Simultaneous comparison of L-NAME and melatonin effects on RAW 264.7 cell line’s iNOS production and activity

Cell culture

The RAW 264.7 cells were provided by Iranian Biological Resource Center at fifth passages. All investigations were performed by 1 h pretreatment of L-NAME and melatonin with/without LPS (5 μg/mL) during an 8 and/or 24 h period. 5 μg/mL dose of LPS was preferred to achieve a maximum induction for iNOS in the target cells.

Cell viability test

Mitochondrial respiration was determined via reduction of MTT to formazan [16]. Briefly, RAW 264.7 Cells were processed by L-NAME and melatonin with or without LPS, then incubated by MTT. The supernatants were discharged and the insoluble formazans were re-suspended using dimethylsulphoxide (DMSO). Evaluation of 540 nm optical density was carried out and viabilities of target cells were expressed in comparison with non-treated group. The following equation describes how the cell viability was calculated:

Cell Viability=(processed samples light absorb) /(light absorb of control group).

Nitrite level evaluation

Analyzing the nitrite level was determined through 540 nm spectrophotometry by the Griess reagent [16] using the sodium nitrite (MERK, cat. no CC634149) as the control. In brief, 100 μL from any sample were transferred to into a 96-well microtiter plate. Then, 100 μL Griess reagent (2% sulfanilamide (MERK, cat. no K3967135) in 5% HCl solution (MERK, cat. no K35384014) and 0.1% N-(1-naphtyl) ethylendiamine (MERK, cat. no K31451237) in water was added to the samples. Also, in order to reduce the nitrate compound into nitrite, 100 μL of vanadium chloride III (Sigma, USA, cat. no S5666093) 0.8% was added into the samples. Afterward samples were incubated for an hour at 37°C. At the next step, the nitrite levels of samples were investigated through colorimetry using ELISA-reader device (Anthos 2020 UK, Biochrom Ltd.) at 540 nm.

Measurement of NOS activity

7.2×10⁶ RAW 264.7 cells were cultivated at wells of 75-cm² diameter flask. After pretreatment with L-NAME and melatonin with/without induction with LPS (5 μg/mL), cells were detached. At the next step, cells were washed by phosphate buffer solution (PBS), re-suspended in 0.3 mL ice-cold homogenization buffer (50 mM HEPES, 0.5 mM EDTA, 1 mM dithiothreitol (DTT), with supplied protease inhibitor cocktail (Sigma-USA) at pH 7.4 followed by sonication on ice with two 30S bursts. Afterward, cells were 15 times passed through a 26-gauge needle to attach to the syringe. Resultant homogenates were sediment at 20,000 g (4°C, 15 min).

After separation of supernatant, protein concentrations were determined via Bradford method. NOS activity was measured according to the protocol described in Ultrasensitive Colorimetric Assay for NOS kit (Oxford Biomedical Research, UK). Briefly, after incubation of 300 μg of cell lysates with reaction mixture (HEPES/EDTA, NADP⁺/Glucose 6-Phosphate/L-Arginine and Glucose 6-Phosphate dehydrogenase) in a 250 μL final volume for 5 h at 37°C, the reconstituted nitrate reductase was added to the sample and kept at room temperature for 20 min. Concentration of the nitrite was measured by Griess reagent.

To analyze the direct effect of L-NAME and melatonin on iNOS activity, homogenates of LPS-stimulated cells (LPS, 5 μg/mL, 24 h) were treated by various loads of L-NAME (0.1–4 mM) and melatonin (0.1–4 mM), separately, and iNOS activity was investigated.

Western blotting

2.4×10⁶ RAW 264.7 cells were treated and centrifuged, then the pellet was washed twice using PBS. Then samples were centrifuged at 250 g (five min, 4°C). Resultant pellet was dissolved at 100 μL lysis buffer (50 mM Tris-HCl with pH 7.5), 1% Triton X-100, 1 mM EDTA, 2% sodium deoxycholate, and protease inhibitor cocktail (Sigma-USA). About 60 μg total proteins were separated through 8% SDS-PAGE gel electrophoresis. After transferring the protein bands to a nitrocellulose membrane, the membrane was immersed in the diluted primary antibodies 1:1000 and 1:2000 of iNOS and beta-actin, respectively for 2 h at 37°C. Then, it was processed by diluted horseradish peroxidase-conjugated goat anti-rabbit antibody at 37°C for an hour.

The target protein bands were inspected through application of the Amersham enhanced chemiluminescence kit (GE Healthcare) and exposed to Kodak X-ray autoradiography films inside a film cassette. Equal protein loadings were confirmed through beta-actin immunoblotting. In order to ascertain the changes, each test was repeated three times.

