When simulating ADMA-like preeclampsia, a significant increase in the level of homocysteine in the blood of pregnant females was observed, compared to that in the intact ones – 6.5 ± 1.4 and 5.0 ± 1.2 µmol/l, respectively.

When correcting PE with the combination of ademethionine 150 mg/kg and taurine 260 mg/kg, a significant decrease in the level of homocysteine was observed to 4.9 ± 1.3 µmol/l (Figure 1).

Figure 1: Effect of ademethionine, taurine on the level of homocysteine in the blood of pregnant rats against the background of simulated ADMA-like preeclampsia (M±m).

Modeling ADMA-like preeclampsia by intraperitoneal administration of L-NAME at a dose of 25 mg/kg/day resulted in an increase in platelet aggregation, which is evidenced by an increase in the maximum plasma light transmission (Table 3). When using ADF as an inducer, the light transmission increased from 24.3±1.09% to 53.8±2.60%, when using collagen – from 50.2±1.66% to 81.3±2.68%, when using ristomycin – from 71.8±1.65% to 85.7±0.80% and when using adrenaline – from 30.1±1.97% to 52.6±2.23%. When administering the studied drugs, ademetionine and taurine, to the animals with ADMA-like preeclampsia, aggregation of platelets with individual peculiarities was corrected. (Table 3). When induced with ADP, collagen and adrenaline against the use of taurine, there was a statistically significant decrease in platelet aggregation relative to that in the control (p<0.05), but it did not reach the target level and was statistically different from that in the group of intact animals (p<0.05). When inducing with ristomycin, the platelet aggregation level was statistically lower (p<0.05) than that of the ”untreated” animals and reached the target level, as evidenced by there being no statistical difference from the platelet aggregation level of the animals of the intact group (Table 3).

Table3: The effect of ademethionin and taurine on the time of induced platelet aggregation in modeling ADMAlike preeclampsia (M ± m; N = 10).

With the development of preeclampsia, events occur in different conditions. It should be noted that platelet activation can be caused not only by proteins of subendothelial structures, but also by adrenaline, norepinephrine, ADP, immune complexes, peroxide radicals, hypoxia, arterial hypertension, prostaglandins PgG2 and PgH2, arachidonic acid, thromboxane A2, platelet activating factor (PAF), phorbol esters, latex, lectins, A23187 ionophore, bacteria and bacterial lipopolysaccharide, tumor cells, increased shear stress, etc. [9, 10, 11]. It is ischemic placenta that excretes a large number of these humoral factors, which leads to platelet activation. Depending on the inducers and their combination, the activation of platelets, as well as the humoral factors secreted by them and their ratios are of a different nature. Unlike natural conditions, there is no exposed subendothelial layer, nor is there platelet adhesion at the site of activation. The mass of activated platelets goes into the systemic circulation, and it can be said that there is a kind of endocrine gland, located throughout the circulating blood and directly contacting with endothelium. Therefore, even if the action of each individual platelet is considered locally, their mass location determines the generalization of their effects. When passing through the ischemic placenta, more and more new platelets are activated. When a critical number is reached, the level of humoral factors secreted by platelets becomes sufficient to cause systemic endotheliosis.

There is even a parallel between the DIC-syndrome, systemic inflammatory response syndrome and preeclampsia. One of the diagnostic criteria for the severity of preeclampsia is the sFlt-1/PlGF ratio. sFlt-1 is a variant of the vascular endothelial growth factor receptor (VEGFR-1). The higher this ratio, the higher the risk of preeclampsia or its severity. The source of sFlt-1 is not only an ischemic placenta, but also a plateletmonocine plateletmonocine complex, which is obtained because of aggregation of platelet-activated monocyte.

During pregnancy, platelet sensitivity to adrenaline, collagen, ristomycin, and other factors increases. This accurately determines polyetiology and another point of application of provoking factors in the pathogenesis of preeclampsia in such conditions as: infections, psychoemotional stress, conditions accompanied by excessive formation of peroxide radicals, metabolic syndrome, conditions accompanied by an increased content of ADMA and homocysteine, etc.

