Inflammatory responses are widely implicated in vast kinds of acute and chronic human diseases, including cancer, atherosclerosis, and diabetes [1
]. Macrophages play a critical role and are involved in the self-regulating cycle of inflammation, as macrophages produce multiple pro-inflammatory cytokines and mediators that are involved in inflammation, such as the TNFα and the IL-6 [2
]. Interference therapy that target macrophages and related cytokines may be some new approaches for controlling inflammatory diseases.
Regulation of the inflammatory response depends on a variety of potential mechanisms, including peroxisome proliferator-activated receptors (PPARs) actions [3
]. PPARs are activated by their synthetic or natural ligands/modulators, which lead to the PPARs to bind to their specific DNA response elements, as heterodimers, with the retinoid X receptor (RXR) [4
]. PPARs have been found to have three subtypes, which are named PPARα, PPARβ/δ, and PPAR. They play crucial roles in the regulation of lipid and glucose metabolism. In addition, accumulating evidence reveals that activation of the PPARs are involved in the various types of inflammatory processes, due to the inhibition of pro-inflammatory genes expression and negative regulation of pro-inflammatory transcription factor signaling pathways, in inflammatory cells [5
]. Furthermore, activation of PPARs shows the anti-inflammatory effect by inhibiting the activation of nuclear factor-κB (NF-κB), leading to a decrease of pro-inflammatory cytokines and mediators [6
]. Therefore, PPARs have been shown to be the drug targets to treat various related inflammatory diseases, such as vascular diseases, cancer, and neurodegenerative diseases [7
]. Searching for the effective ligands or modulators of PPARs, for the prevention and clinical therapeutic options, is of great interest.
The natural product nuciferine ((R
)-1,2-dimethoxyaporphine; Nuci) is an alkaloid found within the leaves of Nymphaea caerulea
and Nelumbo nucifera
, which is widely planted in Asia, the Middle East, and some countries in Africa [8
]. Especially in China, lotus leaves are usually commercially available for tea due to its pharmacologic effects, such as losing weight, heat-clearing, and detoxifying, according to the traditional theory of Chinese medicine [9
]. Recent studies showed that nuciferine, an important component of lotus leave extracts, can improve hepatic lipid metabolism [10
], increase the glucose consumption, and stimulate insulin secretion [11
]. Anti-inflammation activity of nuciferine was also reported in potassium oxonate-induced kidney inflammation [12
], as well as Fructose-induced inflammatory responses [13
], in vivo. However, the underlying molecular mechanisms of its anti-inflammatory effects are not fully understood. Based on the inflammatory-related functions of PPARs and the differences of the distinct tissue-specific expression, physiology, and ligand specificity of the PPARα, PPARβ/δ, and the PPARγ, the aim of this study was to investigate the effect of nuciferine on inflammation in lipopolysaccharide (LPS)-induced RAW264.7 cells and to observe if this effect is mediated by the three PPAR subtypes.
Our present study has shown that treatment with nuciferine ameliorates the LPS-induced inflammation in RAW264.7 cells. Importantly, it was found that the protective effect of nuciferine is mediated by PPARs activation. These results highlight the potential use of nuciferine for preventing inflammation.
Overexpression of the inflammatory mediators is closely associated with systemic injury. There is evidence that anti-inflammatory treatment has become an important component of inflammatory diseases [18
]. Inflammatory mediator inhibitors can be shown to have beneficial effects in improving the severity of inflammation-related diseases. A wide variety of phytochemicals derived from natural plant have anti-inflammatory effects, such as phenolics, terpenolids, and alkaloids [19
]. Nuciferine, an alkaloid found in the lotus leaves, exerted a protective effect against inflammation, in vivo [20
] and in vitro [21
]. Our results showed that nuciferine decreased the expression of inflammatory cytokines IL-6 and TNFα, in both protein and gene levels, dose dependently, in the LPS-treated RAW264.7 cells, indicating that nuciferine had potential anti-inflammatory effects.
