoResearch Lab., West Grove, PA, USA). After washing with PBS, nuclei were counterstained with 4,6diamidino-2-phenyllindile (DAPI) solution (Sigma-Aldrich Chemical Co.) for 15 min in dark, and fluorescence was visualized using a fluorescence microscope (Carl Zeiss, Jena, Germany).
FIG. 2. Inhibition of NO and PGE2 production by HDMPPA in LPSstimulated BV2 microglia. BV2 microglial cells were pretreated with various concentrations of HDMPPA for 30 min before incubation with LPS (500 ng/mL) for 24 h. (A) Nitrite content was measured using the Griess reaction, and (B) PGE2 concentration was measured in the culture medium using the commercial ELISA kit. Each value indicates the mean – SD and is representative of results obtained from three independent experiments. *P < .05, significantly different from the values obtained for cells treated with LPS in the absence of HDMPPA. ELISA, enzyme-linked immunosorbent assay; NO, nitric oxide.
4
JEONG ET AL.
Statistical analyses Data are expressed as means – standard deviations of three separate experiments and were analyzed for statistical significance using analysis of variance, followed by Scheffe’s test for multiple comparisons. Values of P < .05 were considered to indicate statistical significance. We used the Sigma Stat software. RESULTS Effect of HDMPPA on BV2 cell viability The effect of HDMPPA on BV2 cell viability was measured in the concentration range of 50–200 lM using an MTT-based viability assay after incubating the cells for 24 h in the absence or presence of LPS. As shown in Figure 1, cell viability was not significantly affected by HDMPPA treatment compared with the control. Cotreatments with HDMPPA and LPS were also not cytotoxic at any of the concentrations used. Thus, concentrations of HDMPPA from 50 to 200 lM were used in subsequent experiments.
FIG. 3. Downregulation of LPS-induced iNOS and COX-2 mRNA and protein expression by HDMPPA pretreatment in BV2 microglia. (A) Cells were pretreated with different concentrations of HDMPPA for 30 min, then with LPS (500 ng/mL), and incubated for 24 h. Total RNA was prepared for RT-PCR analysis of COX-2 and iNOS gene expression. The amplified PCR products were run on a 1% agarose gel and visualized by ethidium bromide staining. (B) Total cellular proteins (30 lg) were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and detected with anti-COX-2 and iNOS antibodies, and an ECL detection system. GAPDH and actin were used as internal controls for the RT-PCR and Western blot assays, respectively. COX-2, cyclooxygenase-2; ECL, enhanced chemiluminescence; GAPDH, glyceraldehyde 3-phophate dehydrogenase; iNOS, inducible NO synthase; RT-PCR, reverse transcription–polymerase chain reaction; SDS, sodium dodecyl sulfate.
HDMPPA reduces LPS-induced NO production and iNOS expression To characterize the nature of the anti-inflammatory actions of HDMPPA, we initially examined its effects on the production of NO. As shown in Figure 2A, LPS alone was able to induce NO production markedly from BV2 cells; however, HDMPPA decreased LPS-induced NO production in a dose-dependent manner. To examine whether the inhibitory effect of HDMPPA on NO production was associated with decreased iNOS expression, we investigated the levels of iNOS mRNA by reverse transcription–polymerase chain reaction (RT-PCR). HDMPPA inhibited the levels of iNOS mRNA induced by LPS stimulation in a dosedependent manner (Fig. 3A). Consistent with these results, Western blot analysis showed that iNOS protein expression also decreased following HDMPPA pretreatment (Fig. 3B). HDMPPA inhibits LPS-induced PGE2 production and COX-2 expression To investigate the effects of HDMPPA on the production of PGE2, the amount of PGE2 released by BV2 cells was measured using ELISA. The results showed that exposure of BV2 cells to LPS increased the production of PGE2;
FIG. 4. Inhibition of LPS-induced IL-1b and TNF-a production by HDMPPA in BV2 microglia. BV2 cells were pretreated with various concentrations of HDMPPA for 30 min before exposure to LPS (500 ng/mL). Following incubation for 24 h, the levels of IL-1b (A) and TNF-a (B) present in the supernatants were measured. Each value indicates the mean – SD and is representative of results obtained from three independent experiments. *P < .05, significantly different from the value obtained with cells treated with LPS in the absence of HDMPPA. IL-1b, interleukin-1b; TNF-a, tumor necrosis factor-a.