Real-time PCR

The total RNA content was provided through application of the RNeasy mini kit. cDNA was synthesized by cDNA synthesis kit in accordance with the manual provided by manufacturer (Qiagen, Germany). Real-Time PCR analysis was carried out in triplicate using SYBR Premix Ex Taq polymerase II (TaKaRa, Japan). All exon-exon junction designed primers were supplied by Qiagen: beta-actin (Cat. no. QT01136772), iNOS (Cat. no. QT00100275). As an endogenous reference, β-actin protein was used to make the comparison possible.

A standard curve containing five dilution points was evaluated in triplicate. The results of PCR tests displayed efficiencies between 1.8 and 2.0. Gene expression fold changes were evaluated by comparative C_T method [17].

Statistical analysis

Graph pad prism software was used for analysis and presentation of data. Data are presented as the mean±SD. Diversities among groups have been evaluated through analysis of variance (ANOVA) ensuing with post hoc Tukey test for sub-two groups contrast. p-Value <0.05 were considered meaningful.

Ethical considerations

The conducted research was not related to either human or animal use.

Melatonin and L-NAME influences on cell supernatants NO levels

RAW 264.7 cells displayed an increased nitrite concentration upon processing with LPS during 8 and 24 h in a time-dependent manner. Pretreatment of L-NAME (0.1 mM and 1 mM) and melatonin (0.1 mM and 1 mM) with LPS showed an inhibition at nitrite levels in culture media dose-dependently (p<0.05) (Figure 1). In LPS treated cells, at 1 mM concentration of L-NAME, the mean of nitrite concentration was 2.26 μM at 8 h compared with 7.51 μM at 24 h. In comparison, at the same concentration of melatonin, the nitrite concentration at 8 h was 4.96 μM, when at the 24 h this concentration was 12.6 μM. Inhibitory effects of nitrite by 1 mM L-NAME compared with untreated LPS-induced were 78% and 80% at 8 and 24 h, respectively. The inhibitory effects for 1 mM melatonin compared with untreated LPS-induced group were 53% and 67% in 8 and 24 h, respectively (p<0.05). In parallel, when the cells were treated by 1 mM L-NAME and melatonin without LPS, L-NAME led to an increased NO level up to 8 μM in 24 h, but melatonin sustained NO level at its basic which was similar to that of non-treated controls.

Figure 1:

L-NAME and melatonin effects on the nitrite concentration and RAW 264.7 cells viability (MTT assay).

Inhibitory effect of nitrite by 1 mM L-NAME compared with LPS control was 78% and 80% during 8 and 24 h, respectively. The inhibitory effect for 1 mM melatonin was 53% and 67% in 8 and 24 h, respectively. The cell viability in all samples was up to 90%. Data are presented as mean±SD of triplicates versus with untreated control (CTRL): *p<0.05; vs. LPS treated control (LPS): **p<0.05.

MTT assay showed that both L-NAME and melatonin with/without LPS resulted in no considerable influence on the viability of target cells. Figure 1 illustrates how the samples cell viability was up to 90% (p<0.05). Therefore, the reduced NO production or iNOS suppression in LPS-induced samples were not because of the supplemented components toxic effects.

Analysis of iNOS mRNA expression by L-NAME and melatonin

RAW 264.7 LPS-induced cells showed higher iNOS mRNA level compared with non-induced controls in 8 and 24 h. Without LPS-induction, the fold changes with 1 mM of L-NAME were 1.9, and 0.3 in 8 and 24 h relative to the untreated peer control group, respectively, while for melatonin they were 1.07, and 0.8, respectively. In LPS-induced condition, 1 mM of L-NAME caused 10.7, and 7.9-fold changes at 8, and 24 h, respectively, while 1 mM of melatonin 2.7 and 1.7-fold changes, respectively. Nevertheless, a suppressive effect on iNOS mRNA in 24 h rather than 8 h was observed (Figure 2).

Figure 2:

Real-Time PCR analysis of L-NAME and melatonin effects on iNOS mRNA expression.

It was indicated that in LPS-treated group, 1 mM of L-NAME could result in 14% increase of iNOS-mRNA in comparison with control group during 8 h, but no significant inductive effect was demonstrated in 24 h period. A similar pattern was shown for the group without LPS treatment. Data are presented as Mean ± SD of triplicates versus untreated control (CTRL): *p<0.05; vs. LPS treated control (LPS): **p<0.05.

The Real-Time PCR analysis indicated that L-NAME pretreatment in presence of LPS stimulated a considerable augment in iNOS mRNA in comparison with LPS-induced group (p<0.05). However, without LPS, it led to induction of iNOS mRNA in 8 h and suppression in 24 h compared with untreated control (p<0.05). iNOS mRNA analysis with melatonin showed that melatonin dose-dependently suppressed gene expression of LPS-induced iNOS, while the presence of melatonin in cultures lacking LPS resulted in no influence on translation of iNOS mRNA.

