Anti-Inflammatory Activity of Miodesin™: Modulation of Inflammatory Markers and Epigenetic Evidence

Purpose To investigate the effects of a combined herbal medicine Miodesin™ on the inflammatory response of key cells involved in the acute and chronic inflammatory processes as well as the possible epigenetic involvement. Methods After the establishment of the IC50 dose, the chondrocyte, keratinocyte, and macrophage cell lines were pretreated for 2 hours with Miodesin™ (200 μg/mL) and stimulated with LPS (1 μg/mL) for 24 hours. The supernatant was used to measure the levels of cytokines (IL-1β, IL-6, IL-8, and TNF-α) and chemokines (CCL2, CCL3, and CCL5), and the cells were used to extract the mRNA for the transcription factor (NF-κβ), inflammatory enzymes (COX-1, COX-2, PLA2, and iNOS), and chemokines (CCL2, CCL3, and CCL5). Results Miodesin™ inhibited the release of LPS-induced cytokines (IL-1β, IL-6, IL-8, and TNF-α; p < 0.01) and chemokines (CCL2, CCL3, and CCL5; p < 0.01) and the expression of the transcription factor (NF-κβ; p < 0.01), inflammatory enzymes (COX-1, COX-2, PLA2, iNOS; p < 0.01), and chemokines (CCL2, CCL3, and CCL5; p < 0.01). In addition, the evaluation of epigenetic mechanism revealed that Miodesin™ did not induce changes in DNA methylation, assuring the genetic safeness of the compound in terms of the inflammatory response. Conclusions Miodesin™ presents anti-inflammatory properties, inhibiting hyperactivation of chondrocytes, keratinocytes, and macrophages, involving epigenetics in such effects.


Introduction
Although the inflammatory process is a natural response to an offending agent aiming to promote healing and repair, an exacerbated and/or unresolved inflammatory process underlies several acute and chronic diseases [1]. The inflammatory process is complex and involves a group of glycoproteins called cytokines, which coordinate, amplify, and regulate the magnitude and duration of inflammatory events [1].
Acute dermatitis and atopic dermatitis are examples of acute and chronic skin diseases, respectively, in which kerati-nocytes present a key role [2]. In this context, it has already been demonstrated that keratinocytes express different surface alarming receptors against pathogens, being the trigger for cytokines and reactive oxygen species (ROS) and reactive nitrogen species (RNS) release [2,3]. In addition, lipopolysaccharides (LPS) are among the main players for skin infection, which may be installed during skin acute and chronic inflammatory processes [3]. Beyond keratinocytes, skin macrophages also represent the first line of defense during skin infections, contributing to the inflammatory process, releasing, for instance, cytokines and ROS and RNS [4].

Culture and Cytotoxicity Evaluation of Miodesin by MTT
Assay. The human skin keratinocyte cell line (HaCaT) and human chondrocyte cell line (CHON-001) were obtained from the American Type Culture Collection (ATCC, USA). The murine macrophage (RAW 264.7) cells were obtained from the Rio de Janeiro Cell Bank (UFRJ, RJ, Brazil). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM high glucose) supplemented with 10% v/v fetal bovine serum (FBS), 1% L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin and maintained at 37°C in a humidified atmosphere of 5% CO 2 . The cells were trypsinized every 72 h using 0.01% trypsin and 1 mmol ethylenediaminetetraacetic acid (EDTA). For all the experiments, the Miodesin™ was dissolved in the culture medium in appropriate concentrations. The cell viability of the control and Miodesin™ (1-1.000 μg/mL)-treated chondrocyte, human keratinocyte (HaCaT), and macrophage RAW 264.7 cells was measured using a standard MTT assay. Briefly, 5 × 10 4 viable cells were seeded into clear 96-well flat-bottom plates (Corning) in the RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and incubated with different concentrations of the extract for 24 h. Then, 10 μL/well of MTT (5 mg/mL) was added and the cells were incubated for 4 h. Following incubation, 100 μL of 10% sodium dodecyl sulfate (SDS) solution in deionized water was added to each well and left overnight. The absorbance was measured at 595 nm in a benchtop multimode reader (Molecular Device).