ANTI-INFLAMMATORY EFFECTS OF HDMPPA IN BV2 MICROGLIA
however, HDMPPA decreased the levels of PGE2 production significantly from LPS-stimulated BV2 cells in a dosedependent manner (Fig. 2B). HDMPPA also suppressed mRNA expression and protein induction of COX-2 in LPS-stimulated BV2 cells in a concentration-dependent manner (Fig. 3). HDMPPA attenuates LPS-induced proinflammatory cytokines production and expression Next step, we investigated HDMPPA’s effect on the production of proinflammatory cytokines, such as IL-1b and TNF-a, by ELISA. Unstimulated BV2 cells produced low amounts of these cytokines. However, in LPS-stimulated cells, a significant increase in the secretion of IL-1b and TNF-a was observed and it was significantly inhibited, in a concentration-dependent manner, by HDMPPA pretreatment (Fig. 4). Similar to the ELISA result, stimulation with LPS increased the mRNA and protein expression of IL-1b and TNF-a. Meanwhile, pretreatment with HDMPPA markedly attenuated their expression (Fig. 5).
5
stress under our experimental conditions. As shown in Figure 6A, the high fluorescence intensity of the DCF product generated by LPS-induced intracellular ROS was decreased significantly by pretreatment with 200 lM HDMPPA, but HDMPPA itself had no effect on ROS production, relative to untreated control cells. These results were confirmed by flow cytometry, which showed a reduction in DCF fluorescence in HDMPPA-treated and LPS-stimulated cells compared with LPS-stimulated cells (Fig. 6B).
HDMPPA reduces LPS-induced ROS generation To further understand the possible underlying mechanisms involved in the anti-inflammatory effects of HDMPPA, we measured the formation of ROS in cells with the DCF-DA assay to determine whether HDMPPA reduced oxidative
FIG. 5. Downregulation of LPS-induced IL-1b and TNF-a mRNA and protein expression by HDMPPA pretreatment in BV2 microglia. (A) Cells were pretreated with different concentrations of HDMPPA for 30 min, then with LPS (500 ng/mL), and incubated for 24 h. Total RNA was prepared for RT-PCR analysis of COX-2 and iNOS gene expression. The amplified PCR products were run on a 1% agarose gel and visualized by ethidium bromide staining. (B) Total cellular proteins (30 lg) were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and detected with anti-COX-2 and iNOS antibodies, and an ECL detection system. GAPDH and actin were used as internal controls for the RT-PCR and Western blot assays, respectively.
FIG. 6. Scavenging effects of HDMPPA on intracellular ROS generation in LPS-stimulated BV2 microglia. (A) Cells were pretreated with or without 200 lM HDMPPA for 30 min before treatment with LPS (500 ng/mL) for 30 min, and then ROS generation was measured using a flow cytometer. The results are presented as the mean of two independent experiments. (B) Representative fluorescent images illustrate that the fluorescence intensity of DCF produced from DCF-DA by ROS was elevated in LPS-treated BV2 cells compared with the control untreated cells. HDMPPA had no effect on its own but lowered the fluorescence intensity of DCF in LPS-treated cells. DCF-DA, 20 ,70 dichlorofluorescein diacetate; ROS, reactive oxygen species. Color images available online at www.liebertpub.com/jmf
6
JEONG ET AL.