L-NAME and melatonin effects on iNOS protein with or without LPS

Western blot analysis confirmed the results of the NO assay (Figure 3). Without LPS induction, by adding 1 mM of L-LNAME, and melatonin, the mean of protein level relative to the untreated peer groups were 2.8 and 0.9, respectively in 24 h. In LPS-induced samples, 1 mM treated L-NAME caused 9.1 relative fold change compared with non-treated peer group, when these levels for melatonin was 2.5 in 24 h. L-NAME in 24 h incubations with/without LPS increased iNOS levels in comparison with LPS or non-treated control, respectively (p<0.05). In the same conditions, pretreatment of 1 mM melatonin with LPS in a 24 h time, reduced the iNOS protein levels, while no notable changes were detected without LPS (p<0.05).

Figure 3:

Western blotting analysis of iNOS protein expression.

(A) Typical Western blot analysis of iNOS gene expression. (B) Semi-quantitative analysis of iNOS protein levels through image J (NIH) for densitometry analysis and then normalized against β-actin as the endogenous control. In LPS-induced group, 1 mM of L-NAME resulted in 1.4 and 2.84 times increase at iNOS protein level in comparison with LPS induced and non-induced groups, respectively. Data are provided as mean±SD of triplicates in comparison with untreated control (CTRL): *p<0.05; compared with LPS treated control (LPS): **p<0.05.

At 8 h period, in LPS-induced cells, 1 mM of L-NAME produced 10.1 nmol nitrite per mg of protein, while in the peer group with melatonin treatment, this production was 24 nmol. These concentrations in 24 h treatment were 15.6, and 22 nmol for L-NAME, and melatonin, respectively. Activity analysis of iNOS protein (Figure 4A,B) showed that pretreatment of L-NAME with LPS significantly increased the iNOS activity in 24 h (Figure 4B) (p<0.05), but not in 8 h (Figure 4A). In contrast, nitrite measurement in media supernatants of these samples showed an inhibitory effect on nitrite by L-NAME (p<0.05). When cells were processed with L-NAME, but without LPS it increased both basal nitrite and iNOS activity in 24 h (p<0.05).

Figure 4:

Effects of L-NAME and melatonin on the iNOS activity and its related nitrite levels in eight (A) and 24 h (B).

Melatonin did not significantly inhibit iNOS activity in presence of LPS during 8 and 24 h without important effects on its activity without LPS. Pretreatment of L-NAME with LPS in 8 h treated group did not pose any considerable changes (A), but during 24 h period, changes were significant (B). When cells were processed with L-NAME but without LPS, it increased both basal nitrite and iNOS activity in 24 h (B). Data are expressed as mean±SD of duplicates. Compared with untreated control (CTRL): *p<0.05; vs. LPS treated control (LPS): **p<0.05.

Moreover, melatonin did significantly inhibit iNOS activity in presence of LPS during 8 and 24 h without important effects on its activity in absence of LPS (p<0.05). Nitrite measurement in media supernatants related to melatonin was in agreement with the activity assay (Figure 4A,B).

Direct effects of L-NAME and melatonin on iNOS extracted from LPS-induced RAW 264.7 cells

As shown in Figure 5, the mean of iNOS activity at 0.1 mM L-NAME was 35.3 nmol nitrite/min/mg protein, but at 4 mM L-NAME, it 10.9 nmol nitrite/min/mg protein. In comparison, at 0.1 mM melatonin, the activity caused 47 nmol nitrite/min/mg protein while at 4 mM of melatonin it was only reduced to 34 nmol nitrite/min/mg protein. Dose-dependent inhibition of iNOS in LPS-induced cells from non-treated to 4 mM L-NAME showed 79% reduction while at the same concentrations of melatonin this decrease was 32% (p-value <0.05).

Figure 5:

Direct influences of L-NAME and melatonin on iNOS extracted from LPS-induced RAW 264.7 cells.

Melatonin’s High micro molar concentration (>1 mM) decreased the enzyme activity in iNOS extracted from LPS-induced RAW 264.7 cells. L-NAME showed a strong dose-dependent activity inhibition of iNOS extracted from LPS-stimulated cells. Activity of iNOS was calculated versus non-treated LPS control (CTRL LPS). Data are expressed as mean±SD of triplicates versus CTRL LPS: *p<0.05.

Analysis of L-NAME and melatonin direct influences on synthetic nitrite and iNOS extracted from LPS-stimulated RAW 264.7 cells showed that L-NAME could have a stronger dose-dependent activity inhibition of iNOS than melatonin.