Ursolic Acid Derivatives Down Regulate Inflammatory Mediators

Ursolic acid (UA) is being investigated due to its anti-inflammatory potential, and structural modifications can enhance its biological activities. The aim of this study was to evaluate the immunomodulatory effect of the ursolic acid derivatives (UAD) in macrophages and in the carrageenan-induced paw edema model. RAW264.7 cells were cultured in the presence or absence of UA or UAD (1-18). Nitric oxide (NO), nuclear factor kappa B (NF-κB), tumor necrosis factor (TNF), and cellular viability were measured. 30 min before the carrageenan-induced paw edema, the UAD1 and UAD2 (200 mg kg-1) were administered intraperitoneally. The results showed that UAD2-4, UAD7, UAD9-11 had half maximal inhibitory concentration (IC50) greater than 90 µM and were able to reduce NO, NF-κB and TNF production. Moreover, UAD1 and UAD2 reduced paw edema and IL-6 production. In conclusion, the results obtained demonstrated a variation in the response between the derivatives due to the chemical modifications, showing potential to reduce the inflammatory mediators, deserving further investigations.


Introduction
The inflammation processes are associated with the development of several diseases, acute or chronic, many of them of unclear origin, such as rheumatoid arthritis, multiple sclerosis and Crohn's disease. 1,2The activation of macrophages is related to the production of inflammatory cytokine and transcription factor expression, such as tumor necrosis factor (TNF) and nuclear factor kappa B (NF-κB), respectively, playing a major role in the establishment of the inflammation.
The cytokines are involved in the activation of endothelial cells and leukocyte infiltration. 3The TNF is a pro-inflammatory cytokine that has several functions, such as inducing the production of other cytokines and lipid mediators of inflammation, proliferation, cell differentiation and apoptosis. 4Although the macrophage lineage cells are the main source of TNF, in inflammatory diseases, it can be produced by a wide variety of cells including neutrophils, fibroblasts and endothelial cells. 5he transcription factor NF-κB stands out, which plays a crucial role in the initiation and amplification of inflammation.NF-κB consists of a protein assembly located in the cytoplasm of cells and its activity is controlled by a family of inhibitor proteins, denominated IκB, which bind to the NF-κB dimmer, promoting its inhibition.][8] Nitric oxide (NO) is a powerful inflammatory mediator, produced by macrophages and is involved in the regulation of several physiological processes.NO can be generated in excess during the host's response against damage caused by pathogens or other substances, contributing to the pathogenesis of various inflammatory disorders including tissue damage, septic shock, and rheumatoid arthritis. 9,10Therefore, lipopolysaccharide (LPS)-induced NO production may reflect the degree of inflammation.The inhibition of NO production may demonstrate the ability of a substance to act as possible anti-inflammatory agent, although other mediators needed to be evaluated. 10,11acrophages, when sensing pathogen-associated molecular patterns (PAMPs) by their pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), induce the synthesis of endogenous pyrogenic cytokines, among them interleukin 6 (IL-6).IL-6 constitutes an important inflammatory marker secreted by macrophages in response to pathogen invasion during acute inflammation. 12,13hus, IL-6 has been considered as an important mediator of fever and the acute phase response. 14,15he up-regulation of these inflammatory mediators, produced by the defense, is related to signs and symptoms, such as pain, fever, edema and, sometimes, irreversible tissue damage. 16,17Thence, the down-modulation of the inflammatory mediators can be valuable to prevent and control the development of inflammatory diseases. 1,2,18In this context, a disordered and excessive production of proinflammatory mediators may be related to the pathogenesis of several diseases and, therefore, these mediators are possible targets of therapeutic substances. 191][22][23][24] Among the different mechanisms of action of UA, it is possible to highlight its inhibitory action on the expression of NF-κB. 25,26aking into consideration the promising activity of UA, the aim of this study was to synthesize ursolic acid derivatives (UAD) and to evaluate the improvement in its biological activity.The chemical modifications in the UA structure, create semisynthetic derivatives that demonstrated enhanced cytotoxic activity against tumor cell lines. 27In a continuation of the work of Scherrer et al., 27 this study evaluated the immunomodulatory effect of the UAD in macrophage response and in carrageenan-induced paw edema model.