Cell Viability after LPS Treatment.
Briefly, cells were seeded in 96-well culture plates at a density of 5 × 10 4 viable cells/well and incubated for 24 h, then exposed to IC 50 concentration of Miodesin (200 μg/mL), previously determined in Section 2.1 in the presence of LPS (1 μg/mL) for 60 minutes and incubated for another 24 h with Miodesin at 37°C. The MTT solution was added to the final concentration of 0.5 mg/mL and then incubated for 2 h at 37°C followed by the addition of 0.1 mL of dimethyl sulfoxide to dissolve the MTT-formazan. The amount of MTT-formazan was then determined by measuring the absorbance at 595 nm.
2.5. NO Analysis. NO levels were determined by measuring the quantity of nitrite in the cell culture supernatant using a Griess reagent. Chondrocytes, keratinocytes and RAW 264.7 (macrophages) cells were pretreated with LPS (1 μg/mL) for 60 minutes with or without Miodesin (200 μg/mL) for 24 h. Oxidative Medicine and Cellular Longevity The cell culture supernatant (100 μL) was mixed with a 100 μL Griess reagent, and the absorbance was measured at 540 nm. The nitrite concentrations were calculated using a standard calibration curve prepared from different concentrations of sodium nitrite.
Total RNA extracted from cell samples was converted to cDNA using a SuperScript® III RT kit (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol. The concentration of RNA was detected using a NanoDrop 2000 (Thermo Fisher Scientific, Inc.). GAPDH and 18S rRNA were used as the internal control. The thermocycling conditions were as follows: 95°C for 10 min followed by 35 cycles of 95°C for 15 sec and 55°C for 40 sec. The 2 -ΔΔCq method was used to quantify the relative gene expression levels of the target genes. Relative standard curves were generated by serial dilutions, and all samples were run in triplicate. Table 1 indicates the sense and antisense sequences of primers used in qRT-PCR analysis.

Quantification of the 5-mC Content in Genomic DNA.
The genomic DNA cytosine methylation levels in cell lines exposed to Miodesin (200 μg/mL) at 24 h were assessed by using an enzyme-linked immunosorbent assay-based commercial kit (MDQ1, Imprint® Methylated DNA Quantification Kit, Sigma-Aldrich). DNA at a concentration of 150 ng was diluted with 30 μL of binding buffers and incubated at 60°C. The samples were incubated with capture and detection antibodies, and the absorbance was read at 450 nm. Quantification of DNA methylation was obtained by calculating the amount of methylated cytosines in the sample relative to methylation in a positive control, which was provided by the manufacturer.

Statistical
Analysis. The obtained results were expressed as mean ± standard error of mean ðSEMÞ from at least three independent experiments, unless stated otherwise. Paired data was evaluated by Student's t-test. One-way analysis of variance (ANOVA) was used for multiple comparisons, followed by the Bonferroni test for comparison among the groups. A p value of <0.05 was considered significant.

Effects of Miodesin™ on Cell Viability and on LPS-
Induced Cell Cytotoxicity. Figure 1 shows the different concentrations of Miodesin™, which were tested for cell toxicity to determine the IC 50 value. The dose of 200 μg/mL was defined as the study dose. Figures 1(a)-1(c) show the results for keratinocytes, chondrocytes, and macrophages, respectively. In addition, from the IC 50 dose (200 μg/mL), Miode-sin™ was able to reverse the cytotoxicity caused by LPS in all these cell lines ( Figure 1(d)).

LPS-Induced Inflammatory Cytokine and Chemokine
Release in Cell Lines Is Regulated by Miodesin™. Figure 2 shows In addition, Figure 3 shows that all cell types responded similarly to LPS stimulation, since LPS increased the levels of CCL2 (Figure 3