Nuciferine, a natural alkaloid from the lotus leaves, have been reported to exert multiple beneficial effects, in vivo and in vitro, such as anti-tumor [22
] and insulin stimulatory effects [23
]. Our recent results showed that nuciferine improved the hepatic steatosis in high-fat diet/streptozocin-induced diabetic mice [24
]. Some studies reported that nuciferine suppressed the inflammation by regulating inflammatory signaling through different signal pathways. For example, in hyperuricemia mouse model, nuciferine inhibited renal inflammation through suppression of Toll-like receptor 4/myeloid differentiation factor 88/NF-κB signaling and a NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome [12
]. Similarly, in vitro studies, nuciferine exerted the anti-inflammatory and antilipemic effects, as well as the siRNA Per-Arnt-Sim kinase treatment group in oleic acid-induced hepatic steatosis, in HepG2
cells, indicating a potential molecular pathway of the anti-inflammation effect of nuciferine [21
]. As we know, the three subtypes of PPARs exert anti-inflammatory effects in vivo and in vitro by several different molecular mechanisms [25
]. PPARα [28
] and PPARγ [29
] were shown to repress some other transcription factors, such as NF-κB signal pathway, to reduce the release of inflammatory cytokines including IL-6 and TNFα, when they were activated by their ligands. The anti-inflammatory effects of PPARβ are mediated by ligand-independent repression [30
]. Owing to the anti-inflammatory effect of PPARs, we used the luciferase reporter assay and the target gene transcription of PPARα/PPARγ/PPARδ to test if the PPAR family is involved in the anti-inflammatory effect of nuciferine. The results showed that nuciferine activated the PPAR family, especially the PPARα and the PPARγ. Moreover, the antagonists of the PPAR family GW6417/GSK0660/GW9662 were treated in the cells to block the PPARs activities, before the nuciferine treatment. What’s interesting is that all the antagonist treatment increased the inflammation markers. The protective effect of nuciferine was remarkably diminished by the inhibition of PPARα and PPARγ, indicating that the anti-inflammatory effect of nuciferine, at least in part, went through the PPARs receptor activation. To confirm these results, the protein expression of the activated PPARs and the total PPARs should also be tested, using immunoblotting, in the further studies. Our recent in vivo results also clarified that nuciferine-activated PPARα in the liver tissues, in a diabetic mouse model [24
]. Nuciferine is hydrophobic, consistent with the structures of most PPAR agonists. It could interact with the ligand-binding domain of PPARs, in theory, leading to the stabilization of the configuration of the hydrophobic core and subsequently the activation of PPARs to regulate the gene transcription [31
]. However, more binding mechanisms between the nuciferine and the PPARs should be further studied.
It is well known that NF-κB is an important target for inflammatory therapeutic strategy [32
]. PPARs have recently been shown to exert the anti-inflammatory activity by reducing the DNA-binding activity of NF-κB and suppressing its nucleus translocation, which attenuates the cytokine production and reduces tissue injury [32
]. NF-κB is a crucial factor to activate the inflammatory genes transcription, including pro-inflammatory cytokines, such as TNFα and IL-6 [35
]. In addition, IκBα expression was accompanied by a decrease in NF-κB DNA binding activity [36
]. Our results showed that nuciferine treatment alters the IκBα cellular content in LPS stimulation. Moreover, the specific inhibitors for PPARs reversed the effect of nuciferine, partially or completely, indicating that nuciferine could prevent IκBα degradation via PPARs activation, under the LPS stimulated conditions.
Overall, our studies demonstrated that nuciferine, with a concentration of 10 μM, attenuated the LPS-induced inflammation through activation of PPARs, especially PPAR-α and-γ, in RAW264.7 cells. These findings suggest that nuciferine may be a potentially important candidate for inflammatory diseases.
4. Materials and Methods
Nuciferine (purity by HPLC > 98.0%) was purchased from APP-CHEM (YHI-039, Xi’an, Shanxi, China). Dulbecco’s modified Eagle’s medium-high glucose (DMEM), fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Lipofectamine 2000 reagent were purchased from Invitrogen (Carlsbad, CA, USA). Lipopolysaccharide (LPS), PPARs agonists WY14643, GW501516, rosiglitazone (Rosi) were purchased from Sigma (St. Louis, MO, USA). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA). IL-6 and TNF-α Mouse ELISA Kit was obtained from Elabscience Biotechnology Co. Ltd. (Wuhan, Hubei, China). Super Script II Rnase H Reverse Transcriptase kit was purchased from Invitrogen (Carlsbad, CA, USA).
