Correction of morphofunctional disorders in experimental preeclampsy by combined use of trimetazidine and purified micronized flavonoid fraction as well as their combinations with methylampsy

Full Text

Abbreviations: L-NAME – L-Nitro-Arginine Methyl Ester; VEGF – vascular endothelial growth factor; CED – coefficient of endothelial dysfunction; SBP – systolic blood pressure; DBP – diastolic blood pressure.

INTRODUCTION

For many decades, preeclampsia has remained an important medical and social problem in the countries all over the world. It occupies a leading place among the causes for maternal morbidity rate and mortality and, according to various authors, is from 9% to 25% [1, 2]. In Russia, this pathology stably occupies the 3–4^th places [3]. The incidence ranges from 2% to 10% of all pregnancies and has no tendency to decrease [1, 4, 5]. In addition, preeclampsia leads to the development of pathological conditions not only in women, but also in the fetus, contributing to the disability of mothers and children [4, 6].

The problem of preventing and treating preeclampsia, as well as assessing the severity of its course and perinatal risks, is to a great extent due to the lack of consensus among the medical community about its etiology and pathogenesis, although an extraordinary number of studies around the world are devoted to the investigation of this pregnancy complication [7, 8]. Herewith, preeclampsia is increasingly considered from the point of view of endothelial dysfunction [9–11], and “an oxidative stress” as a result of depletion of the antioxidant system under the conditions of placental ischemia, is one of its development mechanisms [12, 13]. The endothelial dysfunction developing against this background, leads to impaired microcirculation and tissue hypoxia and, as a result, to the development of multisystemic lesions that constitute the clinical manifestations of preeclampsia [14–16]. Therefore, the search for new drugs for the prevention and treatment of preeclampsia, is an urgent task of modern pharmacology.

In experimental studies on the model of L-NAME-induced preeclampsia in rats, the protective properties of resveratrol [17, 18], recombinant erythropoietin [19], and tadalafil [20, 21], which have endothelioprotective properties, have been demonstrated. Another promising area of prevention and correction of morphofunctional disorders that occur in preeclampsia, is the use of drugs with anti-ischemic and antioxidant properties. As a result of the previous studies, pronounced protective effects of trimetazidine and a purified micronized flavonoid fraction in the correction of morphofunctional disorders in experimental preeclampsia have been revealed, however, it should be notified that the target level has not been achieved [22–24].

Preeclampsia is a multifactorial disease. It is obvious that the increase in the effectiveness of therapy can be achieved by the complex use of drugs. Therefore, the urgent task of modern pharmacology is not only the search for new drugs for the treatment and prevention of preeclampsia, but also the study of their effectiveness in combined uses, as well as the drugs included in the standards for treatment.

THE AIM of the experiment was to determine the effectiveness of the combined use of trimetazidine and a purified micronized flavonoid fraction, as well as their combinations with methyldopa, in comparison with monotherapy with the same drugs in the correction of morphofunctional disorders arising under the conditions of experimental preeclampsia.

MATERIALS AND METHODS

Compliance with ethical principles

The study has been carried out at the Center for Preclinical and Clinical Research of the Belgorod State National Research University. The experiment was accomplished in accordance with legislative acts and guidelines regulating the conduct of experimental research in the Russian Federation: Order of the Ministry of Health of Russia dated 01.04.2016 No.199n “On the approval of the Rules of good laboratory practice” and “Guidelines for conducting preclinical studies of new drugs” ed. by A.N. Mironov. The ethical principles of handling laboratory animals were in accordance with Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. The study was approved of at the meeting of the Bioethical Commission of the Research Institute of Pharmacology of Living Systems of the Belgorod State National Research University, Protocol No.D2019/13. Keeping the animals was regulated by the norms of the Decree of the Chief State Sanitary Doctor of the Russian Federation dated 08.29.2014 Nо.51, GOST 33044-2014. The animals were kept in plastic cages, equipped with steel lattice lids, with a feeding recess, with a free access to food and water, on a balanced diet appropriate for the animal species. The ambient temperature was maintained at the level of 20–25°C with a relative humidity of 60–65%. The wood sawdust that had undergone preliminary UV sterilization, was used as a litter.

Modeling ADMA-like preeclampsia

The experiment was carried out on 200 white Wistar female rats weighing 250–300 g. An ADMA-like agent, a non-selective NO synthase blocker N-nitro-L-arginine methyl ether (L-NAME), was injected intraperitoneally at the dose of 25 mg/kg/per day for seven days (from the 14^th to the 20^th days of pregnancy) [25, 26]. To assess the effectiveness of the studied drug combinations, the animals had been divided into several groups (n=10).