Discussion
The present study is the first to demonstrate the mechanism of action of Miodesin™ in reducing inflammatory markers such as interleukins, tumor necrosis factor-alpha (TNF-α), chemokines, and nitric oxide (NO) that are triggered by the chondrocytes, keratinocytes, and inflammatory cells [18]. In addition, a probable involvement in epigenetic mechanisms may be involved in the anti-inflammatory action of Miode-sin™. The presence of the phytocomplex formed by Uncaria tomentosa (cat's claw), Endopleura uchi, known as uxi, and astaxanthin, the xanthophyll carotenoid, guarantees Miode-sin™ anti-inflammatory and antioxidant activity.
The presence of Uncaria tomentosa in the formulation gives Miodesin™ a variety of bioactive secondary metabolites, including tetra-and pentacyclic oxindole alkaloids, glycosides, polyoxygenated triterpenes, and procyanidins [19]. Most investigators attribute the biological effects of this plant to the oxindole alkaloids, an assumption that has been corroborated by studies of several such alkaloids that indicated their antioxidant, immunomodulatory, and antineoplastic properties [20]. The anti-inflammatory effects of Uncaria tomentosa could be due to its inhibition of lipopolysaccharide-(LPS-) dependent expression of tumor necrosis factoralpha (TNF-α) [11] by reduction of the expression of the transcription factor nuclear factor κ light-chain enhancer of activated B cells (NF-κβ) [21,22], an effect that, in turn, regulates the expression of tumor necrosis factor-alpha (TNF-α). By reducing the expression of NF-κβ, Uncaria tomentosa also reduces the TNF-α levels and intensifies its anti-inflammatory action [21,23].
Phytochemical studies with Endopleura uchi, which is an Amazon species traditionally used for the treatment of inflammations and female disorders, indicate that the compound bergenin (coumarin) is responsible for the antiinflammatory action [12,24], although it indicates the presence of tannins and saponins in the barks [25,26]. Bergenin, the most abundant molecule, has anti-inflammatory action, apparently through mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κβ) inhibition (N), in addition to inhibiting the action of cycloxygenase-2 [27].
Astaxanthin is a well-known carotenoid, since many studies in recent years have demonstrated its inhibitory role against oxidative stress and inflammation, dangerous processes at the basis of many chronic diseases [28]. Astaxanthin exerts its anti-inflammatory effect by inhibiting nuclear translocation of NF-κβ p65 and by preventing ROS    Oxidative Medicine and Cellular Longevity accumulation in NRF2-dependent and NRF2-independent mechanisms [29]. The treatment of chondrocytes with Miodesin™ reduced the secretion of interleukins and chemokines, corroborating data found in the literature that show the action of Uncaria tomentosa in osteoarthritis [30][31][32][33]. It is worth mentioning the reduction in the secretion of two other interleukins, IL-17 and IL-23, that have been found in osteoarthritis joints which also cause destructive proteases and induction of the synthesis of NO [30], in addition to being considered those which play crucial roles in the induction of local inflammation and cartilage destruction diseases such as rheumatoid arthritis [34]. The effects of Miodesin™ on the expression of mRNA of inflammatory enzymes, NF-κβ, and chemokines in cell lines in the presence or absence of LPS were also evaluated. NF-κβ, PLA2, iNOS, and CCL5 showed significantly reduced mRNA levels, while COX-1, COX-2, and CCL3 showed a reduction, but not significant. It is noteworthy that CCL3 secretion was significantly reduced when chondrocytes were challenged with LPS. The reduction in mRNA expression of the iNOS enzyme, associated with a reduction in NO in the treated chondrocytes, suggests an important role of Miodesin™ in NO modulation [35]. Our results also showed that treatment with Miodesin™ reduced the levels of NF-κβ mRNA. Thus, we suggest that Miodesin™ can regulate the function of NF-κβ, which has a mechanism associated with the destruction of cartilaginous tissue [36]. Also, in this sense, NF-κβ regulates the production of matrix metalloproteinases by chondrocytes that are released promoting matrix degradation [37]. Chemokine CCL5 was increased in chondrocytes after in vitro treatment with LPS. However, when chondrocytes were previously treated with Miodesin™, they showed a significant reduction in the secretion of this chemokine, as well as mRNA levels, suggesting an important role of Miodesin™ in the modulation of this chemokine, which has a primary function of attracting lymphocytes and monocytes as well as other cell types [38,39] and of activating rheumatoid arthritis synovial fibroblasts (RASFs) to promote MMP-1 and MMP-13 mediated ECM destruction [40].