Spectral data
Nuclear magnetic resonance (NMR) spectra were recorded in CDCl 3 on an AC200 (Bruker Corporation, Billerica, USA) at 200 MHz for 1 H and 50 MHz for 13 C, using TMS as internal reference for both nuclei.For each peak, chemical shift values are expressed in parts per million, followed by multiplicity, relative peak integration (when appropriate) and coupling constants (J) in hertz.High resolution mass spectra (HRMS) were obtained using a QSTAR XL spectrometer (Applied Biosystems, Foster City, USA).The spectra in the infrared (IR) were obtained in Spectrum One apparatus (PerkinElmer, Wellesley, USA) coupled to the diffuse reflectance accessory (ATR).The specific rotational power values [α] D 25 were measured on a 241 polarimeter (PerkinElmer, Wellesley, USA) at 20 ºC.Column chromatography was performed on Silica Gel 60 (230-400 mesh, Merck, Darmstadt, Germany), whereas thin-layer chromatography was carried out on Silica Gel 60 F254 plates (0.25 mm thick, Merck, Darmstadt, Germany).Solvents and reagents were used directly from the manufacturer or purified by standard procedures when required.

MTT assay
Cellular viability was measured using the MTT assay.After 48 h of culture, the supernatants were removed and the cells were incubated with 100 μL of supplemented RPMI-1640 and 10 μL of MTT (5.0 mg mL -1 ), during 4 h at 37 ºC in a 5% CO 2 atmosphere.After purple formazan crystal formation, the supernatants were gently removed and crystal products were solubilized with DMSO.Complete solubilization was obtained by shaking the plates.The optical density (OD) values were determined in the Multiskan microplate reader (Multiskan™ FC Microplate Photometer, Thermo Scientific, Waltham, MA, USA) at 560 nm wavelength.The cellular viability was presented as the half maximal inhibitory concentration (IC 50 ) calculated using Prism software (GraphPad Prism 5.00, San Diego, CA, USA). 28tric oxide concentration in RAW264.7 The NO concentration was measured by the Griess method, in the supernatant of the 48 h of culture.To perform the test, 100 µL of the supernatant from each well was transferred to 96-well plates and an equal volume of Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine hydrochloride, 5% H 3 PO 4 ) was added.The NO concentration was determined by comparison with a standard sodium nitrite solution and the values were determined by the Multiskan microplate reader (Multiskan™ FC Microplate Photometer, Thermo Scientific, Waltham, MA, USA) at 540 nm wavelength.

NF-κB concentration in RAW264.7
The cells that were cultured for 3 h were detached after this period and stained for the analysis of the p65 expression (indirectly NF-κB), 27 following the instructions of the manufacturer.The cells were acquired in the FACSVerse (BD, Biosciences Pharmingen, San Diego, CA, USA) and analysed in the FCS Express software (De Novo software, Pasadena, CA, USA). 29duction of acute inflammation by carrageenan-induced paw edema BALB/c (female) mice 6-8 weeks old (n = 6 per group) were obtained from the animal care facilities of the Federal University of Minas Gerais, Belo Horizonte, MG, Brazil, and maintained in micro isolator cages.All procedures were in accordance with the principles of the Brazilian Code for the Use of Laboratory Animals and were approved by the committee on the use of laboratory animals from UFVJM (Protocol No. 03/2018r).The mice were monitored for clinical signs of toxicity after the treatments.Initially, the mice were weighed (26.57± 0.31 g) and their right and left paws were measured (0 h) with a pachymeter (Mitutoyo, Kawasaki, Japan).The treatments, phosphate buffered saline (PBS), dexamethasone (0.5 mg kg −1 ), UA (200 mg kg −1 ), UAD 1 (200 mg kg −1 ) and UAD2 (200 mg kg −1 ), were administered intraperitoneally (100 μL) 30 min before the induction of the edema.Dexamethasone was used as a positive control treatment due to its antiinflammatory activity.Carrageenan (2.5%) was dissolved in PBS, and 20 μL injected into the left footpad, and 20 μL of PBS into the right footpad of all groups.The left and the right paws were measured after 1, 2, 3 and 4 h after the injection of carrageenan and the differences were calculated.The magnitude of the carrageenan-induced paw edema was determined as follows: [paw edema / mm] = [footpad thickness of carrageenan / mm] − [footpad thickness of PBS / mm]. 30

Cytokine production
Cytokine production was assayed by enzyme-linked immunosorbent assay (ELISA) using commercially available antibodies according to the instructions of the manufacturer (BD Biosciences Pharmingen, San Diego, CA, USA).The plates were read at the Multiskan microplate reader (Multiskan™ FC Microplate Photometer, Thermo Scientific, Waltham, MA, USA) at 450 nm wavelength. 18Supernatants of the 48 h macrophage culture were used to evaluate the TNF.The TNF production (%) was calculated using the formula (X1/X2) × 100, considering X1 the TNF production (pg mL -1 ) of stimulated and treated cells and X2 the mean TNF production (pg mL -1 ) of stimulated and untreated cells.The IL-6 productions were measured in the mice paw and lymph nodes, after euthanasia.Paw tissue and lymph nodes were removed and homogenized (100 mg mL -1 ) in the extraction solution containing 0.4 M NaCl, 0.05% Tween 20, 0.5% BSA, 0.1 M PMSF, 0.1 M benzethonium chloride, 10 mM EDTA and 20 kIU mL -1 aprotinin.The homogenate was centrifuged at 2000 ×g for 15 min at 4 °C and supernatants were collected to determine the concentration of IL-6.

