The Aqueous Extract of Radio-Resistant Deinococcus actinosclerus BM2T Suppresses Lipopolysaccharide-Mediated Inflammation in RAW264.7 Cells
1Department of Bio & Environmental Technology, College of Natural Science, Seoul Women’s University, Seoul 01797, Republic of Korea, 2Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon 34054, Republic of Korea, 3College of Health Sciences, Kangwon National University, Samcheok 25949, Republic of Korea, 4Department of Convergence Biomedical Science, Cardiovascular and Metabolic Disease Center, College of Medicine, Inje University, Busan 47392, Republic of Korea
J. Microbiol. Biotechnol. 2020; 30(4): 583-590
Published April 28, 2020
Copyright © The Korean Society for Microbiology and Biotechnology.
Inﬂammation occurs in response to any type of bodily injury, including infection, and is marked by neutrophil and macrophage recruitment leading to the production of proinflammatory cytokines and chemokines [1, 2]. Inflammation is controlled by complex regulatory mechanisms . However, if poorly resolved, it may result in chronic inflammation, dubbed as the major organ dysfunction associated with fibrosis [4, 5]. Macrophages play an important role in resistance against bacterial pathogens, tissue remodeling, repair, and inflammation resolution. Furthermore, resting macrophages (M0) can be polarized to M1 and M2 in response to different stimuli, such as cytokines, microbes, and other modulators . M1 macrophages (killer type cells) are activated by interferon-γ and/or lipopolysaccharides (LPS), gram-negative bacterial endotoxins that can stimulate the secretion of a variety of proinflammatory cytokines and enzymes, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) . M1 macrophages then secrete proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-12. M1 macrophages are also involved in the generation of reactive oxygen species (ROS), such as nitric oxide via iNOS . In contrast, M2 macrophages (repair type cells), which are induced by IL-4, IL-13, or glucocorticoid exposure, are involved in inflammation resolution
Extremophiles, microorganisms with the ability to survive in extreme environmental conditions, may produce radiation-responsive metabolites, pigments, and enzymes for their survival . Their biological ability to adapt to harsh environmental conditions has been extensively exploited for the development of therapeutic drugs, especially anti-cancer drugs, antibiotics, and agricultural products of commercial significance [9-11]. It is speculated that radio-resistant genus
Materials and Methods
Chemicals and Reagents
Dulbecco’s modified Eagle media (DMEM), fetal bovine serum (FBS), trypsin-EDTA, penicillin, and streptomycin were purchased from WelGene (Korea). Lipopolysaccharides from
BM2U Extract Preparation
DPPH Radical Scavenging Activity
Different concentrations of BM2U were adjusted to 100 μl with reaction mixture, and then reacted with 100 μl of 0.4 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution in 99% EtOH. After vigorous shaking, reaction mixtures were allowed to reach a steady state at room temperature for 30 min. DPPH decolorization was determined by measuring the absorbance at 517 nm, using a microplate reader VICTOR X3 (PerkinElmer, USA). The half-maximal inhibitory concentrations (IC50) of DPPH radical formation were calculated from the graph by plotting the inhibition percentages against the tested BM2U concentrations.
Oxygen Radical Absorbance capacity (ORAC) Assay
The oxygen radical absorbance capacity (ORAC) assay was performed as previously described . Trolox is a water-soluble analog of vitamin E, commonly used as the standard. Briefly, 20 μl of BM2U or Trolox at equal concentrations was incubated with 10 μM of β-PE and 50 mM of AAPH in a total volume of 200 μl. β-PE and AAPH were used as the fluorescent probe and a peroxy radical generator, respectively. The determination of a decreasing amount in fluorescence was followed at 2 min intervals for 60 min at 37°C. All ORAC analyses were performed on a microplate reader VICTOR X3 at 37°C, with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. After obtaining the area under the curve (AUC) for each sample and standard, the BM2U ORAC values at different concentrations were expressed as a μM of Trolox Equivalents (TE), by comparing to the standard curve.
RAW264.7 (murine macrophage) cells were purchased from the American Type Culture Collection; ATCC (USA), and maintained in DMEM medium containing 10% FBS and 1% penicillin/streptomycin (10,000 U pen/ml and 10,000 μg strep/ml) in a humidified atmosphere of 5% CO2 at 37°C.
RAW264.7 cells were attached to a 96-well microplate and treated with different doses (6.25, 12.5, 25, 50, and 100 μg/ml) of BM2U for 24 h. After incubation with MTT (150 μg/ml) for 4 h, the formazan crystals formed were dissolved in DMSO and the absorbance was measured at 540 nm using a microplate reader.
Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
The mRNA expression levels of IL-1β, IL-6, and TNF-α were determined using reverse transcription-polymerase chain reaction (RT-PCR). RAW264.7 cells were attached to a 6-well microplate and treated with different doses (0.2, 2, and 20 μg/ml) of BM2U, with or without LPS (1 μg/ml), for 16 h. Total RNA was extracted from RAW264.7 macrophages using the TRIzol reagent from Invitrogen (USA), and processed using a cDNA Synthesis kit from TAKARA (Japan). SYBR master mix kit from TAKARA was used for RT-PCR, and cDNA was amplified using specific primers (Table 1). Quantitative real-time RT-PCR reactions were performed on a Light Cycler 96 Instrument from Roche (Basel, Switzerland). Relative quantitative evaluation of each gene was performed by the comparative cycle threshold method .
RAW264.7 cells were attached to a 6-well microplate and treated with different doses (0.2, 2, and 20 μg/ml) of BM2U, with or without LPS (1 μg/ml), for 15 min (p-p65, IκBα, and MAPKs) or for 24 h (COX-2, iNOS, and HO-1). The cells were then harvested, washed with cold PBS, and lysed with a homogenized PRO-PREP™ Protein Extraction Buffer from Intron Biotechnology (Korea), for 1 h on ice. After centrifugation at 15,000 ×
Data are presented as mean ± standard error of the mean (SEM), from at least three independent experiments performed in triplicates. Statistically significant differences between control and experimental values were calculated with analysis of variance (ANOVA) followed by Tukey’s test, using GraphPad Prism 5 from GraphPad Software Inc. (USA). A
Antioxidant Capacities of BM2U
ROS suppression via quenching free oxygen radicals is critically important in attenuating inflammatory responses. Thus, we determined the antioxidant capacities of BM2U (6.25-100 μg/ml), via the DPPH radical scavenging activity (Fig. 1A) and peroxy-radical scavenging ORAC assay (Fig. 1B). The IC50 values of BM2U on DPPH radical scavenging and ORAC were 349.3 μg/ml and 50.24 μg/ml, respectively. These results indicate that BM2U possesses radical scavenging capacity, favorable for direct or indirect resolution of ROS-mediated inflammation.
Effect of BM2U on DPPH radical scavenging activity, ORAC, and cell cytotoxicity.The antioxidant capacities of BM2U were determined through the DPPH radical scavenging activity (A) and ORAC (B) assays. RAW264.7 macrophages were incubated with different doses of BM2U for 24 h, and cell viability was determined using the MTT assay (C). * p <0.05 or ** p< 0.01 vs. the untreated control. Values represent mean ± SEM ( n= 4, per group). BM2U; an aqueous extract of D. actinosclerusBM2T (GenBank: KT448814).
BM2U cytotoxicity in RAW264.7 macrophage cells was evaluated at 24 h, by MTT assay. There was no significant toxicity up to 25 μg/ml, but cytotoxicity was observed above 50 μg/ml of BM2U (Fig. 1C). Thus, the treatment doses of BM2U for the cells were set at 0.2, 2, and 20 μg/ml, to avoid toxicity.
Suppressive Effect of BM2U on LPS-Mediated TNF-α, IL-1β, and IL-6 Expression
LPS-induced monocyte and macrophage stimulation induces many genes that express proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 [7, 21]. As depicted in Fig. 2A, the LPS-challenge on RAW264.7 macrophages led to significant induction of TNF-α, IL-1β, and IL-6 mRNA (
Effect of BM2U on LPS-mediated TNF-α, IL-1β, and IL-6 mRNA expression, and COX-2 and iNOS expression.RAW264.7 macrophage cells were challenged with LPS (1 μg/ml) in the presence or absence of BM2U (0.2-20 μg/ml) for 16 h, and the mRNA levels of TNF-α, IL-1β, and IL-6 were quantified using qRT-PCR. Values represent the relative ratio to β-actin as mean ± SEM ( n= 4, per group). COX-2 and iNOS expression levels at 24 h were quantified by immunoblotting. ## p <0.01 vs. the untreated control; ** p<0.01 vs. the LPS-only group. BM2U, an aqueous extract of D. actinosclerusBM2T (GenBank: KT448814).
Suppressive Effect of BM2U on COX-2 and iNOS Expression
NO and prostaglandin E2 (PGE2) are important inflammatory mediators produced by iNOS and COX-2, respectively. No significant COX-2 and iNOS expression was observed at baseline. However, upon LPS-induced stimulation of RAW264.7 macrophage cells, iNOS and COX-2 expression were significantly upregulated, as shown in Fig. 2B (
Suppressive Effect of BM2U on NF-κB Activation
As shown in Fig. 2, BM2U treatment significantly inhibited LPS-mediated upregulation of TNF-α, IL-1β, and IL-6 mRNA, as well as COX-2 and iNOS. Considering the involvement of the transcription factor NF-κB in the upregulation of those proinflammatory cytokines, and the induction of iNOS and COX-2, we determined the effect of BM2U on LPS-mediated NF-κB activation. NF-κB activation has been associated with increased p65 phosphorylation (p-p65) and downregulation of the NF-κB inhibitory protein, IκBα. As depicted in Fig. 3, the LPS challenge on RAW264.7 macrophages led to significantly increased expression of p-p65, and decreased expression of IκBα. However, at 2 and 20 μg/ml, BM2U significantly reversed the increased expression of p-p65, and decreased expression of IκBα. These results suggest that LPS-induced NF-κB activation can be attenuated, at least in part, in the presence of BM2U. This suppressive effect of BM2U on NF-κB activation may result in the suppression of proinflammatory cytokines, and induction of COX-2 and iNOS.