Keratinocytes form the first line of defense against microorganisms, physical or chemical tissue damage, in addition to producing cytokines that regulate the migration of inflammatory cells, activation of immune responses, and proliferation and differentiation of keratinocytes and fibroblasts [3]. In addition, these cells mediate the skin's immune response by secreting various proinflammatory cytokines [41] and recruit immune cells to the site of insult [42]. Regarding keratinocytes, Miodesin™ reduced the secretion of IL-8, IL-1B, and IFN-γ. IL-17 showed a reduction after 72 hours of treatment. Regarding the evaluated chemokines, although all showed a reduction, only CCL2 (MCP-1) showed a significant reduction compared to the control (cells without treatment). CCL2 mRNA levels were also significantly reduced after previous treatment with Miodesin™. Monocyte chemoattractant protein-1 (MCP-1/CCL2) is one of the key chemokines that regulate migration and infiltration of monocytes/macrophages [43]. Upregulated expression of chemokines such as RANTES/CCL5 and MCP-1/CCL2 has been detected in the epidermis of patients with both atopic dermatitis and psoriasis [44]; in addition, with TNF-α being a potent inducer of IL-8, whose high production is associated with psoriasis, but not in the skin of patients with atopic 7 Oxidative Medicine and Cellular Longevity dermatitis or healthy skin, we can suggest that, due to the reduced production of TNF-α by Miodesin™, the phytocomplex could present itself as an interesting adjunctive therapy option in the treatment of psoriasis [45,46]. Interestingly, a reduction in IFN-γ levels was observed, since IFN-γactivated keratinocytes express a wide range of cytokines, chemokines, and membrane molecules that direct the recruitment, activation, and retention of specific leukocyte subpopulations in the skin [47].
Innate immunity cells, such as macrophages, trigger a rapid immune response, being able to secrete various types of cytokines [48]. In this way, we use RAW 264.7 (macrophages) cells associated with LPS to stimulate these macrophages to produce inflammatory mediators. The results found showed that Miodesin™ inhibited NO production and suppressed iNOS mRNA levels in LPS-stimulated cells. In addition, Miodesin™ also significantly reduced the secretion of cytokines, such as TNF-α, IL-6, IL-8, and IL-1β. The results of the evaluated chemokines showed that CCL2/MCP-1 CCL3/MIP-1α were significantly reduced when compared to RAW 264.7 (macrophage) cells stimulated with LPS. The levels of NF-κβ, COX-1, PLA2, CCL2, and CCL5 mRNA were also reduced in RAW 264.7 (macrophage) cells when compared to cells stimulated with LPS. The results obtained suggest that the anti-inflammatory action of Miodesin™ is due, at least in part, to the action of the components of its formulation, either by the action of astaxanthin, reducing proinflammatory cytokine secretion, e.g., IL-1β, IL-6, and TNF-α, and reducing NF-κβ nuclear expression [49,50]; by the action of Uncaria tomentosa, promoting the inhibition of interleukins such as IL-1β, IL-17, and TNFα, in addition to the inhibition of NF-κβ [10,51,52]; or by the action of Endopleura uchi [27,53], inhibiting the action of cyclooxygenases (COX-1 and COX-2) and phos-pholipase A2 (PLA2), reducing the production of proinflammatory cytokines (IFN-γ and TNF-α). Our results, therefore, show the anti-inflammatory activity of Miode-sin™, by regulating the expression and/or secretion of several important inflammatory biomarkers.
Finally, we evaluated if Miodesin™ promotes changes in the global DNA methylation and in the levels of mRNA of the enzymes called methylases (Dnmt1, Dnmt3A, and Dnmt3B). The results obtained indicated no significant alterations in global DNA methylation in all cell lines evaluated. The levels of mRNA of the enzymes called methylases were not statistically reduced, corroborating the findings of global DNA methylation. These findings seem to be in accordance with the current evidence which suggests a potential role of epigenetics on the level of inflammatory markers reporting on the association of inflammation with global DNA methylation showing a hypomethylation trend [54][55][56]. The evidence reinforces the role of epigenetic changes in the modification and modulation of transcription factors, leading to the deregulation of multiple cascades of intracellular signaling [57]. In this sense, the literature shows that some bioactive molecules present in components of the formulation of Miodesin™ could have an epigenetic effect, as in the case of hirsutine from the genus Uncaria, such as Uncaria rhynchophylla [58] and Uncaria tomentosa [59], which exerts its epigenetic action inhibiting the activation of NF-κβ [58]. Thus, NF-κβ could become a possible target in antiinflammatory therapy, since it is able to regulate epigenetic changes associated with inflammation [60]. The correct understanding of these aspects may generate strategies for the development of new therapeutic approaches, opening space for future studies with the aim of investigating the participation of Miodesin™ in epigenetic mechanisms related to the control of different inflammatory processes.

Data Availability
The authors declare that all raw data presented in this manuscript will be available upon request.

Conflicts of Interest
All authors disclose any influence of companies or manufacturers in the present study. In addition, all authors declare that the results of the study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation.