Telmisartan directly ameliorates the neuronal inflammatory response to IL-1β partly through the JNK/c-Jun and NADPH oxidase pathways

Cell-culture media and supplements were obtained from Invitrogen (Carlsbad, CA, USA). Recombinant rat IL-1β was purchased from R&D Systems (Minneapolis, MN, USA). Telmisartan, losartan, CGP 42112, PD 123319, pioglitazone, diphenyleneiodonium chloride (DPI), SP600125, GW9662 and T0070907 were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Candesartan was a kind gift from Astra-Zeneca (Mőlndal, Sweden). Angiotensin II was purchased from Bachem (Torrance, CA, USA). Primers for real-time PCR were synthesized by BioServe (Beltsville, MD, USA). SYBR Green PCR Master Mix for qPCR was purchased from Applied Biosystems (Foster City, CA, USA). The remaining reagents for RNA isolation and reverse transcription were from Invitrogen. Primary antibodies used for western blot analysis were: rabbit polyclonal anti-nuclear factor-kappa B (NF-κB)-p65 antibody (1:2000, Millipore, Billerica, MA, USA); mouse polyclonal anti-cyclooxygenase-2 (COX-2) (1:1000, Cayman Chemical, Ann Arbor, MI, USA); rabbit anti-phospho-p38 mitogen-activated protein kinase (MAPK) (1:1000), rabbit anti-phospho-extracellular signal-regulated kinases (ERK)1/2 (1:1000), rabbit anti-phospho-JNK (1:1000), rabbit anti-phospho-c-Jun (1:1000), rabbit anti-IκB-α (1:1000), rabbit anti-β-actin (1:1000), and rabbit anti-histone H4 (1:1000), all from Cell Signaling Technology (Danvers, MA, USA). Secondary horseradish peroxidase-conjugated antibodies for western blot analysis were: donkey anti-rabbit IgG (1:5000, Amersham BioSciences, Piscataway, NJ, USA) and goat anti-mouse IgG (1:10,000, Jackson ImmunoResearch, West Grove, PA, USA). Protease inhibitor cocktail and SuperSignal West Dura Substrate for chemiluminescent detection were purchased from Thermo Fisher Scientific (Pittsburg, PA, USA). All other chemicals were obtained from Sigma-Aldrich unless otherwise stated.

Human SK-N-SH neuroblasts were obtained from the American Type Culture Collection (HTB-11, Rockville, MD, USA) and grown in MEM with Earle’s salts and HEPES, supplemented with 10 % fetal bovine serum and 100 U/ml penicillin/streptomycin. Cells were cultured at 37°C in a humidified atmosphere of 5 % CO₂/95 % air until they reached 80 % confluence, then confluent monolayers were passaged routinely by trypsinization. Cells between passages 3 and 10 were used in this study, and before each experiment, they were starved overnight in a serum-free medium.

All animal care and experimental procedures in the present study were approved by the National Institute of Mental Health Animal Care and Use Committee (Bethesda, MD, USA). All efforts were made to minimize the number of animals used and their suffering (National Institutes of Health Guide for the Care and Use of Laboratory Animals, Publication number 80–23, received 1996). Primary cortical neuron cultures were obtained from fetal Sprague–Dawley rats (Charles River Laboratories, Wilmington, MA USA) at embryonic day 18 (E18) [42]. Fetal cerebral cortices were collected and placed in ice-cold Hank’s balanced salt solution. After removal of the meninges, the cortices were dispersed into the same buffer containing 0.25 % trypsin, and digested for 15 minutes at 37°C. Trypsin digestion was stopped by adding a two-fold volume of DMEM, supplemented with 10 % FBS and 0.1 mg/ml DNase I. After gentle trituration, digested tissues were separated by centrifugation at 200 × g for 5 minutes. The cell pellets were resuspended in complete Neurobasal culture medium supplemented with 2 % B27 and 0.5 mmol/l GlutaMax. After filtration through a 70 μm cell restrainer (BD Falcon, Vernon Hills, IL, USA), cells were plated at a density of 1 × 10⁶ cells/ml onto poly-D-lysine coated plates (Becton Dickinson and Co., Franklin Lakes, NJ, USA). Cultures were incubated in a humidified atmosphere of 5 % CO₂/95 % air at 37°C. Only mature cultures (10–14 days in vitro) were used in this study. Immunocytochemical validation with anti-microtubule-associated protein 2 (MAP-2) antibody and 4',6-diamidino-2-phenylindole (DAPI) showed that more than 95 % of the cells in the culture system were neurons (data not shown).

