Molecular evaluation of anti-inflammatory activity of phenolic lipid extracted from cashew nut shell liquid (CNSL)

LDT11 is a derivative designed from cashew nut shell liquid (CNSL) phenolic lipid. Compounds from library of the Laboratory of Development of Therapeutic Innovations (LDT), part of the University of Brasília (Brazil) were used in this research. LDT11 synthesis was performed as follows: to a solution of the mixture of anacardic acids (5 g, 14.5 mmol for average molecular wt 344) in ethanol (50.0 mL) was added 10% palladium-carbon (0.2 g) and shaken in a Parr apparatus (Parr Instrument Company©, Moline, IL, USA), under hydrogen atmosphere (4 atm, 60 psi) at room temperature. After six hours, the mixture was filtered and the solvent was evaporated under reduced pressure. The residue was recrystallized from hexane to afford a saturated anacardic acid (LDT11) as a white solid (4,55 g, 90%, mp 81 °C–83 °C, Rf 0.48 – Hex:AcOEt 4:1). IR (KBr) ν_máx cm^− 1: 3326 (ν_OH); 2954 (ν_asCH3); 2920 (ν_asCH2); 2850 (ν_sCH2); 1610 (ν_C=O); 1560, 1542, 1498 e 1466 (ν_C=C); 1287(ν_asC-O); 1086 (ν_sC-O); ¹H NMR (300 MHz, CDCl₃): δ 0.89 (t, J = 6.5 Hz, 3H, 15); 1.26 (m, 24H, 3–14); 1.60 (qi, J = 6.6 Hz, 2H, 2); 3.00 (t, J = 7.7 Hz, 2H, 1); 6.79 (d, J = 7.4 Hz, 1H, 3′); 6.89 (d, J = 8.0 Hz, 1H, 5′); 7.37 (t, J = 7.9 Hz, 1H, 4′); 10.72 (s, ArOH); ¹³C NMR (75 MHz, CDCl₃): δ 14.3 (CH₃, 15); 22.9 (CH₂, 14); 29.6–30.0 (CH₂, 3–12); 32.1 (CH₂, 13); 32.2 (CH₂, 2); 36.7 (CH₂, 1); 110.6 (C, 1′); 116.1 (CH, 3′); 123.0 (CH, 5′); 135.6 (CH, 4′); 148.1 (C, 6′); 163.8 (C, 2′); 176.5 (ArCO₂H).

The cytotoxicity of the macrophages treated with synthetic phenolic derivatives was determined using the WST-8 assay (Cell Counting kit-8, Sigma-Aldrich, St. Louis, MO, USA). Cells were grown in complete Dulbecco’s Modified Eagle’s Medium (DMEM) at a concentration of 1 × 10⁵ in 96-well plates and incubated at 37 °C in an atmosphere of 5% CO₂ for 48 h. The culture medium was subsequently exchanged for 100 μL of DMEM supplemented with 5% colorless fetal bovine serum (FBS), and 100 μL of LDT11 was added to the wells at concentrations of 25 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, totaling a volume of 200 μL per well. After that, the plate was once more incubated under the previously described conditions.

The assay was performed using samples and controls in triplicate. After 48 h, the medium was discarded, and 100 μL of colorless DMEM, supplemented with 5% FBS was added, followed by the addition of 10 μL of WST-8 to each well. Non-stimulated cells were used as positive controls. Cell death control was performed using cells treated with 10 μL of 1% Triton-X. After an incubation of four hours with WST-8, the absorbance of the samples was measured in a TP-Reader microplate spectrophotometer (Thermoplate, Palm City, FL, USA) using a 450 nm wavelength filter.

The Neutral red uptake assay was performed following the protocol described by Tanner et al. [26], with some modifications. The assay was carried out under the same conditions as the WST-8 assay. After 48 h of incubation with the LDT11, the medium was discarded, the cells were washed twice with phosphate buffered saline solution (1X PBS, pH 7.4), and 100 μl of DMEM supplemented, and 50 μg/mL of neutral red was added to the wells. The plate was incubated under the conditions described in the item 2.2. The medium was then discarded, and the cells were washed five times with 1X PBS to remove the excess of dye; which was followed by the addition of 100 μl of alcohol-acid solution (50% ethanol, 1% acetic acid and 49% distilled water) to each well to fix the neutral red to the cells. The plate was shaken for 10 min, and the absorbance of the samples was read in a TP-Reader microplate spectrophotometer (Thermoplate, Palm City, FL, USA) with a 492 nm filter. The results were expressed as a percentage, the value obtained for the positive control (untreated cells), being considered as 100% viability. The equation used was: viability (%) = (number of viable cells / total number of untreated cells) × 100.

