1. Introduction
Increase in cell production of reactive oxygen species (ROS) leads to activation of intracellular signaling pathways, which in turn induce transcriptional changes that enable a cell to activate expression of a number of genes encoding antioxidant proteins, DNA repair proteins, stress-regulated chaperones, and antiapoptotic proteins. These genes are generally regulated by transcription factors whose structure, subcellular localization, or affinity for DNA is directly or indirectly regulated by the level of oxidative stress [1]. In such a way, ROS may serve as messenger molecules to activate adaptive responses, such as redox-sensitive nuclear factor kappa B (NF-κB) signaling, which enhance gene expression of antioxidant enzymes in oxidatively stressed tissue [2, 3]. In addition, NF-κB may participate in regulation of nitric oxide synthase (NOS) isoforms expression including eNOS [4, 5].
The transcription factor NF-κB has been shown to be cardioprotective after permanent coronary occlusion and late ischemic preconditioning. However, cell injurious effect of this factor after ischemia/reperfusion was shown in the heart as well. Tranter et al. identified 16 NF-κB dependent cardioprotective genes that might contribute to understanding the mechanism of NF-κB-induced myocardial salvage after permanent coronary occlusion [6].
NF-κB belongs to the Rel family of transcriptional activator proteins and it exerts a variety of actions [7]. Sen et al. have found that NF-κB responds directly to oxidative stress and its activation is controlled by the cell glutathione disulphide/glutathione (GSSG/GSH) ratio [8]. On the other hand, in vitro inhibition of the transcriptional activity of NF-κB may lead to accumulation of reactive oxygen species following oxidative damage [9]. The contradictory findings on the role of NF-κB signaling reflect the diversity of cellular processes on molecular level and should be taken into account in different therapeutic settings.
It is evident that the model of nonobese hereditary hypertriglyceridemic (HTG) rats selected from the Wistar strain [10] represents a suitable model for the study of metabolic disturbances in relation to blood pressure as well as in the search for genetic determinants of these abnormalities [11]. Hereditary HTG rats exhibit insulin resistance, hyperinsulinemia, disturbances in glucose metabolism, hypertension, and different signs of oxidative stress, for example, increased lipoprotein oxidability and lipid peroxidation [12]. According to our knowledge, the role of NF-κB signaling in response to increased oxidative damage in HTG rats was not studied as yet.
Shin et al. [13] showed that aromatic diamine, 4-methyl-
N
1
-(3-phenyl-propyl)-benzene-1,2-diamine (JSH-23), had an inhibitory effect on NF-κB transcriptional activity in lipopolysaccharide- (LPS-) stimulated macrophages RAW 264.7. JSH-23 had inhibitory effects, in parallel, on LPS-induced DNA binding activity and nuclear translocation of NF-κB p65. However, the compound JSH-23 did not influence LPS-induced inhibitory kappa B alpha protein (IκBα) degradation. These results indicate that the JSH-23 could inhibit nuclear translocation of NF-κB p65 without affecting IκBα degradation, which is a very rare mode of action, lending JSH-23 a specific character of NF-κB inhibition.
In this study we investigated the effect of NF-κB inhibition (caused by JSH-23 injection) on heart reactive oxygen species level, superoxide dismutase and nitric oxide synthase activities, and blood pressure regulation in hereditary hypertriglyceridemic rats.
2. Material and Methods
2.1. Animals and Treatment
Male 12-week-old normotensive Wistar rats and Prague hereditary hypertriglyceridemic (HTG) rats (bred in the Institute of Physiology AS CR, Prague) were used in this study. All animals were kept under standard laboratory conditions (12 h light, 12 h darkness,
23
±
1
°C, pelleted ST-1 diet, drinking ad libitum). All procedures and experimental protocols were approved by the Animal Care Ethical Committee of the Institute of Physiology AS CR in Prague and conformed to the European Convention on Animal Protection and Guidelines on Research Animal Use. Adult 12-week-old Wistar (
n
=
18
) and HTG (
n
=
18
) rats were included in the study. Nine Wistar rats and 9 HTG rats were taken as controls, whereas the remaining rats (9 Wistar and 9 HTG) were injected with JSH-23 (bolus, 10 μmol, i.v.).