The cells were pre-incubated for 2 hours with telmisartan, candesartan, losartan, CGP 42112, PD 123319, DPI, SP600125, pioglitazone, T0070907, GW9662, or vehicle before exposure to IL-1β. Most of the experiments were performed with the maximum stimulatory concentration of 10 ng/ml IL-1β, and the exposure times were 2 hours for ROS determination, 3 hours for RT-PCR analysis, and 24 hours for COX-2 protein and PGE₂ determinations. The SK-N-SH neuroblasts were incubated with 100 μmol/l H₂O₂ for 3 hours to determine the protective effect of telmisartan. Activation of MAPKs, c-Jun, and NF-κB was determined by western blotting at various time intervals up to 2 hours. All concentrations used and time intervals are indicated in the figure legend for each particular experiment. All drugs were initially prepared as 1000-fold concentrated stock solutions, and were added directly into the cell-culture medium. Telmisartan, DPI, SP600125, pioglitazone, T0070907, and GW9662 were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in experimental conditions was 0.1 %. Candesartan was initially dissolved in 0.1 mol/l Na₂CO₃, and further diluted to stock concentration with isotonic saline, at a final pH of 7.5 to 8.0. All other drugs were dissolved in isotonic saline. Control cells were treated with the corresponding vehicle in all experiments.

Total RNA was isolated using TRIzol reagent followed by purification using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s instructions. Synthesis of complementary DNA (cDNA) was performed with 0.6 μg of total RNA and Super-Script III first-Strand Synthesis Kit (Invitrogen, Carlsbad, CA, USA). Quantitative real-time PCR was performed on DNA Engine Opticon™ (MJ Research, Waltham, MA) with SYBR Green PCR Master Mix. PCR was performed in a 20 μl reaction mixture containing 10 μl SYBR Green PCR Master Mix, 2 μl cDNA and 0.3 μmol/l of each primer for a specific target (Table 1). The amplification conditions consisted of 1 denaturation/activation cycle at 95°C for 10 minutes, followed by 40 to 45 cycles at 95°C for 15 seconds and 60°C for 60 seconds. Serial dilutions of cDNA from the same source as samples were used to obtain a standard curve. The individual targets for each sample were quantified by determining the cycle threshold (Ct) and by comparison with the standard curve. The relative amount of the target mRNA was normalized to the level of GAPDH mRNA.

For the determination of NFκB-p65 nuclear translocation, nuclear protein extracts were prepared using Nuclear Extraction Kit (Pierce, Rockford, IL, USA) in accordance with the manufacturer’s instructions. For other proteins, the whole-cell lysates were prepared in Tris-Glycine SDS Sample Buffer (Invitrogen). The protein extracts were separated by electrophoresis on 10 % SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked for 1 hour and incubated overnight at 4°C with the primary antibodies, followed by washing and exposure to secondary antibodies for 1 hour at room temperature. The membranes were exposed to SuperSignal West Dura Substrate for chemiluminescent detection.