To evaluate the influence of LDT11 ​​on RAW 264.7 gene expression, after stimulation with LPS, quantitative real-time polymerase chain reaction (qPCR) was performed. The RNA from cells was extracted, purified and quantified, and the resulting complementary DNA was prepared for the qPCR reaction, as follow: RAW 264.7 cells were cultured in six well plates at a concentration of 5 × 10⁶ cells/well, each containing 3 mL 10% complete DMEM medium. The plates were incubated (approximately 24 h) until an estimated confluence of 90% was obtained. The medium was discarded, and 3 mL of colorless DMEM medium without FBS supplementation was added in order restrain the cells growth rate. The immune-protective properties of LDT11 were analyzed in: (a) untreated cells (NT); (b) cells stimulated exclusively with LPS; (c) cells treated with LDT11, and subsequently exposed to LPS; (d) cells treated with ASA, and then exposed to LPS; (e) cells treated with DEX, and exposed to LPS.

For the extraction and purification of RNA, Direct-zol ™ RNA Miniprep Kit (R2051, Zymo Research, Orange, CA, USA), was used following the manufacturer’s recommendations. The quantification of extracted RNA was determined by using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA synthesis was performed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA), according to manufacturer’s instructions.

Genes involved in the inflammatory response were selected to test the biological activity of LDT11. Seven primers synthesized by Integrated DNA Technologies (Skokie, IL, USA) were used, as described in Table 1. The design of the primer pairs was performed using the Primer Express Program (Applied Biosystems, Waltham, MA, USA) based on sequences obtained from the Mouse Transcriptome Database (http://​www.​informatics.​jax.​org).

The result, expressed in CT value, refers to the number of cycles in qPCR required for the fluorescent signal to reach the threshold detection. The analysis of the gene expression of the inflammatory markers was obtained by the relative quantification of their transcripts by the Delta-Delta Ct (ΔΔCt) method, which allows a relative comparison with the group that did not receive treatment (NT) and cells stimulated with LPS. The melting curve was used as quality control of amplification products.

For the data analysis, the values ​​found for the control group, LPS- stimulated cells, were considered 100% inflamed. The other tests, with their respective treatments (LDT11, ASA and DEX) were analyzed comparing with the inflamed group.

For the analysis of Nitric Oxide (NO), was used the Griess method, by adding 100 μL of Griess reagent (1% [w/v] sulfanilamide, in a 5% phosphoric acid, and 0.1% [w / v] N-1- naphthyl-ethylenediamide dihydrochloride (NEED) in water). Samples of culture supernatants were analyzed by microplate reader (TP-Reader microplate spectrophotometer, Thermoplate, Palm City, FL, USA), using the spectrum 450 nm, and the results expressed in μmol/L of NO - comparing the optical density (OD) obtained with a standard curve of NO - ranging from 1.56 μM to 100.0 μM.

The quantification of Interleukin-6 (IL-6) was performed by applying the competitive immunoenzymatic assay, as established in the manufacturer’s kit (#27768 Mouse IL-6 Kit – Immuno-Biological Laboratories Co., Ltda, Hamburg, Germany). The same treatment used in the gene expression assessment was applied in this assay, using the supernatants of RAW 264.7 cells treated with the synthetic phenolic derivative LDT11 at 50 μM concentration.

The determination of the cytotoxic effect of LDT11 by the WST-8 and Neutral Red methods was performed using the following concentrations: 25 μM, 50 μM, 75 μM, 100 μM, 125 μM, and 150 μM.

LDT11 showed approximately 100% cell viability in a concentration of 25 μM. The cell viability declined to 90% with a LDT11 concentration of 50 μM. Concentrations equal or over 75 μM ensued a cell viability equal or lower than 60% and were considered cytotoxic (Fig. 3).

Neutral Red Uptake Assay was performed to confirm the cell viability obtained by the WST-8 assay on the RAW264.7 cell line, using similar LDT11 concentrations. The assay showed results similar to those found by the WST-8 method (Fig. 4).

From the results of the cell viability assays, toxic concentrations of LDT11 were excluded from the study. The standardized concentration for the other tests was 50 μM. For further comparison, the following assays with ASA and DEX were also made using the same concentration established for LDT11, 50 μM.

Gene modulation of the inflammatory process was analyzed after interaction with the CNSL derivative, LDT11. Gene expression was evaluated by the relative quantification of TNF-α, COX-2, iNOS, NF-kB, IL-1β and IL-6 genes in RAW264.7 cells. Inflammation was induced with 1 μg/mL LPS, both in control cells and in LDT11 treated cells. The analysis of the results was carried out 6 and 24 h post- treatment.