HDMPPA blocks LPS-induced NF-jB activation
HDMPPA inhibits LPS-induced Akt and MAPKs activation
To understand whether HDMPPA blocked the NF-jB pathway, implicated in the transcriptional regulation of inflammatory mediators and cytokines in LPS-activated BV2 microglia, cytosolic and nuclear extracts prepared from cells were assayed for the degradation and phosphorylation of IjB-a and subsequent translocation of NF-jB to the nucleus. As shown in Figure 7A, the amount of NF-jB p65 in the nucleus was increased markedly on exposure to LPS alone. However, the expression levels of NF-jB p65 protein in the nuclear fractions were significantly attenuated by pretreatment with HDMPPA. Because the degradation of IjB-a is a key step in NF-jB activation with LPS, we examined the effect of HDMPPA on LPS-induced degradation and phosphorylation of IjB-a protein. Our results indicated that IjB-a proteins were degraded and phosphorylated in BV2 cells by a 30-min treatment with LPS, and the degradation and phosphorylation were noticeably prevented by HDMPPA pretreatment, indicating that HDMPPA decreases the NF-jB activity in LPSstimulated BV2 cells by suppressing the phosphorylation or degradation of IjB-a. Additionally, an immunocytochemical analysis revealed that the NF-jB p65 protein was located primarily in the cytosol in the untreated condition (Fig. 7B). When BV2 cells were exposed to LPS, the NF-jB p65 protein appeared in nuclei within 1 h; however, HDMPPA pretreatment reduced NF-jB p65 nuclear immunoreactivity, suggesting that HDMPPA inhibited the translocation of NF-jB p65 protein from the cytosol to the nucleus.
Because there is accumulating evidence indicating roles for PI3K/Akt and MAPKs signaling pathways in mediating inflammatory responses, we investigated whether the antiinflammatory potentials of HDMPPA were mediated through these pathways. As indicated in Figure 8, LPS alone treatment strongly activated Akt and MAPKs, including ERK, JNK, and p38 MAPK (their phosphorylated forms are shown), within 1 h. However, pretreatment of the cells with HDMPPA suppressed the LPS-induced phosphorylation of the Akt and MAPKs markedly, while their unphosphorylated forms remained the same, indicating that signal transduction by Akt and MAPKs may effectively be blocked by HDMPPA. DISCUSSION It is generally accepted that excess production and accumulation of NO, PGE2, and ROS by activated microglial cells can contribute to neurodegeneration. After LPS stimulation, microglia show an increase in iNOS and COX-2, key enzymes for the production of NO and PGE2, respectively, during inflammation, and contributors to the development of chronic neurological disorders.15,16 Thus, agents with the ability to inhibit iNOS and COX-2 expression may be beneficial in the treatment of conditions associated with NO and PGE2 overproduction, including inflammatory diseases. Accumulating evidence also indicates that oxidative components contribute to LPS-induced iNOS and COX-2
FIG. 7. Inhibition of LPS-induced nuclear accumulation of NF-jB p65 and degradation of IjB-a by HDMPPA pretreatment in BV2 microglia. Cells were pretreated with or without 200 lM HDMPPA for 30 min before treatment with LPS (500 ng/mL) for 30 min. (A) Nuclear and cytosolic proteins were separated on 10% SDS-polyacrylamide gels, followed by Western blotting using anti-NF-jB p65, anti-IjB-a, and anti-p-IjB-a antibodies. Nucleolin and actin were used as internal controls for the nuclear and cytosolic fractions, respectively. (B) Cells were pretreated with 200 lM HDMPPA for 30 min, LPS (500 ng/mL) was then added, and cells were incubated for 30 min. Localization of NF-jB p65 was visualized with fluorescence microscopy after immunofluorescence staining with NF-jB p65 antibody (red). Cells were stained with DAPI for visualization of the nuclei (blue). A representative example of three independent experiments is shown. DAPI, 4,6-diamidino-2-phenyllindile; NF-jB, nuclear factor-jB. Color images available online at www.liebertpub.com/jmf
ANTI-INFLAMMATORY EFFECTS OF HDMPPA IN BV2 MICROGLIA
FIG. 8. Effect of HDMPPA on LPS-induced MAPKs and Akt activation in BV2 microglia. BV2 cells were pretreated with 200 lM HDMPPA for 30 min before exposure to LPS (500 ng/mL), and total proteins were isolated at the times indicated following exposure to LPS. The proteins were separated on SDS-polyacrylamide gels followed by Western blot analysis using the indicated antibodies and an ECL detection system. Actin was used as an internal control for Western blot assays. MAPKs, mitogen-activated protein kinases.