At the end of the experiment, one week after JSH-23 injection, blood pressure was measured by a direct puncture of the carotid artery under light ether anesthesia. Heart was dissected and left ventricle (LV) was taken for determination of biochemical parameters.
2.2. Biochemical Parameters
The concentration of conjugated dienes (CD) was measured in lipid extracts of heart homogenates [14]. After chloroform evaporation under inert atmosphere and addition of cyclohexane, conjugated diene concentrations were determined spectrophotometrically (
λ
=
233
nm, GBC 911A, Bio-Rad Laboratories).
Reduced glutathione (GSH) level was determined according to Ellman [15]. Samples of LV were homogenized in 1 mL of ice-cold 3% sulphosalicylic acid and, after centrifugation at 3.000 ×g for 5 min, GSH concentration was determined spectrophotometrically in the acid-soluble fractions (
λ
=
412
nm, GBC 911A, Bio-Rad Laboratories).
Total NO synthase (NOS) activity was determined in crude LV homogenates by measuring L-[3H]citrulline formation from L-[3H]arginine (Amersham, UK) as previously described by Bredt and Snyder [16] with minor modifications [17, 18].
Superoxide dismutase (SOD) activity was analyzed in LV homogenates spectrophotometrically using the SOD assay kit (Fluka, Switzerland). The absorbance was measured at 450 nm using a microplate reader (Thermo Scientific Multiscan FC, Finland). SOD activity was expressed in U/mg of protein in the tissues.
For Western blot analysis, samples of the LV were used and probed with polyclonal rabbit anti-eNOS (Santa Cruz Biotechnology, USA), anti-SOD1 (Santa Cruz Biotechnology, USA), anti-NFκB (p65) (BioLegend, USA), and anti-β-actin (Santa Cruz Biotechnology, USA) antibodies.
2.3. Statistical Analysis
Results are expressed as means ± SEM. One-way ANOVA and Bonferroni test were used for statistical analysis.
p
<
0.05
value was considered statistically significant.
4. Discussion
In the present study, the aromatic diamine JSH-23 compound was used for the first time to inhibit NF-κB transcriptional activity in the model of hereditary hypertriglyceridemic rats. In this model we detected increased BP and heart hypertrophy together with increased CD concentration, SOD activity, and SOD1, eNOS, and NF-κB (p65) protein expressions. On the other hand, NOS activity was decreased significantly. NF-κB inhibition led to additional increase in blood pressure and CD concentration, decrease in SOD1 expression and SOD activity, and, interestingly, increase in eNOS expression followed by elevated NOS activity. These results suggested rather regulatory than pathological role of NF-κB in HTG rats.
Heart hypertrophy observed in HTG rats in our experiments is in accordance with previous studies, in which increase in blood pressure about 20–40 mmHg represents a hemodynamic overload that induced left ventricular hypertrophy [19]. The serious role of superoxides in blood pressure maintenance of moderately hypertensive HTG rats was demonstrated by Kuneš et al. using acute i.v. tempol administration in conscious animals [20]. Zicha et al. reported that the concentration of conjugated dienes, a marker of oxidative membrane damage, was significantly increased in the kidney of both 3- and 7-month-old HTG rats in comparison with Wistar rats. On the other hand, baseline GSH/GSSG ratio, as a marker of redox control, was significantly higher in 12-week-old HTG rats than in age-matched Wistar rats [11]. This increase was probably caused by activation of antioxidant mechanisms in hypertriglyceridemic animals, in which higher production of reactive oxygen species was documented [20, 21]. Oxidative stress manifestation was further enhanced by high-sucrose diet, as demonstrated by increased TBARS and conjugated diene concentration, decreased GSH levels, and decreased glutathione peroxidase activity in blood and liver of this respective animal model [22].
