A novel compound C12 inhibits inflammatory cytokine production and protects from inflammatory injury in vivo

Abstract

Inflammation is a hallmark of many diseases. Although steroids and cyclooxygenase inhibitors are main anti-inflammatory therapeutical agents, they may cause serious side effects. Therefore, developing non-steroid anti-inflammatory agents is urgently needed. A novel hydrosoluble compound, C12 (2,6-bis(4-(3-(dimethylamino)-propoxy)benzylidene)cyclohexanone), has been designed and synthesized as an anti-inflammatory agent in our previous study. In the present study, we investigated whether C12 can affect inflammatory processes in vitro and in vivo. In mouse primary peritoneal macrophages, C12 potently inhibited the production of the proinflammatory gene expression including TNF-α, IL-1β, IL-6, iNOS, COX-2 and PGE synthase. The activity of C12 was partly dependent on inhibition of ERK/JNK (but p38) phosphorylation and NF-κB activation. In vivo, C12 suppressed proinflammatory cytokine production in plasma and liver, attenuated lung histopathology, and significantly reduced mortality in endotoxemic mice. In addition, the pre-treatment with C12 reduced the inflammatory pain in the acetic acid and formalin models and reduced the carrageenan-induced paw oedema and acetic acid-increased vascular permeability. Taken together, C12 has multiple anti-inflammatory effects. These findings, coupled with the low toxicity and hydrosolubility of C12, suggests that this agent may be useful in the treatment of inflammatory diseases.

Figure 1. The structure and NO-inhibitory activity of C12.

A. Chemical structures of C12 and its water-soluble form C12-HCl; B. C12 inhibitd nitrite production. MPMs were pretreated with vehicle (H₂O) or C12 (2.5, 5, and 10 µM) for 2 h and then stimulated with 1 µg/mL LPS for 18 h. The nitrite levels in medium were detected as described in Materials and Methods. Bars represent the mean±SD of three independent experiments performed in duplicate, and asterisks indicate significant inhibition (* p<0.05, ** p<0.01).

Figure 2. C12 inhibited LPS-induced inflammatory mRNA expression in MPMs.

Cells were pretreated with compounds at 2.5 µM or vehicle (H₂O) control for 2 h and treated with LPS (0.5 µg/mL) for 6 h. The mRNA levels of inflammatory cytokines IL-12, IL-1β, TNF-α, IL-6 and COX-2 were quantified by real-time quantitative PCR. The mRNA value was normalized to internal control β-actin mRNA and was expressed as a ratio to H₂O. Bars represent the mean±SD of three independent experiments (* p<0.05, ** p<0.01).

Figure 3. C12 inhibited LPS–induced NF-κB activation and MAPK phosphorylation.

A. Cultured MPMs were pretreated with vehicle (H₂O) or 10 µM C12 for 2 h and then stimulated with 0.5 µg/mL LPS. After 1 h of treatment, the cells were incubated with p65 antibody and Cy3 fluorescein-conjugated secondary antibody, and nuclei were stained with DAPI. The images (200×) were obtained by fluorescence microscope and overlay. Similar results were obtained with five independent experiments. B–F. MPMs were pretreated with vehicle (H₂O) or C12 (10 µM) for 2 h followed by incubation with LPS (0.5 µg/mL) for 1 h. The protein levels of p-IκBα, IκBα, p-ERK, ERK, p-JNK, JNK, p-p38, and p38 were examined by western blot with actin as a loading control (values on the top of western blots represent the mean optical density ratio in three independent experiments, * p<0.05, ** p<0.01, vs LPS group). G. MPMs were pretreated with vehicle (H₂O) or C12 (5 µM) for 2 h, and then stimulated with 0.5 µg/mL LPS for 12 h in the absence or presence of PD98059 (20 µM). The TNF-α level in medium were detected by ELISA as described in Materials and Methods. TNF-α expression is reported as the fold-expression relative to the LPS-treated group (n = 3, * p<0.05, ** p<0.01).

Figure 4. Inhibition of plasma TNF-α (A), IL-6 (B) and NO (C) by C12 in LPS-injected mice.

C12 (15 mg/kg, i.v.) was given to B6 mice 15 min before LPS (10 mg/kg, i.v.) administration. Mice were killed and blood samples were collected 1 or 6 h after administration of LPS. Plasma TNF-α, IL-6 and NO concentrations were measured as described in Materials and Methods. Data point represent the mean±SD (n = 6). (* p<0.05, ** p<0.01, vs LPS group).

Figure 5. Inhibitory effects of C12 pretreatment (15 mg/kg, i.v., 15 min before LPS injection) on the induction of inflammatory gene mRNAs in B6 mouse livers 1 or 6 h after LPS administration (10 mg/kg, i.v.).

Liver mRNA from vehicle control, LPS, and LPS+C12-treated mice was reverse transcribed for quantitative real-time PCR analysis of the expression of TNF-α, IL-6, IL-1β, COX-2, iNOS, and PGES. Expression is normalized to actin and reported as the fold-expression relative to one of samples in the vehicle control group. Values shown are the mean ± SD of 3 mice from the saline control group (white), and 4–6 mice each from the LPS (black)- and LPS+C12 (yellow)- treated groups. (* p<0.1, ** p<0.05, compared to LPS group).

Figure 6. Beneficial Effects of C12 against acute inflammatory injury and shock in B6 mice.

A. Mice were treated with C12 (15 mg/kg, i.v.) 15 min before LPS (10 mg/kg, i.v.) administration. After 48 h, lung histopathological analysis was performed using H&E staining as described in Materials and Methods (magnification ×200; n = 3; Con, control). B and C. C12 improves survival of mice subjected to a lethal dose of LPS. Mice were pretreated with vehicle (saline) or 15 mg/kg C12 (i.v.) 15 min before the injection of 20 mg/kg of LPS (i.v.). Survival (B) and body weight (C) were recorded for 7 days after the LPS injection at the interval of 12 h. Results from the summary of two different experiments are shown. n = 12 animals in each group. C12 improved survival rate at 12–48 h (*** p<0.0001).

Figure 7. Effects of C12 (5 mg/kg or 15 mg/kg, i.p.) on the chemical-induced inflammatory response.

A. Paw oedema models induced by carageenan; B. acetic acid-induced pain model; C. acetic acid-vascular permeability models; D. formalin-induced nociception mice (first-inflammatory phase, left; second-inflammatory phase, right). Control animals received saline (0.9% NaCl). Dexamethasone (5 mg/kg) was used as a positive control. Each value represents the mean±SD of 6 animals, and asterisks indicate significant difference of the paw thickness in relation to the corresponding saline group (* p<0.05, ** p<0.01, *** p<0.001).