1. Intact (Int.) (animals with oral administration of NaCl in equivalent doses from the 14^th to the 20^th days of pregnancy).
2. Modeling of ADMA-like preeclampsia (L-NAME) (N-nitro-L-arginine-methyl ester, Sigma-Aldrich), 25 mg/kg/per day.
3. L-NAME + methyldopa (Dopegit®, ZAO “Pharmaceutical Plant EGIS”, Hungary), 86 (2×43) mg/kg/per day.
4. L-NAME + trimetazidine (Preductal® MB, ZAO “Servier”, Russia), 6 mg/kg/per day.
5. L-NAME + purified micronized flavonoid fraction (Detralex®, ZAO “Servier”, Russia), 260 mg/kg/per day.
6. L-NAME + trimetazidine 6 mg/kg/per day + purified micronized flavonoid fraction 260 mg/kg/per day.
7. L-NAME + trimetazidine 6 mg / kg + methyldopa 86 (2x43) mg/kg.
8. L-NAME + purified micronized flavonoid fraction 260 mg/kg/per day + methyldopa 86 (2×43) mg/kg/per day.

Assessment of the degree of endothelial dysfunction development in pathology modeling

The development of endothelial dysfunction in experimental animals, as well as the degree of its correction by the studied drugs and their combinations, were assessed by the calculated coefficient of endothelial dysfunction (CED) [27, 28].

$CED=\frac{S_{NP}}{S_{ACH}}$

The endothelial dysfunction coefficient is the ratio of the triangle area above the blood pressure recovery curve in response to 30 μg/kg nitroprusside (S_NP) to the triangle area above the blood pressure recovery curve in response to 40 μg/kg acetylcholine (S_ACH). The legs in the both triangles were indicators of the blood pressure recovery time (reaction duration) and the changes in the blood pressure in response to the intravenous administration of acetylcholine and nitroprusside, respectively.

Placental microcirculation assessment

To obtain the data on the state of microcirculation in the placenta, the equipment manufactured by Biopacsystems was used: polygraph MP100 with an LDF100C Laser Doppler Flowmetry (LDF) module and an invasive TSD144 needle probe. On the 21^st day of pregnancy, under anesthesia, the level of microcirculation was measured in the projection of the placental disc at a distance of 1 mm at 4 points. The registration and processing of LDF results were carried out using the AcqKnowledge version 3.8.1. The microcirculation values were stated in perfusion units (PU) [29].

Proteinuria research

Urine collection was carried out for 12 hours using special metabolic cells. The determination of the protein amount in daily urine, was carried out by the pyrogall method. It is based on the determination of the optical density of a solution of a colored complex, formed by the interaction of protein molecules with the molecules of the complex of the pyrogallol red dye and sodium molybdate. The color intensity of the solution is proportional to the protein content. The measurements were carried out using a PE-5400 V spectrophotometer at the wavelength of 600 nm [30].

Study of the greater omentum сальник edema

On the 21^st day of pregnancy, the greater omentum was removed under anesthesia, and weighed. Within 24 hours it was dried at 37°C, then weighed again. From the difference in the mass of the greater omentum before and after drying, the amount of the evaporated water was obtained in each specific piece. The water content was stated in %, relative to the total weight of the gland while the first weighing [31].

Morphological methods for assessing changes in placenta when modeling experimental gestosis

A histological study of the placenta was carried out in all the series of the experiment for a morphological confirmation of the development of modeled pathological processes and giving a comprehensive assessment of the drugs’ effectiveness. The material was fixed in 10% formalin with subsequent embedding in paraffin. Histological sections of the placenta were made in a strictly vertical direction through the middle of the placental disc with the capture of all layers of the placenta and the wall of the uterine horn. The study of microslides, photorecording and morphometry, were carried out on a Leica DM4000B microscope with a video recording and an image processing system. All morphological studies were stained with hematoxylin and eosin [25].

Fetus study

The fetuses were removed from the uterine cavity, weighed, and the fetus size (craniocaudal size) was measured, followed by the calculation of the statural-weight coefficient [32].

Statistical processing of research results

Descriptive statistics was applied to all the data: the data were checked for a normal distribution. The distribution type was determined by the Shapiro-Wilk test. In case of a normal distribution, the mean (M) and a standard error of the mean (m) were calculated. The intergroup differences were analyzed by parametric (Student’s t-test) or nonparametric (Mann-Whitney U-test) methods, depending on the type of the distribution. The calculations were performed using statistical programs Microsoft Excel 7.0. The groups were compared in pairs.

RESULTS

Effect of the combined trimetazidine and detralex use, as well as their combined use with methyldopa, for the correction of morphofunctional disorders in ADMA-like preeclampsia

The combined use of preductal and detralex, as well as their combined use with dopegit for the correction of morphofunctional disorders in ADMA-like preeclampsia, led to a more pronounced decrease in blood pressure. Thus, when the combined administration of preductal and detralex took place, the systolic and diastolic kinds of blood pressure decreased to 145.7±3.93 mm Hg and 100.1±3.59 mm Hg, respectively. When detralex was used individually, it decreased to 169.3±5.40 mm Hg and 125.7±4.91 mm Hg. When preductal was used individually, it decreased to and 152.5±1.99 mm Hg and 112.3±3.90 mm Hg. Herewith, the decrease of diastolic pressure was statistically significant in comparison with the reference groups (Table 1). The combined use of preductal and dopegit led to a statistically significant decrease in systolic blood pressure compared with monotherapy, and when detralex and dopegit were combined, both systolic and diastolic kinds of blood pressure decreased statistically significantly (p<0.05) compared with monotherapy (Table 1).