Thus, platelet activation is an intermediate pathogenetic link between the immaturity of the spiral arteries, ischemia of the placenta and systemic endothelial dysfunction. The chain of events described above looks logical in other pathological conditions as well, such as: sepsis, atherosclerosis, arterial hypertension, metabolic syndrome, varicose veins, etc. Summarizing the analyzed data, it can be said with a certain degree of accuracy that vascular-coagulation-immunological disorders in pregnant women have a common pathogenetic interaction in various diseases and, depending on the provoking factors and individual characteristics, determine the development of DIC-syndrome, preeclampsia or eclampsia [5].

An increase in platelet aggregation capacity and activation of the plasma-coagulation component of hemostasis, oxidative stress and mitochondrial dysfunction in modeling ADMA-like preeclampsia repeat the patterns observed in the clinic in women with preeclampsia. L-NAME is an analogue of ADMA, which, in turn, is an eNOS inhibitor [18, 19, 20, 21, 22]. That is why the main task of pharmacology is the search and study of active pharmacological agents aimed at the prevention [23, 24] and correction [25, 26, 27] of ischemic processes occurring in various organs and tissues. the search for new substances and ways to increase the resistance of tissues to ischemia remains relevant, despite the rapid development of experimental pharmacology [28, 29, 30, 31], the spread of peptide drugs [32, 33, 34, 35, 36], the improvement of targeted synthesis methods that allow the creation of highly selective drugs [37, 38, 39].

Therefore, the introduction of L-NAME results in a reduced activity of eNOS ,, as well as a reduced formation of potent antiaggregants – NO – by endothelium. At this stage, the most sensitive are the placental vessels. The resulting endothelial dysfunction leads to ischemic events in it. When passing through the placenta, platelets get activated due to ischemia, as well as due to humoral factors released in response. Activated platelets not only have an increased aggregation capacity by themselves, but also activate intact platelets, secreting aggregation inducers into the bloodstream. Modeling ADMA-like preeclampsia leads to a pronounced persistent increase in blood pressure. There is a change in shear rate, which can also activate platelets. According to the literature, in women with preeclampsia, on the surface of the platelet, the expression of receptors to different aggregation inducers increases compared with that in normal pregnancy. This contributes to increased sensitivity of platelets in them. No concrete literature data on the number of receptors for aggregation inducers in animals with ADMA-like preeclampsia have been found, but taking into account the comparable pathogenetic mechanisms of both pathological conditions, the probability of this mechanism is rather high.

The time of the blood clot formation until the complete cessation of blood flow in the artery is an integral sum of vascular-platelet and plasma-coagulation components of hemostasis. Therefore, its shortening is a logical consequence of a shift of hemostasis towards hypercoagulation. The pronounced positive effects of ademetionine in the correction of platelet aggregation disorders caused by the ADMA-like preeclampsia model are accounted for by several direct and indirect mechanisms. The literature describes various positive effects of ademetionine: improvement of mitochondrial respiration, increased the intraplatelet concentration of glutathione, enhanced the antiaggregant effect both red blood cells and leukocytes of ademetionine (SAMe), as did simultaneous incubation with L-arginine [40], antiiinflammatory effects by changing the concentration of interleukins: the content of tumor necrosis factor (TNF) a decreases and the synthesis of interleukin-10 (IL-10) is activated. In addition, the ratio of SAM to SAH (S-adenosyl homocysteine) changesalso was by SAMe [41].