The levels of intracellular ROS were determined by the change in the fluorescence resulting from the oxidation of the fluorescent probe H₂DCFDA using OxiSelect™ ROS Assay Kit (Cell Biolabs, San Diego, CA, USA) in accordance with the manufacturer’s instructions. After preincubation with telmisartan or DPI, the cells were loaded with H₂DCFDA for 30 minutes at 37°C and exposed to IL-1β for an additional 2 hours. The level of fluorescence, corresponding to intracellular ROS, was determined using a plate reader (VICTOR3; Perkin-Elmer, Torrance, CA, USA) with 485 nm excitation and 535 nm emission filters.

PGE₂ release was determined in cells culture medium by enzyme immunoassay (EIA) (PGE₂ EIA Kit; Cayman Chemical) in accordance with the manufacturer’s instructions.

The lucigenin method was used to determine NADPH oxidase activity in SK-N-SH cells. Cells were collected by scraping, and pelleted by centrifugation at 500 × g for 5 minutes. The pellets were resuspended and homogenized in ice-cold buffer containing 50 mmol/l Tris, pH 7.4, 1 mmol/l EDTA, 1 mmol/l DTT, 0.5 mmol/l phenylmethylsulfonyl fluoride (PMSF) and 1× protease inhibitor cocktail. The crude membrane fraction was pelleted by centrifugation at 16,000 × g for 90 minutes at 4°C, and the pellets were resuspended in 200 μl of assay buffer containing 8 mmol/l sodium phosphate, pH 7.4, 140 mmol/l NaCl, 10 mmol/l KCl, 2 mmol/l MgCl₂, 50 mmol/l triethanolamine, 1 mmol/l DTT, and 1× protease inhibitor cocktail. The total protein concentration was determined by the Bradford assay and adjusted to 1 mg/ml. An aliquot (200 μl) of protein sample (100 μg of membrane proteins) were incubated in the presence of 5 μmol/l lucigenin and 100 μmol/l NADPH. The luminescence was monitored at 2-minute intervals using a plate reader (VICTOR3; Perkin-Elmer) to determine relative changes in NADPH oxidase activity.

Ang II concentration in the cell-culture medium was measured using a commercial kit (Ang II EIA Kit; Cayman Chemical) following the manufacturer’s instructions. The limit of sensitivity of the assay was 1.5 pg/ml.

Incubation of SK-N-SH neuroblasts in the presence of IL-1β induced COX-2 mRNA expression in a dose-dependent and time-dependent manner (Figure 1A,B). Maximum stimulation of COX-2 mRNA was obtained with 10 ng/ml IL-1β, and it reached a peak after 3 hours of exposure (Figure 1A and 1B). Thus, this dose of IL-1β was selected for all subsequent experiments.

Telmisartan, candesartan and losartan reduced IL-1β induction of COX-2 mRNA with equal potency (Figure 1C). All three ARBs dose-dependently reduced IL-1β-induced PGE₂ release, but telmisartan was significantly more potent than candesartan or losartan (Figure 1D). Telmisartan dose-dependently decreased IL-1β-induced COX-2 mRNA expression (Figure 1E) and COX-2 protein expression (Figure 1F).

SK-N-SH neuroblasts expressed AT₁ receptor mRNA, and the receptor expression was not affected by IL-1β or telmisartan, either alone or in a combination (Figure 2A).

AT₂ receptor mRNA was not detectable in our preparation of SK-N-SH neuroblasts. Incubation in the presence of the AT₂ receptor agonist CGP 42112 did not change IL-1β stimulation of COX-2 gene expression (Figure 2B) or PGE₂ release (Figure 2C). Similarly, incubation in the presence of the AT₂ receptor antagonist PD 123319 did not change IL-1β stimulation of PGE₂ release, and did not alter the inhibitory effect of telmisartan (Figure 2C).

