INTRODUCTION

It is known that dinitrosyl iron complexes (DNICs) with thiol-containing ligands are one of the main natural forms of NO deposition in biosystems [1–9]. Such stabilized NO forms ensure the transfer and accumulation of NO in the body, and as a result of their decomposition, NO, S-nitrosothiols, and divalent iron ions are released [7, 8]. It is also known that DNICs have their own antioxidant and cytoprotective effects [4, 10–13] and can also be a natural regulator of the NO level in organs and tissues [14].

It is also known that DNICs with small molecule thiol-containing ligands (glutathione, cysteine) can be relatively easily synthesized in laboratory or industrial conditions for subsequent introduction into the body. Previously, in [15, 16], we presented data on the effect of DNIC with glutathione ligands (DNIC-Glt) under various ways of administration of this complex into the body. It was established that the formation of DNIC with protein ligands in the blood and their hypotensive effect are recorded in cases where DNIC-Glt was injected into the body, namely intravenously, subcutaneously, intraperitoneally and intramuscularly.

In this work, an attempt to analyze the biological activity of DNIC, in which the thiol-containing ligand is N-acetyl-L-cysteine (DNIC-Acc), was made. This compound is known to be nontoxic and is widely used as a mucolytic agent that easily penetrates cell membranes. It can be assumed that DNIC-Acc will have similar properties. We previously showed that these amphiphilic complexes and/or their half-life products penetrate into the body as a result of transdermal [17] or inhalation [18] administration and act as NO sources in organs and tissues.

The aim of this work is to analyze the effect of the stabilized NO form DNIC-Acc under sublingual administration of this compound into the rat body.

MATERIALS AND METHODS

Synthesis of DNIC-Acc was carried out under laboratory conditions in accordance with the procedure we described earlier [17]. The formation of these complexes, as before, was controlled by electron paramagnetic resonance (EPR) [17]. Next, the resulting aqueous solution of this compound was frozen, stored in liquid nitrogen, and thawed immediately before the beginning of the experiment. After thawing, glycerol was added, the final concentration of which was 20%.

The experiments were carried out in normotensive Wistar rats (males weighing 350–450 g). The animals were kept at the bioclinic of the National Research Center for Cardiology, Ministry of Health of the Russian Federation, in cages of five individuals with free access to dry food and water. The light regime was controlled in the 12 h : 12 h (light : dark) regime with a sufficient change in air volumes and a temperature of 19–23°С.

At the beginning of the experiment, rats were anesthetized with Zoletil-100 (5 mg/kg of body weight). Further, to record the mean arterial blood pressure (BPm) and heart rate (HR), the animals were injected with a PE-50 polyethylene catheter into the left carotid artery, which was connected to a P23 Db manometric sensor (Gould Statham, United States). The obtained BP signal was fed through a Biograph-4 amplifier (St. Petersburg State University of Aerospace Instrumentation, Russia) to an NI-USB 6210 ADC (National Instruments, United States) and processed on a computer using special programs for recording and processing signals of physiological parameters of the heart (the author is E V. Lu-koshkova). Blood samples were taken through the same catheter after the administration of DNIC-Acc to the animals.

Further, all the animals were divided into two experimental groups of five or six animals each. Animals from the first group received DNIC-Acc in glycerol by sublingual administration. In this case, this drug was administered into the oral cavity or into the cheek pouch by drip using an insulin syringe with a blunt needle for 35–40 min. The total volume of DNIC-Acc was 1.0 mL, and its dose was standard and amounted to 5.0 μmol per animal. The other group included control animals, which were not treated with this drug. During the experiments, BPm and heart rate were recorded in rats both before and within 1 h after the introduction of DNIC-Acc.

Further, in a half of the experiments, the animals were slaughtered with an overdose of urethane, after which samples of organ tissues (heart, lung, liver, kidney, skeletal (femoral) muscle), as well as whole blood, were taken. The resulting samples were placed in plastic tubes 5.0 mm in diameter, which were immediately frozen, stored in liquid nitrogen, and used to record EPR spectra.

In other experiments in both experimental groups, the total level of NO in organ tissues was studied by EPR. For this, lipophilic nitric oxide spin traps such as complexes of iron ions and diethyldithiocarbamate (Fe-DETC2) were used with the registration of formed NO-Fe-DETC2 spin adducts by EPR. One hour after sublingual administration of DNIC-Acc, the animals were injected with the components of this NO spin trap: diethyldithiocarbamate (DETC, 620 mg/(kg body weight) in 1.0 mL saline, intraperitoneally) and FeSO4⋅7H2O with sodium citrate (25 and 125 mg/(kg of body weight), respectively, in 1.0 mL saline, subcutaneously in the area of the left shoulder). After 20 min, the animals were sacrificed and their heart, lungs, liver, kidneys, and skeletal (femoral) muscle were isolated. These organs were immediately washed in physiological saline and mechanically grinded. Ground tissue samples of these organs were placed in plastic tubes 5.0 mm in diameter, which were immediately frozen, stored in liquid nitrogen, and used to record EPR spectra. The EPR spectra of the synthesized DNIC-Acc in an aqueous medium were recorded at room temperature in glass capillaries. The amplitude of the high-frequency modulation of the magnetic field was 0.4 mT. In this case, the microwave field power of the spectrometer was always set at a level of 10 mW at a frequency of 9.14 GHz. Scanning of the magnetic field, as before (see [17]), was carried out with the center at g = 2.03.

EPR spectra of all obtained organ tissue samples were recorded on an E-109E X-band EPR spectrometer (Varian, United States) at liquid nitrogen temperature. The amplitude of the high-frequency modulation of the magnetic field was 0.4 mT (when examining samples of organ tissues and blood without Fe-DETC2) or 0.2 mT (when recording signals of spin adducts in the tissue) at a frequency of 100 kHz. The microwave field power of the spectrometer was set at 10 mW, and its frequency was 9.33 GHz. Scanning of the magnetic field during recording of EPR signals of the samples was carried out with the center at g = 2.03. After recording the signals, all samples were thawed and the weight of the tissue in the active zone of the spectrometer resonator was determined.

Statistical data processing and plotting were performed using the Origin 8 software package (OriginLab Corporation, United States). All data in the article are presented as the mean ± error of the mean. The repetition rate of the experiments was at least five to six experiments. The two-tailed Student’s t test was used to test the statistical significance of the effects.

RESULTS AND DISCUSSION

In the course of the experiments under laboratory conditions, the synthesis of DNIC-Acc was carried out and its effect was studied when administered sublingually to the body of rats. Figure 1 shows the EPR spectrum of the resulting compound at room temperature. It can be seen that it is represented by an asymmetric singlet signal with the center at g = 2.03, which is characteristic of these compounds and was previously described by many authors [7, 8, 17].

Fig. 1.
figure 1

EPR spectrum of synthesized DNIC-Acc complexes in an aqueous solution at room temperature.

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In the other part of this work, the parameters of BPm and HR were monitored during the entire experiment. It was found that in the initial state (before the introduction of DNIC-Acc) BPm was 137 ± 6 mm Hg, and HR was 394 ± 27 bpm. Five minutes after the beginning of instillation of this drug under the tongue, a slight rise in BPm (up to 156 ± 8 mm Hg) was usually observed, while HR dropped slightly (up to 378 ± 29 beats/min). After 10–15 min from the beginning of DNIC-Acc administration, BPm returned to the norm, and starting from the 40th minute it slightly decreased, reaching 135 ± 7 mm Hg at the 50th minute and 123 ± 10 mm Hg at the 60th minute of the experiment. At the same time, HR increased to 426 ± 28 beats/min.

Consequently, only slight changes in BPm and HR were recorded during the experiments, but no significant changes in the values of these parameters were found as a result of DNIC-Acc introduction, thus, it did not have a noticeable effect on the general hemodynamics of the body., There is probably no significant increase in the level of free NO and/or its stabilized forms in the bloodstream of the animal under this method of drug administration, which is why there is also no pronounced hypotensive effect. At the same time, it cannot be ruled out that, under sublingual DNIC-Acc administration, its transport in the body is largely carried out by the lymph flow [16, 17], and the content of these complexes and/or secondary products of their breakdown in the bloodstream is low and insufficient to initiate a noticeable decrease in the mean BP.

In addition, the analysis of organ tissues and whole blood by EPR was performed, while samples frozen 1 h after sublingual administration of DNIC-Acc to experimental animals were examined. The characteristic EPR spectra of the samples are shown in Fig. 2. This figure shows that free radical signals (g = 2.00), which are represented by a superposition of the spectra of ubisemiquinone and flavosemiquinones, were recorded in tissue samples [19].