Effect of BM2U on LPS-mediated NF-κB activation.RAW264.7 macrophage cells were challenged with LPS (1 μg/ml) in the presence or absence of BM2U (0.2-20 μg/ml) for 15 min, and the expression levels of p-p65 and IκBα were quantified by immunoblot analyses. Values represent the relative ratio to β-actin as mean ± SEM ( n= 3, per group). ## p< 0.01 vs. the untreated control; * p< 0.05 or ** p< 0.01 vs. the LPS-only group. BM2U, an aqueous extract of D. actinosclerusBM2T (GenBank: KT448814).
Effect of BM2U on LPS-Mediated Activation of MAPKs
MAPKs play a critical role in the expression of various cytokines and chemokines; thus, LPS-induced MAPKs inhibition can act as another restriction point for inflammatory responses. In addition, MAPKs are involved in the activation of the NF-κB signaling pathway . As depicted in Fig. 4, the LPS challenge on RAW264.7 macrophages caused significant activation of ERK, JNK, and p38 MAPK (
Effect of BM2U on LPS-mediated JNK, ERK, and p38 MAPK activation.RAW264.7 macrophage cells were challenged with LPS (1 μg/ml) in the presence or absence of BM2U (0.2-20 μg/ml) for 15 min. The phosphorylated JNK, ERK, and p38 MAPK levels, and their total form, were quantified by immunoblot analyses. Values represent the relative ratio to β-actin as mean ± SEM ( n= 3, per group). ## p< 0.01, vs. the untreated control; * p< 0.05 or ** p< 0.01 vs. the LPS-only group. BM2U, an aqueous extract of D. actinosclerusBM2T (GenBank: KT448814).
Effect of BM2U on HO-1 Expression
Apart from the NF-κB signaling pathway, the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway contributes to repressing oxidative stress and exerts its anti-inflammatory effects via the induction of HO-1 expression, because HO-1 attenuates inflammatory responses, and drives the phenotypic shift to M2 macrophages . Hence, we examined if BM2U could induce HO-1 expression in RAW264.7 macrophage cells, increasing the anti-inflammatory potential of BM2U (Fig. 5). A significant induction of HO-1 was observed at 20 μg/ml BM2U (
Effect of BM2U on the induction of HO-1. RAW264.7 macrophages were treated with BM2U (0.2-20 μg/ml) for 24 h.HO-1 levels were then quantified by immunoblot analyses. Values represent the relative ratio to β-actin as mean ± SEM ( n= 3, per group). ** p< 0.01 vs. the untreated control. BM2U, an aqueous extract of D. actinosclerusBM2T (GenBank: KT448814). HO-1, heme oxygenase-1.
In this study, we have demonstrated the anti-inflammatory potential of BM2U, an aqueous extract of BM2T strain, on LPS-challenged RAW264.7 macrophages. BM2U treatment suppressed LPS-mediated NF-κB activation, resulting in the suppression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6), and iNOS and COX-2 proteins involved in inflammatory mediator production. Three MAPKs (ERK, JNK, and p38 MAPK), which are major contributors to the progression of inflammation, were significantly inhibited by BM2U treatment (
Inflammation caused by infection or non-infectious etiologies, has a major impact on health and quality of life, and may trigger many chronic diseases . There is a strong public need for natural and less expensive, but more effective anti-inflammatory drugs with less adverse effects . In addition to classical synthetic drugs, plants and other natural products are the best sources of anti-inflammatory drugs . Microbial-based therapies, such as beneficial bacterial transplants and bacterial intake as probiotics, have demonstrated the ability to reduce both dysbiotic environments and inflammatory mediator production; thus, inducing remission, specifically in ulcerative colitis . To obtain novel applicability for BM2T on a biological system, we investigated the anti-inflammatory potential of BM2U in LPS-challenged RAW264.7 macrophages. Inflammatory cytokines, chemokines, and interferons are major triggers of inflammatory responses, and these mediators are produced by interactions with pathogen and membrane receptors [3, 7]. Many studies have shown that the increase in inflammatory factors, such as NO, PGE2, iNOS, COX-2, TNF-α, and IL-6, in LPS-induced RAW264.7 cells, is mediated through the MAPK pathways [24-26]. BM2U treatment reduced mRNA expression levels of IL-1β, IL-6, and TNF-α (Fig. 2). The decreased production of these cytokines in the LPS-challenged RAW264.7 macrophages appeared to be associated with the suppression of NF-κB activation in the presence of BM2U (Fig. 3). Since NF-κB activation
In conclusion, BM2U, the aqueous extract of
This work was supported by a research grant from Seoul Women’s University (2020).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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