The study of the effect of SAM on the expression of the antioxidant stress proteins heme oxygenase-1 and ferritin in endothelial cells showed that the induction of the oxygenase / ferritin system leads to the protection of tissues from inflammatory factors. Ademetionine increases the levels of mRNA and oxygenase in endothelial cells. The induction of oxygenase gene expression is associated with an increase in ferritin levels and is regulated at the transcriptional level through an increase in promoter activity. The activation of oxygenase under the influence of SAM is associated with a decrease in the NADP-mediated release of reactive oxygen species. Thus, the HO-1 / ferritin system can be another target for the antioxidant action of ademetionine .Ademetionine is able to prevent the toxic effects of xenobiotics in various tissues. It was shown that its use improved the growth of animal embryos, reducing the embryotic effect of ethanol. Ademetionine does not possess embryophetotoxicity, does not have any negative effect on reproductive function, and does not cause any developmental abnormalities [44]. It not only eliminates the symptoms of cholestasis in the mother and prevents possible complications of pregnancy, but also reduces the incidence of premature birth in patients with cholestasis, neu-tralizes the toxic embryopathic effects of xenobiotics and maintains the normal functioning of the placenta. Direct infusion of ademenionine (S-adenosylmethionine, AdoMet) to the brain cortex of thioacetamide (TAA) rats decreased extracellular concentration of dimethylarginines. The possible explanation of this phenomenon is associated with a feedback inhibition of PRMT by S-adenosylhomocysteine (AdoHcy), formed from AdoMet upon trans-methylation [45, 46]. The intensity of this inhibition was reflected by the diminished dimethylarginine levels, the feedback inhibition of dimethylarginines (DMAs) synthesizing enzymes (PRMTs). PRMT enzyme by AdoHcy seems the most reliable explanation of the observed decrease in asymmetric dimethylarginine (ADMA) concentration. Increased circulating ADMA levels, which inhibits NO synthesis, may be associated primarily with endothelial dysfunction that somehow can be translated on changes of CBF considered as a causative and a predictive factor of overt hepatic encephalopathy (HE). This study reveals the ability of ademetionine to directly block the formation of asymmetric dimethylarginine. DNA methylation is altered in preeclampsia placentas determined the methylation index by measuring placental S-adenosylmethionine (SAM) and Sadenosylhomocysteine (SAH) levels hypomethylation state of the placenta in early onset preeclampsia (EOPE), which is reflected by lower SAM and placental growth factor (PlGF) DNA hypomethylation underlines the possible role of placental DNA hypomethylation in the pathophysiology of EOPE, which needs further investigation the use of ademetionine in correction EOPE.

Taurine appears to have a protective effect against the hyper-coagulative state in edema, proteinuria and hypertension gestosis. Plasma and platelet taurine levels are reduced in patients with type 1 diabetes mellitus. The pronounced positive effects of taurine in the correction of platelet aggregation disorders caused by an ADMA-like model of preeclampsia are also associated with direct and mediated mechanisms. The literature also describes the positive effects of taurine in pregnant women in particular, its ability to reduce platelet aggregation, including not through the Ca2+-dependent mechanism. Summing up, it should be noted that the proposed methodological complex of functional, biochemical and morphological changes linked with the development of NO deficiency due to the blockade of NO-synthase in male rats and pregnant female rats against the background of preeclampsia makes it possible to quite objectively detect and evaluate the endotheliotropic effect of pharmacological agents.Thus, the most pronounced endothelioprotective effect on the model of ADMA-like preeclampsia was observed with the combined use of ademethionine and taurine, which was reflected in a decreased CED.The maximum reduction in ?ED to the level of that in the intact animals, against the background of simulated L-NAME-induced NO deficiency in male rats, was recorded when using taurine 260 mg/kg and amounted to 1.3 ± 0.1.

