Vaccaria hypaphorine alleviates lipopolysaccharide-induced inflammation via inactivation of NFκB and ERK pathways in Raw 264.7 cells

Vaccaria hypaphorine was purchased from Shanghai Shifeng technology Co., Ltd.,China. Vaccaria hypaphorine was solubilized in sterile phosphate buffer saline (PBS). The cells including mouse macrophage Raw264.7, human lung adenocarcinoma cell line A549 and mouse lung cancer cell Lewis were purchased from American Type Culture Collection (Rockville, MD, USA). Human microvascular endothelial cells HMEC-1 was obtained from the Health and Medicine Research of French National Institute. Human umbilical vein endothelial cells EA · hy926, mouse fibroblast cells L929, human breast cancer cell line MCF-7 and mouse melanoma cells were gifts from Tang Zhongying Institute of Hematology of Soochow University. Mouse liver cancer cell H22 was a gift from Department of pharmacology, Shanghai Pharmaceutical Industry Research Institute. Cell culture supplies were purchased from Costar (Corning Inc., Cypress, CA, USA). Sulforhodamine B (SRB), MCDB 131, rhodamine B, DAPI, lipopolysaccharide and aspirin (Asp) were purchased from Sigma Chemical Co. (St Louis, MO, USA). RPMI-1640 medium and DMEM were obtained from Hyclone (Logan, UT, U.S.A.). Dexamethasone (Dex) was purchased from Shanghai biological engineering Limited by Share Ltd. Antibody against β-tublin was purchased from Abcam (Cambridge, MA, USA). Antibodies against total or phosphorylated nuclear factor (NF)κB, IκBα, IκB-kinase β (IKKβ) and ERK as well as iNOS, COX-2 were obtained from Cell Signaling Technology (Beverly, MA, USA). The goat anti-rabbit secondary antibody was purchased from SangonBiotech (Shanghai) Co.,Ltd. (Shanghai, China). M-PER Mammalian Protein Extraction Reagent was purchased from Thermo scientific (USA). RNAiso Plus reagent, SYBR® Premix Ex Taq™ and PrimeScript™RT reagent Kit with gDNA Eraser were purchased from Takara Co. (Takara, Otsu, Shiga, Japan). Dex and Asp were served as positive controls.

Raw264.7, EA · hy926, L929 and MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, U.S.A.) and 1 × Antibiotic-Antimycotic Solution. A549, Lewis, B16 and H22 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1 × Antibiotic-Antimycotic Solution. HMEC-1 cells were cultured in MCDB 131 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1 × Antibiotic-Antimycotic Solution. These cells were incubated at 37 °C in a humidified air containing 5% CO₂. The growth medium was replaced every 2–3 day and the cells were seeded onto petridishes or multiwell plates at a ratio of 1–3 upon 80% confluency.

The sulforhodamine B (SRB) assay was used to assess the cell viability as our previous report [21]. The cell growth was arrested by incubation of the cells in 2% serum medium for 24 h before treatment. The indicated cells were then seeded in 96-well culture plates at a density of 5 × 10³ cells/well, and stimulated with different concentrations of vaccaria hypaphorine (6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM) at 37 °C in 5% CO₂ saturated humidity condition for 24 h. Finally, the optical density (OD) was measured at 540 nm with the aid of a Multiskan MK3 (Labsystem company). The results were expressed as the ratio by normalizing the targeted OD level to that of control OD.

The levels of NO were analyzed using Griess reaction as previously described [26, 27]. Equal volumes of N-(1-naphthyl) ethylenediamine and sulfanilic acid were combined to form the Griess reagent. The 100 μl supernatant from each well was collected in all samples, and reacted with 100 μl of the Griess Reagent for 30-min incubation at room temperature. The absorbance was measured at 540 nm. The standard curves were plotted from sodium nitrite standards (ranging from 10 to 60 μM).

The mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10 and monocyte chemoattractant protein 1 (MCP-1) were detected by a fluorescence quantitative LightCycler 480 Real Time PCR system (Roche, Basel, Sweden) [28]. In brief, total RNA of each sample was extracted by Trizol reagent according to the manufacturer’s instructions. The equal RNA was reverse transcribed to cDNA using PrimeScript™RT reagent Kit with gDNA Eraser (Takara, Otsu, Shiga, Japan). The real-time quantitative PCR was performed in triplicates by using SYBR® Premix Ex Taq™ (Takara, Otsu, Shiga, Japan). The average cycle thresholds (Ct) were employed to quantify fold-change. The relative quantification of gene expression was reported as a relative quantity to the control value by using 2^-△△Ct methods. The sequences of primers were listed in the supplemental table (Additional file 1: Table S1).

Commercial ELISA kit was used for the measurement of TNF-α,IL-1,IL-6,PGE2 levels (R&D Systems, Inc., Minneapolis, MN, USA) following the manufacturer’s protocols. The cell culture supernatants in each sample were collected and stored -20 °C prior to use. The standards or samples diluent were added into appropriate well of specific antibody pre-coated microtiterplate and incubated for 30 min at 37 °C. The reacted microtiterplate was washed by diluted washing buffer for 5 times. Conjugate was added and incubated for 1 h at 37 °C and then re-washed. The reactions were stopped with 50 μl stop solution, and the absorbance was read at 450 nm with a microtiter plate reader (STNERGY/H4, BioTek, Vermont, USA).

Whole cell lysates were obtained and homogenized by RIPA buffer. The supernatant was collected by centrifugation at 12,000 g for 10 min at 4 °C. Total protein concentration in the homogenate was measured with a protein assay kit (Santa Cruz, Dallas, TX, USA). Total 25 μg of total protein were subjected to SDS-PAGE and electro-transferred onto nitrocellulose membrane (Millipore, USA). The membranes were blocked with 5% nonfat milk for 1 h at room temperature before overnight incubation with indicated primary antibodies at 4 °C. The primary antibodies were as follows: COX-2 (1:1000), iNOS (1:1000), phosphorylation of ERK (1:1000), ERK (1:1000), p-IκBα (1:1000), IκBα (1:1000), p-IKKβ (1:1000), IKKβ (1:1000), p-NFκB (1:1000), NFκB (1:1000), and β-tublin (1:1000). The membranes were washed three times with TBST for 5 min and subsequently incubated with HRP-conjugated secondary antibody. The positive bands were captured using the enhanced chemiluminescent.

The stimulated Raw264.7 cells were fixed with 4% formaldehyde for 30 min, and then permeabilized with 0.1% Triton X-100 in PBS for 15 min. After incubation with 10% goat serum for 30 min, and incubated with indicated primary antibody rabbit anti-NFκB overnight at 4 °C. After three washes with PBS, cells were incubated with FITC-labeled secondary antibodies for 30 min. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) after immunofluorescence staining. Immunofluorescence signals were visualized on a fluorescence microscope (80i, Nikon, Japan).

All results were defined as mean ± S.D.. Comparisons within two groups were made by Student’s t test. Statistical analysis was performed by ANOVA/Dunnet t-test for multiple group comparisons. A difference of P < 0.05 was considered statistically significant.

The different cell lines RAW264.7, L929, A549, Lewis, H22, B16, MCF-7, HMEC-1 and EA · hy926 were treated with various doses of vaccaria hypaphorine (6.25, 12.5, 25, 50, 100 and 200 μM) for 24 h. SRB assay then showed that vaccaria hypaphorine had no significant effect on the growth of tumor cells and endothelial cells. Vaccaria hypaphorine had no cytotoxicity on these cells including RAW264.7 cells (Additional file 1: Table S2).

To explore the anti-inflammatory effect of vaccaria hypaphorine, vaccaria hypaphorine was treated on LPS-induced inflammation in RAW 264.7 cells. Firstly, the determination of optical density by SRB assay showed that LPS exerted a slight promotion in RAW264.7 cell proliferation, and combination application of LPS with vaccaria hypaphorine, Dex and Asp had no obvious effect on the proliferation of RAW264.7 cells (Additional file 1: Figure S1).