Statistical analysis
The results represent at least three independent experiments and are presented as the mean ± standard error of the mean (SEM).All data were analyzed using two-way analysis of variance (ANOVA) followed by Bonferroni posttests (GraphPad Prism 5.00, San Diego, CA, USA), 28 and the differences were considered significant at p < 0.05.

Biological activity
The cytotoxicity index (IC 50 ) and NO production by RAW264.7 macrophages, treated with UA or the UADs, were shown in the Table 1.The IC 50 was obtained by the MTT test of the unstimulated cells treated with UA or UADs and the respective controls corresponding to untreated cells.Besides the viability, the Table 1 shows the results of NO production by RAW264.7 macrophages treated with UA or the UADs, stimulated with LPS and interferon (IFN)-g after 48 h of culture.
The percentage of TNF produced by RAW264.7 was inhibited by the UAD7, UAD12, UAD13, UAD14, UAD15 and UAD18 in all concentrations tested.The UAD1, UAD10, UAD11 and UAD16 reduced the TNF production in concentrations above 15 µM.All the compounds reduced the production of TNF at 60 and 90 μM (Table 3).
A study 33 has already shown that NF-κB is a wellknown transcription factor in the inflammatory process,  responsible for inducing the transcription of several proand anti-inflammatory mediator genes, including TNF.In the present work, the reduction in the expression of NF-κB was accompanied by the reduction in the production of TNF, probably due to the incorporated transformations to obtain the UADs, which improved the compounds capacities to act on the NF-κB, partly reported by other authors. 20,34Also, these modifications generated UADs with lower cytotoxicity than UA, except for the UAD1.The structural modification of the UAD2, consists of the esterification of both C-3 and C-28 carbons, 35 which may be the factor that improved its capacity to reduce NO, being the compound that best reduced NO at 90 µM, without altering cell viability.Also, it was able to inhibit the expression of NF-κB in a dose response manner and to reduce the production of TNF at concentrations of 60 and 90 µM.
The UAD1 and UAD2 were chosen to be used as a treatment on carrageenan-induced paw edema, for its similar results to UA in inhibiting inflammatory mediators, such as NF-κB and NO, at the lowest concentrations tested.However, it is possible to recognize the potential in others UAD that deserves to be tested in the inflammatory models.
Both, UAD1 and UAD2, showed an effective reduction of paw edema of mice, as well as dexamethasone, compared to carrageenan group, who received only PBS treatment.The reduction in paw edema is already observed in the 2 nd h and persists until the 4 th h (Figure 3).
Regarding the analysis of IL-6 in the supernatant of the paw and lymph node macerates, a reduction of IL-6 was observed in the groups treated with UA and the derivatives UAD1 and UAD2 compared to the carrageenan group (Figures 4 and 5).Different studies [36][37][38] have shown that UA and derivatives are able to reduce the inflammatory marker IL-6.Rapid IL-6 production is also known to contribute to host defense during infection and tissue injury, but excessive IL-6 synthesis and dysregulation of IL-6 receptor signaling are involved in inflammatory and autoimmune disorders.Thus, various therapeutic agents have been evaluated to inhibit the cytokine itself or targets associated with its signaling pathway. 39,40

Conclusions
The results obtained demonstrated a variation in the response between the derivatives, due to their chemical modifications, but with potential to reduce the inflammatory mediators evaluated.Regarding paw edema, the UAD1 and UAD2 proved to be as efficient as dexamethasone, an anti-inflammatory used in clinical practice.Furthermore, the investigation of the other derivatives, in in vivo models of inflammation, deserves to be expanded.

Table 3 .
Percentage of the TNF production by RAW264.7 macrophages treated with UA and UADs a Compounds: ursolic acid (UA) and ursolic acid derivative (UAD); b treatment concentration; c p < 0.05 percentage of the tumor necrosis factor (TNF) production by RAW264.7 macrophages treated with UA and UADs versus RAW 264.7 not treated (100%).