High expression of the NADPH oxidase isoform NOX-4 and substantially lower expression of NOX-5 were found in SK-N-SH neuroblasts (Figure 3A). Expression of NOX-1 and NOX-2 was not detected (Figure 3A). Exposure to IL-1β significantly increased NOX-4 mRNA expression, and this effect was reduced by telmisartan (Figure 3B). IL-1β significantly increased NADPH oxidase activity, an effect also reduced by telmisartan (Figure 3C). IL-1β enhanced ROS production, and this effect was decreased by both telmisartan and DPI (Figure 3D). DPI dose-dependently inhibited IL-1β-induced PGE₂ release (Figure 3E). The reduction in IL-1β-stimulated PGE₂ release was similar for both telmisartan and DPI (Figure 3F).

Telmisartan reduced the enhanced COX-2 mRNA expression produced by H₂O₂ to an extent similar to that resulting from exposure to DPI (Figure 3G).

Exposure to IL-1β enhanced mRNA expression of its receptor, IL-1R1, and this change was reduced to a similar degree by telmisartan and DPI (Figure 3H).

IL-1β time-dependently activated JNK in SK-N-SH neuroblasts, reaching maximum stimulation after 30 to 60 minutes of exposure, and this effect was significantly reduced by telmisartan (Figure 4A). Exposure to IL-1β simultaneously and time-dependently enhanced c-Jun phosphorylation, a change significantly decreased by telmisartan (Figure 4A). The effect of telmisartan was of similar magnitude to that of DPI (Figure 4B). Incubation in the presence of the specific JNK inhibitor SP600125 abrogated the IL-1β-induced phosphorylation of JNK and c-Jun (Figure 4B), COX-2 mRNA expression (Figure 4C), and PGE₂ release, in a dose-dependent manner (Figure 4D).

Incubation in the presence of telmisartan did not modify IL-1β-induced p38 MAPK phosphorylation (Figure 5A) or the ERK1/2 phosphorylation (Figure 5B). Telmisartan did not change the time-dependent IL-1β-induced IκB-α degradation (Figure 6A), the IκB-α mRNA expression (Figure 6B), or the NF-κB-p65 protein nuclear translocation (Figure 6C). DPI was equally ineffective, and did not change IL-1β-induced IκB-α mRNA expression or the NFκB-p65 protein nuclear translocation (Figure 6B and 6C).

Incubation of SK-N-SH neuroblasts with the PPARγ agonist pioglitazone significantly reduced IL-1β-induced COX-2 mRNA expression (Figure 7A), dose-dependently reduced PGE₂ release (Figure 7B), and upregulated the mRNA expression of the PPARγ target genes ABCG1 and CD36, without affecting PPARγ mRNA expression (Figure 7C). Conversely, telmisartan did not alter ABCG1 or CD36 mRNA expression (Figure 7C). Incubation of SK-N-SH neuroblasts in the presence of the PPARγ antagonists T0070907 or GW9662 alone did not significantly alter IL-1β-induced COX-2 mRNA expression (Figure 7D), and neither T0070907 nor GW9662 modified the inhibitory effect of telmisartan on IL-1β-induced COX-2 mRNA and protein expression (Figure 7D,E).

Angiotensin II levels were undetectable in the cell-culture medium (results not shown). Exposure of SK-N-SH neuroblasts to 1 μmol/l Ang II for 24 hours did not alter PPARγ gene expression but strongly decreased gene expression of the PPARγ target genes ABCG1 and CD36 (Figure 8A). Pretreatment of neuroblasts with Ang II for 24 hours did not change basal COX-2 mRNA expression or basal PGE₂ release. Ang II did not affect COX-2 mRNA expression induced by 10 ng/ml IL-1β, but did enhance IL-1β-induced PGE₂ release (Figure 8B,C). Pretreatment with Ang II did not change the inhibitory effect of telmisartan on IL-1β-stimulated COX-2 gene expression and cumulative PGE₂ release (Figure 8B,C).

Exposure of primary rat cortical neurons to IL-1β induced both COX-2 and IL-1R1 mRNA expression and ROS generation, and these effects were significantly reduced by telmisartan (Figure 9A-C).