expression.17,18 Thus, the management of cellular ROS levels could be regarded as key to the regulation of NO and PGE2 production. In this study, HDMPPA significantly inhibited LPS-induced NO and PGE2 productions by suppressing iNOS and COX-2 expression in BV2 cells in a concentration-dependent manner, which was associated with HDMPPA’s ROS scavenging activity. These data indicate that HDMPPA acts by regulating NO, PGE2, and ROS generation and that it could be a suppressor of microglial activation. Additionally, IL-1b and TNF-a are major proinflammatory cytokines that are produced by activated microglia in CNS inflammation, and their excessive production due to several stimuli, such as LPS, b-amyloid, and traumatic brain injury, has linked with many neurodegenerative disorders in the CNS.4,19,20 In this study, we investigated whether HDMPPA inhibited LPS-induced production of these cytokines. The results indicated that HDMPPA inhibits the LPSinduced release of proinflammatory cytokines, IL-1b and TNF-a in BV2 cells. We also found that the inhibitory effects of HDMPPA on the LPS-induced release of proinflammatory cytokines correlated with their ability to suppress the expression of their genes, suggesting that HDMPPA may modulate gene expression levels of cytokines; these levels then control their release.
7
Various intracellular signaling pathways are involved in inflammatory mediator expression. Several inflammatory stimuli, including LPS, commonly activate the NF-jB, MAPK, and PI3K/Akt signaling pathways in microglia. Among them, NF-jB is known to play a critical role in the expression of proinflammatory enzymes and cytokines related to the inflammatory process. It is known that blockade of NF-jB transcriptional activity in microglia can also suppress the expression of iNOS, COX-2, and proinflammatory cytokines, including IL-1b and TNF-a.21,22 NF-jB is present normally in the cytoplasm as a heterotrimer complex consisting of p50, p65, and IjB subunits. Upon activation of the complex, phosphorylation and degradation of IjB exposes nuclear localization signals on the p50/p65 complex, allowing NF-jB translocation to the nucleus and binding to specific regulatory sequences in the genomic DNA, thus controlling gene transcription.23,24 The MAPK family proteins—ERK, JNK, and p38 MAPK—also appear to play key roles in inflammatory processes. Previous studies have shown that the activation of MAPKs has a significant effect on the regulation of COX-2, iNOS, and proinflammatory cytokine gene expression by controlling the activation of NF-jB in microglia.25,26 Akt also can modulate the NF-jB activation through IjB phosphorylation and degradation,27,28 which has been implicated in the inflammatory pathway in LPS-stimulated BV2 microglial cells.29–31 Moreover, excessive accumulation of intracellular ROS triggers the release of inflammatory factors in microglia through the activation of downstream signaling molecules, such as NF-jB and MAPKs.8,32–34 Thus, it is possible that neuroprotective mechanisms are associated with the inhibition of NF-jB, PI3K/Akt, and MAPKs signaling pathways in activated microglia. In the present study, we found that HDMPPA effectively attenuated LPS-induced IjB-a degradation and phosphorylation as well as the nuclear translocation of NF-jB p65. We also demonstrated that HDMPPA inhibited LPS-induced phosphorylation of MAPKs and Akt in LPS-activated BV2 microglia associated with the inhibition of intracellular ROS accumulation. This indicates that the transcriptional downregulation of inflammatory mediators and cytokines by HDMPPA in activated microglia is, at least part, due to the blocking of the NF-jB, MAPKs, and PI3K/ Akt pathways by inhibiting the generation of ROS in LPSactivated BV2 microglia. In conclusion, the present observations identify a potential anti-inflammatory role for HDMPPA against neuroinflammatory toxicity resulting from the presence of inflammatory mediators and cytokines, through the inhibition of NF-jB, MAPKs, and PI3K/Akt activation. Although the neuroprotective actions of HDMPPA need to be examined further using in vivo models, our results indicate that HDMPPA is a novel compound with potential for the treatment of neurodegenerative diseases that involve microglial activation.
