The Effect of Natural-Based Formulation (NBF) on the Response of RAW264.7 Macrophages to LPS as an In Vitro Model of Inflammation

Abstract

Macrophages are some of the most important immune cells in the organism and are responsible for creating an inflammatory immune response in order to inhibit the passage of microscopic foreign bodies into the blood stream. Sometimes, their activation can be responsible for chronic inflammatory diseases such as asthma, tuberculosis, hepatitis, sinusitis, inflammatory bowel disease, and viral infections. Prolonged inflammation can damage the organs or may lead to death in serious conditions. In the present study, RAW264.7 macrophages were exposed to lipopolysaccharide (LPS; 20 ng/mL) and simultaneously treated with 20 µg/mL of natural-based formulation (NBF), mushroom-cannabidiol extract). Pro-inflammatory cytokines, chemokines, and other inflammatory markers were analyzed. The elevations in the presence of interleukin-6 (IL-6), cycloxygenase-2 (COX-2), C-C motif ligand-5 (CCL5), and nitrite response, following exposure to LPS, were completely inhibited by NBF administration. IL-1β and tumor necrosis factor alpha (TNF-α) release were inhibited by 3.9-fold and 1.5-fold, respectively. No toxic effect of NBF, as assessed by lactate dehydrogenase (LDH) release, was observed. Treatment of the cells with NBF significantly increased the mRNA levels of TLR2, and TLR4, but not NF-κB. Thus, it appears that the NBF possesses anti-inflammatory and immunomodulatory effects which can attenuate the release of pro-inflammatory markers. NBF may be a candidate for the treatment of acute and chronic inflammatory diseases and deserves further investigation.

Keywords: cannabidiol; chemokines; cytokines; inflammation; lipopolysaccharide; macrophages; medicinal mushrooms.

Figure 1

Cytotoxicity analysis as assessed by LDH release from RAW264.7 macrophages. LDH levels were measured by O.D. (arbitrary units) and are presented as the mean ± SD. At least 8 replicates were in each group (n = 8 for naïve, n = 16 for vehicle, and n = 14 for NBF). ANOVA with Bonferroni’s post hoc test was performed. p-value = 0.92.

Figure 2

NBF downregulates the release of pro-inflammatory cytokines induced by LPS in RAW264.7 macrophages. RAW 264.7 macrophages were exposed to 20 ng/mL LPS and simultaneously with the NBF, 20 µg/mL. IL-1β (A), IL-6 (B), and TNF-α (C) levels were measured using ELISA. The results are presented as the mean ± SD, (n = 4 in each group). ANOVA followed by Bonferroni’s post hoc test was performed. *** p < 0.001 compared to all other groups.

Figure 3

NBF downregulates the release of CCL5 (RANTES) in RAW264.7 macrophages following LPS stimulation. RAW 264.7 macrophages were exposed to 20 ng/mL LPS and simultaneously with the NBF, 20 µg/mL. CCL5 levels were measured (pg/mL) using standard calibration curves and are presented as the mean ± SD. Four replicates in each group (n = 4). ANOVA followed by Bonferroni’s post hoc test was performed. *** p < 0.001 compared to all other groups.

Figure 4

NBF affects COX-2 protein level in RAW264.7 macrophages following LPS stimulation. RAW 264.7 macrophages were exposed to 20 ng/mL LPS and simultaneously with the NBF, 20 µg/mL. COX-2 levels were measured by ELISA using standard calibration curve and are presented as the mean ± SD. Four replicates in each group (n = 4). ANOVA followed by Bonferroni’s post hoc test was performed. *** p < 0.001 compared to all other groups.

Figure 5

NBF downregulates the secretion of nitrite in RAW264.7 macrophages following LPS stimulation. RAW 264.7 macrophages were exposed to 20 ng/mL LPS and simultaneously with the NBF, 20 µg/mL. Nitrite levels were measured using standard calibration curve with sodium nitrite and are presented as the mean ± SD. Four replicates in each group (n = 4). ANOVA followed by Bonferroni’s post hoc test was performed. *** p < 0.001 compared to all other groups.

Figure 6

NBF upregulates mRNA expression levels of TLR2 and TLR4 but not NF-κB. RAW264.7 cells were treated with either Vehicle + LPS or NBF + LPS for 3 h. mRNA was extracted 3 h after treatment using an mRNA extraction kit and NF-κB, TLR2, and TLR4 mRNA were quantified and normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH)—mRNA expression levels are shown as the fold differences compared to untreated cells. Data are represented as the mean  ±  SEM of three independent experiments (three separate experiments in triplicates) and statistical analyses were performed using ANOVA followed by Bonferroni’s post hoc test. * p < 0.05, *** p < 0.001 vs. vehicle.