Increased level of reactive oxygen species may represent an initial step in the signal cascade of NF-κB activation [2, 5]. Consequently, transcription factor NF-κB enhances gene expression of antioxidant enzymes in oxidatively stressed tissue [2]. In our experiment, NF-κB inhibition by JSH-23 showed decreased SOD1 expression and SOD activity together with increased CD concentration in the heart of HTG rats. On the other hand, GSH level was not affected by this treatment. Previously we have reported that chronic NF-κB inhibition with lactacystin also increased CD concentration in the heart of
N
G
-nitro-L-arginine methyl ester- (L-NAME-) treated rats [23]. Similarly, other authors showed that lactacystin treatment significantly increased oxidative protein damage (measured as the level of protein carbonyls), lipid peroxidation, and concentration of 3-nitrotyrosine in cell culture [24]. Higher levels of lactacystin increased the concentrations of 8-hydroxyguanine (a biomarker of oxidative DNA damage) and decreased GSH levels. Lactacystin treatment also decreased significantly activity of superoxide dismutase 1 and 2 [24]. Previously, Chen et al. documented accumulation of reactive oxygen species after inhibition of the NF-κB transcriptional activity also [9].
It has been suggested that intracellular ROS overproduction may represent one of the causes leading to increased blood pressure in both experimental models and human hypertension [25–27]. Considering that the reactions between NO and superoxide anion are most likely a major cause of impaired endothelium dependent vasorelaxation in hypertension [28]. As mentioned above, a higher production of reactive oxygen species was also documented in HTG rats. In our study, NF-κB inhibition by JSH-23 led to a slight but significant blood pressure increase in HTG rats in comparison with untreated animals. We hypothesize that elevated production of ROS after NF-κB inhibition might play an important role in blood pressure increase observed in this experimental model. Similarly, our recent results indicated that chronic NF-κB inhibition with lactacystin increased blood pressure in L-NAME-treated rats [23]. Moreover, in this study, JSH-23 treatment increased total NOS activity in the heart of HTG rats. Similarly, lactacystin treatment led to increased production of nitric oxide, measured as levels of
N
O
2
-
plus
N
O
3
-
in cells [24]. Finally, we observed increased endothelial NO synthase expression in the heart of HTG rats treated with JSH-23. These changes could be explained as a compensatory mechanism activated due to the ROS and blood pressure increase.
While excessive amounts of ROS can be harmful within the cells [29], their increase to the regulatory level may trigger different signal transduction pathways [30, 31]. Dröge et al. [32] demonstrated that ROS elevated intracellular GSSG level and thereby acted indirectly on the signal cascade of NF-κB activation, because NF-κB activation requires an altered level of GSSG. Activated NF-κB then enhances gene expression of antioxidant enzymes [2]. In our study, the inhibition of NF-κB protein by JSH-23 caused inactivation of NF-κB signaling, blocking presumably also the synthesis of antioxidants. Indeed, we detected decreased superoxide dismutase activity and SOD1 expression in the heart of HTG rats treated with JSH-23. Our present results confirm the role of NF-κB signaling in gene expression of antioxidant enzymes in oxidatively stressed tissue. Cho et al. showed several possible mechanisms responsible for cellular ROS accumulation induced by NF-κB inhibition. The most important ones involve impairment of the activities of antioxidant enzymes and glutathione depletion by NF-κB inhibition [33]. Moreover, the production of antioxidant enzymes, such as ferritin heavy chain, manganese-dependent superoxide dismutase, and metallothionein, was found to exhibit NF-κB-dependent manner [34]. A study by Cho et al. also reported that NF-κB inhibition increased superoxide anion level and decreased GSH level in isolated human CD4+ T cells [33]. Reduced GSH is a major intracellular antioxidant and NF-κB is the most important transcription factor, which induces the gene for glutamylcysteine synthetase, the rate-limiting enzyme for GSH synthesis [35]. NF-κB inhibitors, including pyrrolidine dithiocarbamate [36], parthenolide [37], gliotoxin [38], and proteosome inhibitor [24], were reported to cause GSH depletion. However, our present study did not confirm the decrease of GSH level after JSH-23 treatment. The unchanged GSH level along with increased eNOS protein expression after JSH-23 treatment in the heart may represent a compensatory mechanism activated due to ROS accumulation and blood pressure increase in HTG rats.
In conclusion, our data show that NF-κB inhibition by JSH-23 leads to further increase of oxidative damage followed by increased blood pressure in the model of hereditary hyperglyceridemic rats. Under these conditions, increased NO production represents rather counterbalancing mechanism activated by blood pressure increase in this respective model. Thus, NF-κB inhibition under increased ROS level may not always have a beneficial effect in the heart.