ACKNOWLEDGMENTS This research was supported by Grants from the Globalization of Korean Foods R&D Program (912001-1), funded by
8
JEONG ET AL.
the Ministry of Food, Agriculture, Forestry and Fisheries and the National Research Foundation of Korea (NRF) Grant funded by the Korea government (NRF-2014R1A2A1A09006983 and 2012046358), Republic of Korea. AUTHOR DISCLOSURE STATEMENT No competing financial interests exist. REFERENCES 1. Block ML, Hong JS: Microglia and inflammation-mediated neurodegeneration: Multiple triggers with a common mechanism. Prog Neurobiol 2005;76:77–98. 2. Glezer I, Simard AR, Rivest S: Neuroprotective role of the innate immune system by microglia. Neuroscience 2007;147:867–883. 3. Dheen ST, Kaur C, Ling EA: Microglial activation and its implications in the brain diseases. Curr Med Chem 2007;14:1189–1197. 4. Smith JA, Das A, Ray SK, Banik NL: Role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases. Brain Res Bull 2012;87:10–20. 5. Cheigh HS, Park KY: Biochemical, microbiological, and nutritional aspects of kimchi (Korean fermented vegetable products). Crit Rev Food Sci Nutr 1994;34:175–203. 6. Park H, Kim HS: Korean traditional natural herbs and plants as immune enhancing, antidiabetic, chemopreventive, and antioxidative agents: A narrative review and perspective. J Med Food 2014;17: 21–27. 7. Choi IH, Noh JS, Han JS, Kim HJ, Han ES, Song YO: Kimchi, a fermented vegetable, improves serum lipid profiles in healthy young adults: Randomized clinical trial. J Med Food 2013;16: 223–229. 8. Park KY, Jeong JK, Lee YE, Daily JW 3rd: Health benefits of kimchi (Korean fermented vegetables) as a probiotic food. J Med Food 2014;17:6–20. 9. Lee YM, Kwon MJ, Kim JK, Suh HS, Choi JS, Song YO: Isolation and identification of active principle in Chinese cabbage kimchi responsible for antioxidant effect. Korean J Food Sci Technol 2004;36:129–133. 10. Kim HJ, Lee JS, Chung HY, et al.: 3-(40 -hydroxyl-30 ,50 dimethoxyphenyl)propionic acid, an active principle of kimchi, inhibits development of atherosclerosis in rabbits. J Agric Food Chem 2007;55:10486–10492. 11. Noh JS, Kim HJ, Kwon MJ, Song YO: Active principle of kimchi, 3-(40 -hydroxyl-30 ,50 -dimethoxyphenyl)propionic acid, retards fatty streak formation at aortic sinus of apolipoprotein E knockout mice. J Med Food 2009;12:1206–1212. 12. Noh JS, Choi YH, Song YO: Beneficial effects of the active principle component of Korean cabbage kimchi via increasing nitric oxide production and suppressing inflammation in the aorta of apoE knockout mice. Br J Nutr 2013;109:17–24. 13. Chao CC, Gekker G, Hu S, Peterson PK: Human microglial cell defense against Toxoplasma gondii. The role of cytokines. J Immunol 1994;152:1246–1252. 14. Blasi E, Barluzzi R, Bocchini V, Mazzolla R, Bistoni F: Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus. J Neuroimmunol 1990;27:229–237. 15. Minghetti L, Pocchiari M: Cyclooxygenase-2, prostaglandin E2, and microglial activation in prion diseases. Int Rev Neurobiol 2007;82:265–275.