As seen in Fig. 5, the gene expression of TNF-α, six hours after interaction with LDT11, showed an eight-fold decrease (88%) in comparison with the gene expression disclosed by the control cells (LPS). The results obtained from the interaction with ASA and dexamethasone (DEX) were respectively one and a half times lower (33%) for ASA and two times lower (50%) for DEX (Fig. 5a). The results obtained 24 h after treatment were similar with those obtained after six hours. Both had a decrease in gene expression in all treatments analyzed. Cells treated with LDT11 disclosed 10 times (91%) less gene expression than the positive control, while cells treated with ASA and DEX respectively showed a gene expression two and three times lower (55 and 64%) than control cells, Fig. 5b.

COX-2 was the second gene analyzed (Fig. 6). After six hours of treatment with LDT11, COX-2 gene expression decreased more than five-fold (81%) when compared to control cells (Fig. 6a). Cells treated with ASA did not show a significant difference, and those treated with DEX showed a four-fold decrease (74%) in gene expression when compared to control cells. After 24 h of interaction with LTD11 (Fig. 6b), the gene expression disclosed a six-fold decrease (84%) in treated cells than in control cells. The reduction in gene expression caused by both ASA and DEX was approximately 2-fold higher (30%) than that observed in control cells.

After six hours of LTD11 treatment, the expression of the iNOS gene, (Fig. 7) showed a 200 times decrease (100%) comparing with control cells. Treatment with ASA resulted 20-fold (99%) decreased expression, while cells treated with DEX showed a 60-fold (98%) gene suppression (Fig. 7a). After 24 h (Fig. 7b), both LDT11 and DEX treated cells showed approximately a 2-fold decreased gene expression (50%) while a threefold decrease (68%) was observed in ASA-treated cells.

The action of LTD11 on the NF-kB gene expression can be seen in Fig. 8. In comparison with control group, cells treated with LTD11 resulted an eight-fold (88%) decrease in NF-kB gene expression. The treatment of the cells with ASA and DEX produced respectively a decrease in gene expression equivalent to 1.5-fold (33%) for the first and two-fold (50%) for the second drug (Fig. 8a). In the treatment performed after 24 h (Fig. 8b), both LDT11 and ASA treated cells showed a decrease of about two-fold (55%) in the NF-kB gene expression. On the other hand, cells treated with DEX failed to show any significant difference when compared to the control cells.

The IL-1β gene also showed decreased expression in most of the assays (Fig. 9). After a six-hour interaction with LDT11, IL-1β gene expression decreased more than 14-fold (93%). Treatment with ASA did not cause a significant decrease in its expression whereas DEX led to a three-fold reduction (69%) when compared to control cells (Fig. 9a). After 24 h (Fig. 9b), when compared with control cells, those treated with LDT11 showed an approximately six-fold decrease (83%) in gene expression, whereas ASA and DEX caused respectively a two-fold (39%) and five-fold (75%) decline.

After six hours of treatment with LDT11 there was a 65-fold (98%) decrease in the gene expression of IL-6, in comparison with the control cells. Gene expression of IL-6 decreased respectively 8-fold (88%) in ASA-treated cells and 27-fold (96%) in DEX-treated cells (Fig. 10a). After 24 h (Fig. 10b) the gene expression decreased 5-fold (79%) in the cells treated with LDT11 while the decrease was respectively two-fold (45%) in the cells treated with ASA and three-fold (65%) in the cells treated with DEX.

Nitric oxide, resulting from the presence of oxygen and nitrogen reactive metabolites produced during the inflammatory process, causes an increase in the oxidative stress of the cells. The presence of NO in the supernatant of RAW 264.7 cells was measured to confirm the influence of LDT11 on anti-inflammatory activity. The following assays were performed with the same timing (six and 24 h) of the previous analysis. The LDT11 was able to protect RAW264.7 cells against oxidative stress by reducing NO production by about eight times (95%) after six hours, and about 15 times (100%) after 24 h (Fig. 11).

Additionally, to confirm the results obtained in the analysis of gene expression, the cytosine IL-6 was also assayed. The pre-treatment of the cells with LTD11 resulted in a significant protective effect against inflammation, reducing IL-6 production by more than 1700-fold (76%) after six hours (Fig. 12a) and more than 1400-fold (60%) after 24 h (Fig. 12b). Cells treated with ASA reduced IL-6 production respectively by more than 120-fold (52%), after six hours and by more than 400 (17%) after 24 h. Treatment with DEX reduced the IL-6 production at about 1000-fold (42%) after six hours and at about 1400-fold (60%) after 24 h.