Fig. 2.
figure 2

EPR spectra of frozen samples of organ tissues and whole blood of rats obtained 1 h after sublingual administration of DNIC-Acc: (1) heart, (2) lung, (3) liver, (4) kidney, (5) skeletal muscle, (6) blood. Temperature—liquid nitrogen.

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Figure 2 also shows that the spectrum of frozen liver tissue (signal 3) at g = 2.03 contained a component corresponding to DNIC with a protein thiol-containing ligand, which was absent in the other spectra in this figure. At the same time, in the control group of animals, the components at g = 2.03 were also absent in samples of the heart, lungs, kidney, and skeletal muscle, and only a very weak signal, corresponding to basal DNIC with protein ligands, was observed in samples of liver tissue at g = 2.03 (data not shown), which was significantly less than that observed in this organ after sublingual administration of DNIC-Acc (spectrum 3, see Fig. 2). This result suggests that as a result of the administration of DNIC-Acc, these complexes are converted into DNIC associated with proteins and the accumulation of the latter in the liver tissue, while these compounds are not recorded in other studied organs.

This figure also shows that after the introduction of DNIC-Acc, there was no signal at g = 2.03 in the blood spectrum, which may indicate the absence of a registered amount of paramagnetic DNIC in the bloodstream. In addition, the signal from NO complexes with iron of the heme group of hemoglobin (nitrosylHb) [3] was not recorded in this case, which may indicate no excess of free NO in the bloodstream after the administration of DNIC-Acc.

In the third part of this study, the total NO level was estimated in both experimental groups by EPR and using the Fe-DETC2 spin trap. As is known [20], this trap has hydrophobic properties and is capable of effectively interacting with both free NO and its deposited forms with the formation of stable paramagnetic NO–Fe–DETC2 spin adducts detected by EPR.

Figure 3 shows the characteristic EPR spectra of tissue samples of the studied organs, obtained 20 min after the introduction of the components of this trap into the body. It can be seen from this figure that in the studied samples, free radical EPR signals, as well as components belonging to molybdenum complexes in the high field part of the spectra were also recorded. It should be noted that in all cases, the spectra contained narrow equidistant triplet signals belonging to NO–Fe–DETC2 spin adducts (g = 2.036). The content of these mononitrosyl complexes, as noted above, reflects the level of NO, including its deposited forms.

Fig. 3.
figure 3

EPR spectra of frozen rat organ tissue samples obtained 20 min after the administration of Fe-DETC2 spin trap components to the animals, i.e. 1 h 20 min after the introduction of DNIC-Acc: (1) heart, (2) lung, (3) liver, (4) kidney, (5) skeletal muscle. Temperature—liquid nitrogen.

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Based on these signals, the content of NO-Fe-DETC2 in all tissue samples was estimated as the number of paramagnetic centers in the sample normalized to the tissue weight in the active zone of the spectrometer resonator. To assess the effect of sublingually administered DNIC-Acc on the total level of NO in organ tissues, the values of the NO-Fe-DETC2 content corresponding to animals treated with DNIC-Acc (parameter N+DNIC-Acc) normalized to similar parameters corresponding to the control group (Ncont) are presented in Fig. 4. This figure shows that an increase in the total level of NO was recorded in all organs of the experimental animals as a result of the action of DNIC-Acc. At the same time, these differences are significant for the heart and liver, and the most significant effect was observed for the liver tissue of the animal.

Fig. 4.
figure 4

Values of the content of paramagnetic NO-Fe-DETC2 spin adducts in tissue samples of animal organs (parameter N+DNIC-Acc) treated with DNIC-Acc, normalized to the values corresponding to control animals (Ncontr); *p < 0.05 compared with the value of this ratio equal to 1.

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It should be noted that our results are consistent with the data of an earlier study [17], where it was shown that DNIC-Acc administered transdermally to rats led to a slight decrease in BPm, but caused an increase in the total level of NO in lung and liver tissue. At the same time, the effect of DNIC-Acc differed significantly from the effect of DNIC-Glt administered by injections, which had a pronounced hypotensive effect [15, 16].

Thus, DNIC-Acc, as a stabilized form of NO, can transport NO into the body when it is administered sublingually. As a result of its introduction into the liver tissues, the formation and accumulation of DNIC complexes with protein ligands was registered. Moreover, a significant increase in the total level of NO in the tissue of the heart and liver was recorded as a result of the action of DNIC-Acc, with the most significant effect found for the liver of the animal.