4.2. Cell Culture
Murine macrophage RAW264.7 cells (ATCC, Rockville, MD, USA) and human embryonic kidney cells (HEK293 cells, ATCC, Rockville, MD, USA) were cultured with DMEM containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin. Cells were maintained at 37 °C, in a humidified atmosphere of 5% CO2 and 95% air. RAW264.7 cells were seeded into plates and treated at approximately 80% confluence.
RAW264.7 cells were seeded at a density of 1.5 × 103
cells/well in 96-well plates. After 24 h, cells were treated with different concentrations of nuciferine (0–50 μM), for 24 h, followed by an addition 20 μL MTT solution (5 g/L), to each well, for 4 h. The insoluble formazan product was dissolved in 150 μL/well dimethyl sulfoxide (DMSO), after washing out the supernatant [37
]. Then, the absorbance at 490 nm was measured using a microplate reader (Olympus America Inc., New York, NY, USA). The percentage of cytotoxicity was calculated by the equation: Cytotoxicity (%) = (1 − A490
of control well.
4.4. IL-6 and TNFα Levels Determination
RAW264.7 cells were grown into 12-well plates, treated with different concentrations of nuciferine (0, 1, 10 or 50 μM) and stimulated with LPS (500 ng/mL). Cell-free supernatants were collected and the levels of pro-inflammatory cytokines, TNFα and IL-6 were measured using ELISA kits, by a determination of the absorbance at 450 nm, according to the manufacturer’s instructions. Standard curves were used to calculate the concentration of TNFα and IL-6 in each sample.
4.5. PPARs Luciferase Reporter Assay
HEK293T cells and RAW264.7 cells were plated into 12-well plates at 4 × 105
cells/well without antibiotics. After 24 h at 60% confluence, cells were transfected according to the manufacturer’s instructions. Briefly, PPARs isoforms (PPARα/PPARδ/PPARγ) plasmid (0.9 µg), reporter plasmid PPRE×3-TK-LUC (0.3 µg) and β-gal (0.1 µg) were transfected into the cells, using Lipofectamine 2000 reagents (1:1), for 4 h. Since transfection efficiency is typically low in RAW264.7 cells, more Lipofectamine 2000 was needed (1:2.5) and the transfection time was extended to 24 h. The medium was replaced with a complete media containing DMSO, nuciferine, or PPARs agonists, for 24 h. The cells were harvested and lysed to measure the luciferase activities using a luciferase assay kit, according to the manufacturer’s instructions. The β-gal was transfected to normalize the transfection efficiency [38
4.6. RNA Isolation and Analysis
Cells were cultured into 12-well plate with a density of 4 × 105
cells/well. Total RNA was isolated using TRIzol reagent and reverse transcribed into cDNA. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed as described by Yang et al. [39
]. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh
) was used as an internal control. Ct values of the sample were calculated, and the mRNA levels were analyzed by 2−ΔΔCt
method and normalized to Gapdh
]. The primer sequences were listed in Supplemental Table S1
RAW264.7 Cell lysates were prepared using a lysis buffer containing 0.1% Triton X-100 and proteinase inhibitors (Roche, Nutley, NJ, USA). Protein concentrations were determined using the BCA protein assay kit (Thermo Scientific, PA, USA). Western blot was performed as described by Yang et al. [39
]. After blocking the membranes, primary rabbit antibody against IκBα (Santa Cruz Biotechnology, Dallas, TX, USA) was incubated with a ratio of 1:1000 overnight. β-actin (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA) was used as a loading control. Membranes were then washed with TBST and incubated with the secondary antibodies conjugated to anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (1:3000, Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h. Bands were detected by enhanced chemiluminescence using ECL (Amersham Biosciences, Picataway, NJ, USA) and then visualized by X-ray films.
4.8. Data Statistics
Quantitative data are expressed as mean ± SEM using SPSS 18.0 (IBM Corporation, Chicago, IL, USA). Student t test and ANOVA followed by Tukey’s post hoc test were used to analyze the significant difference between two or more groups, respectively. The rank-based test methods were employed when data were not in a normal distribution or the variances were not homogeneous. All the results were representative of at least three independent experiments.