It should be noted that the introduction of the studied drugs at the studied doses showed their endothelium and cardioprotective activity, but was not accompanied by reaching the target blood pressure indicators. This, on the one hand, indicates that ?ED has independent significance, and, on the other hand, that L-NAMEinduced arterial hypertension on the 7th day involves into the pathogenic process not only the nitroergic system, but also all the elements of the humoral and neurogenic regulation contours of the circulatory system. The study of the NO-producing function of the endothelium confirms the endothelioprotective effects of ademethionine, taurine, and their combination, which shows in preventing the reduction of stable metabolites of nitric oxide under conditions of endothelial dysfunction. On the model of ADMA-like preeclampsia in pregnant rats, the maximum reduction in ?ED was observed when using ademethionine 150 mg/kg and taurine 260 mg/kg. The level of homocysteine, which is a marker of preeclampsia, also decreased to the level of that in the intact animals with the combined use of ademethionine 150 mg/kg and taurine 260 mg/kg in the ADMA-like preeclampsia model. Therefore, the combination of ademethionine with taurine on the model of ADMA-like preeclampsia can be one of the rational approaches for correcting endothelial dysfunction during pregnancy in clinical practice, since the effect of this combination on the nitroergic and neurohumoral systems is pathogenetically justified, and these drugs are approved for use in pregnant women. The way of how damage to the system is caused by L-NAME, as well as points of application of ademethionine, which increases the amount of NO, and taurine, which simulates the synthesis of NO, due to capturing reactive oxygen species. From the literature it is known that ademethionine has an endothelioprotective effect by blocking the protein arginine methyltransferase (P?MT) enzyme – an enzyme that participates in the synthesis of asymmetric dimethylarginine (ADMA), and taurine, in turn, is a powerful antioxidant: it inhibits the production of superoxide radicals, decreases the production of the factor, and decreases the tumor necrosis factor, which is a marker of placenta inflammation, and restores the expression of endothelial NO synthase

SAM or ademethionine is formed endogenously from methionine amino acid using the methionine adenosyl transferase enzyme. Further, S-adenosylhomocysteine is formed from SAM under the action of SAM-dependent methyltransferase enzyme, and it is hydrolyzed to homocysteine by S-adenosylhomocysteine hydrolase. Homocysteine, completing the cycle, is converted to methionine in the transfer reaction of the methyl group from 5-methyltetrahydrofolate, one of the two classes of methionine synthases. With a hereditary defect of enzymes involved in the SAM cycle and with an excessive intake of ademethionine, remethylation processes can be disturbed, which leads to an accumulation of homocysteine in the blood, i.e. hyperhomocysteinemia, which results in oxidative stress and ED.

However, in this L-NAME model of induced endothelial dysfunction, the administration of ademethionine had endothelium protective effects and reduced oxidative stress. This may have happened because most of the administered ademethionine inhibited PRMT, which contributed to an increase in NO. And as known, one of the main vasoactive substances secreted by endotheliocytes is nitric oxide –an endothelial vasodilator.

Taurine is a sulfur-containing amino acid, which is synthesized in the body from cysteine, which is formed from homocysteine with the participation of pyridoxal phosphate. Thus, therapy with ademethionine can be used in case of pathologies with pronounced oxidative stress. It can also be combined with a drug with antioxidant properties – taurine.

The combinatione effects of ademethionine and taurine is explained by the ability to enhance the positive effects of the investigated pharmacological agents in the correction of endothelial dysfunction disorders and hemostasis disorder sin animals with ADMA-like preeclampsia and, thus, to a greater extent lead to a decrease in the number of factors contributing to the transition of platelets into the activated form.

Conclusion

Ademethionin at a dose of 150 mg/kg and taurine at a dose of 260 mg/kg showed an endothelium protective effect on ADMA-like preeclampsia model both in monotherapy and in combination. On the model of ADMAlike preeclampsia in pregnant rats, the maximum reduction in ?ED was observed when using ademethionine 150 mg/kg and taurine 260 mg/kg. The level of homocysteine, which is a marker of preeclampsia, also decreased to the level of that in the intact animals with the combined use of ademethionine 150 mg/kg and taurine 260 mg/kg in the ADMA-like preeclampsia model. When modeling ADMA-like preeclampsia, there is an increase in platelet aggregation from about 19% to 121% when the inducers used are ADP, collagen, ristomycin and adrenaline, as well as a shift in the plasma-coagulation component of hemostasis towards hypercoagulation. The time of blood clot formation was reduced to 70% of the original. The use of ademethionin and taurine resulted in a pronounced correction of the endotyelial dysfunction, disturbance of induced platelet aggregation, indicators of the plasma-coagulation component of hemostasis and the time of thrombus formation in animals with ADMA-like preeclampsia.

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