LPS, a Gram-negative bacterial cell wall component, is responsible for pro-inflammatory cytokines accumulation in activated macrophages [29]. Stimulation of RAW 264.7 macrophages with LPS induced remarkable increases in mRNA levels of TNF-α (Fig. 1a), IL-1β (Fig. 1b), IL-6 (Fig. 1c), IL-10 (Fig. 1d) and MCP-1 (Fig. 1e). Vaccaria hypaphorine markedly prevented LPS-induced productions of IL-1β, IL-6, IL-10 and MCP-1 (Fig. 2) in a dose-dependent manner. Vaccaria hypaphorine effectively counteracted TNF-α mRNA levels in LPS-challenged RAW 264.7 cells, but with dose correlation (Fig. 1a). Both Dex and Asp exhibited an inhibitory effect on LPS-induced inflammation reactions at mRNA level in RAW 264.7 macrophages (Fig. 1). In particular, pro-inflammatory cytokine productions were suppressed more effectively by vaccaria hypaphorine (50 μM) than by both Dex and Asp (Fig. 1). Excessive production of NO may act as pro-inflammatory mediators to evoke chronic inflammation contributing tissue damages [30]. Incubation of RAW264.7 macrophages with LPS (1 μg/ml) significantly increased NO overproduction by more than 5 fold, which was effectively reduced by both vaccaria vypaphorine and Dex as well as Asp (Fig. 1f).

Similar to mRNA results, treatment of RAW 264.7 macrophages with LPS caused enormous increases in protein levels of key inflammatory factors including TNF-α (Fig. 2a), IL-1β (Fig. 2b), IL-6 (Fig. 2c) and PGE₂ (Fig. 2d). The pro-inflammatory effects of LPS were suppressed by vaccaria hypaphorine in a concentration-related fashion. Both Dex and Asp obviously impeded LPS-induced inflammation reactions at protein levels of TNF-α, IL-1β, IL-6 and PGE₂ in RAW 264.7 macrophages (Fig. 2).

The iNOS in macrophages may produce excessive NO to exert toxic effects on cells under inflammatory stimuli such as LPS and cytokines [31]. It is accepted that macrophages may induce the expression of inflammatory enzymes such as iNOS and COX-2 during inflammatory responses [32]. The up-regulated iNOS and COX-2 protein expressions were obviously inhibited by vaccaria hypaphorine (Fig. 3). The reduction in NO release by vaccaria hypaphorine may be attributed to iNOS protein inhibition.

It is well clarified that mitogen-activated protein kinase ERK or NFκB transduction pathways are majorly involved in LPS-induced macrophages inflammation [33]. ERK pathway may be taken as intermediate stage in the regulation of NF-κB activation [34, 35]. Treatment of RAW 264.7 macrophages with LPS promoted the phosphorylation of ERK, which was attenuated by vaccaria hypaphorine at higher dose (Fig. 4), suggesting that the upstream kinases for ERK may be modulated by vaccaria hypaphorine.

Nucleus translocation of p65-NFκB is considered as a prerequisite for the transcription [36]. The phosphorylation of IκBα is identified to be upstream events of p65-NFκB translocation. Activation IKKβ is crucial for phosphorylation of IκBα and translocation of NFκB in a canonical pathway [37]. Exposure of RAW 264.7 macrophages to LPS significantly augmented the phosphorylated protein levels of NFκB, IκBα and IKKβ in RAW 264.7 macrophages, whereas vaccaria hypaphorine (50 μM) markedly inhibited the LPS-induced production of phosphorylated IκBα (Fig. 5a), IKKβ (Fig. 5b) and NFκB (Fig. 5c). Furthermore, LPS promoted the nuclear accumulation of p65-NFκB as evidenced by immunofluorescence staining, and which was effectively attenuated by pretreatment with vaccaria hypaphorine (Fig. 6).