16. Kanwar JR, Kanwar RK, Burrow H, Baratchi S: Recent advances on the roles of NO in cancer and chronic inflammatory disorders. Curr Med Chem 2009;16:2373–2394. 17. Pawate S, Shen Q, Fan F, Bhat NR: Redox regulation of glial inflammatory response to lipopolysaccharide and interferongamma. J Neurosci Res 2004;77:540–551. 18. Munhoz CD, Garcı´a-Bueno B, Madrigal JL, Lepsch LB, Scavone C, Leza JC: Stress-induced neuroinflammation: Mechanisms and new pharmacological targets. Braz J Med Biol Res 2008;41: 1037–1046. 19. Whitney NP, Eidem TM, Peng H, Huang Y, Zheng JC: Inflammation mediates varying effects in neurogenesis: Relevance to the pathogenesis of brain injury and neurodegenerative disorders. J Neurochem 2009;108:1343–1359. 20. Lee CY, Landreth GE: The role of microglia in amyloid clearance from the AD brain. J Neural Transm 2010;117:949–960. 21. Mattson MP, Camandola S: NF-kappaB in neuronal plasticity and neurodegenerative disorders. J Clin Invest 2001;107: 247–254. 22. Zheng Z, Yenari MA: Post-ischemic inflammation: Molecular mechanisms and therapeutic implications. Neurol Res 2004;26: 884–992. 23. Guha M, Mackman N: LPS induction of gene expression in human monocytes. Cell Signal 2001;13:85–94. 24. Li Q, Verma IM: NF-kappaB regulation in the immune system. Nat Rev Immunol 2002;2:725–734. 25. Gosselin RD, Suter MR, Ji RR, Decosterd I: Glial cells and chronic pain. Neuroscientist 2010;16:519–531. 26. Ji RR, Gereau RW 4th, Malcangio M, Strichartz GR: MAP kinase and pain. Brain Res Rev 2009;60:135–148. 27. Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB: NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 1999;401:82–85. 28. Chan CB, Ye K: Phosphoinositide 3-kinase enhancer (PIKE) in the brain: Is it simply a phosphoinositide 3-kinase/Akt enhancer? Rev Neurosci 2012;23:153–161. 29. Lee SH, Lee JH, Oh EY, et al.: Ethanol extract of Cnidium officinale exhibits anti-inflammatory effects in BV2 microglial cells by suppressing NF-jB nuclear translocation and the activation of the PI3K/Akt signaling pathway. Int J Mol Med 2013; 32:876–882. 30. Park HY, Han MH, Park C, et al.: Anti-inflammatory effects of fucoidan through inhibition of NF-jB, MAPK and Akt activation in lipopolysaccharide-induced BV2 microglia cells. Food Chem Toxicol 2011;49:1745–1752. 31. Jeong JW, Lee WS, Shin SC, et al.: Anthocyanins downregulate lipopolysaccharide-induced inflammatory rsponses in BV2 microglial cells by suppressing the NF-jB and Akt/MAPKs signaling pathways. Int J Mol Sci 2013;14:1502–1515. 32. Rosenberger J, Petrovics G, Buzas B: Oxidative stress induces proorphanin FQ and proenkephalin gene expression in astrocytes through p38- and ERK-MAP kinases and NF-kappaB. J Neurochem 2001;79:35–44. 33. Kim BH, Oh I, Kim JH, et al.: Anti-inflammatory activity of compounds isolated from Astragalus sinicus L. in cytokineinduced keratinocytes and skin. Exp Mol Med 2014;46:e87. 34. Kim M, Li YX, Dewapriya P, Ryu B, Kim SK: Floridoside suppresses pro-inflammatory responses by blocking MAPK signaling in activated microglia. BMB Rep 2013;46:398–403.