 

Table 1 – The effect of the Preductal^® MB and Detralx^® combination, as well as their combination with Dopegit^®, on the functional parameters of the cardiovascular system in ADMA-like preeclampsia.

Indicator/Index Group	SBP, mm Hg	DBP, mm Hg	CED, relative units.	Microcirculation, PU
Int	123.4±3.54*	83.8±5.47y*	1.21±0.13*	472.6±22.44*
L-NAME	193.6±6.28#	150.8±4.80#	2.89±0.25#	215.6±9.29#
L-NAME+ Dopegyt® 86 mg/kg	155.5±3.14*#	114.4±7.13*#	2.49±0.28#	297.8±13.41*#
L-NAME+ Preductal® MR 6 mg/kg	152.5±1.99*#	112.3±3.90*#	1.57±0.15*	402.3±15.81*#
L-NAME+Detralex® 260 mg/kg	169.3±5.4*#	125.7±4.91*#	1.79±0.11*#	394.0 ±9.87*#
L-NAME+ Preductal® MR + Detralex®	145.7±3.93*#	100.1±3.6*# y	1.30±0.05*	505.9±17.83*y
L-NAME+ Preductal® MR + Dopegyt®	138.6±3.16*#y	97.6±5.84*	1.32±0.08*	477.4±27.61*y
L-NAME+ Detralex®+ Dopegyt®	145.5±2.75*#y	97.8±2.2*#y	1.49±0.05*y	437.0±19.87*

Note: # – at p <0.05 in comparison with intact pregnant rats; * – at p <0.05 in comparison with the group of pregnant animals treated with L-NAME; y – at p <0.05 in comparison with both monotherapy options

 

When the combinations of preductal + detralex, preductal + dopegit and detralex + dopegit were used, the coefficient of endothelial dysfunction (CED) decreased to 1.30±0.05, 1.32±0.08 and 1.49±0.05, respectively, and the microcirculation improvement was up to 505,9±17.83 PU, 477.4±27.61 PU and 437.0±19.87 PU, respectively. It should be notified that CED reached a statistically indistinguishable value from the group of intact animals when the combinations of preductal + detralex and preductal + dopegit were used. Herewith, when the combination of detralex + dopegit was used, microcirculation improved to the level of intact animals.

In the described groups, a decrease in proteinuria resulted in 1.08±0.09 g/L, 0.92±0.09 g/L, and 0.92±0.09 g/L, respectively (Fig. 1A), and the fluid content in the greater omentum decreased to 46.33±1.08%, 45.98±2.26% and 47.89±1.50%, respectively (Fig. 1B). It should be notified that in comparison with the group with monotherapy, a statistically significant (p <0.05) decrease in proteinuria, was observed when using preductal + dopegit. The fluid content in the greater omentum in all the groups with a combined use of drugs, was at the level of intact animals.

 

Figure 1 – The effect of the Preductal® MB and Detralex® combination, as well as their combination with Dopegit®, on proteinuria and fluid content in the tissue of the greater omentum in ADMA-like preeclampsia

Note: # – at p <0.05 in comparison with intact pregnant rats; * – at p <0.05 in comparison with the group of pregnant animals treated with L-NAME; y – at p <0.05 in comparison with both monotherapy options.

 

A histological examination of the placenta revealed the following: a combined administration of the studied drugs to the animals with experimental preeclampsia, led to a pronounced positive dynamics of the morphological picture, which was approximate to the group of intact animals. There was a statistically significant (p <0.05) increase in the density of the cellular component of the placental tissues of the maternal and fetal parts of the placenta, and the diameter of the chorionic villi, in comparison with the groups of the animals in which the studied drugs had been used in the monotherapy mode (Table 2). Only the diameter of the villi reached the level of the intact animals when the combination of Preductal^® MB + Dopegit^® had been used. In addition, there was a statistically significant (p <0.05) increase in the fetal part of the placenta and a decrease in the maternal part of the placenta (Fig. 2). The exception was the group using Preductal^® MB + Detralex^®, in which there was no statistically significant change in the maternal part of the placenta compared to the groups with monotherapy with the same drugs. It should be notified that the level of the intact animals had not been reached.

 

Figure 2 – The effect of the Preductal® МВ and Detralex® combination, as well as their combination with Dopegit®, on the fetal and maternal size parts of the placenta, in ADMA-like preeclampsia

Note: # – at p <0.05 in comparison with intact pregnant rats; * – at p <0.05 in comparison with the group of pregnant animals treated with L-NAME; y – at p <0.05 in comparison with both monotherapy options

 

The study of the height-weight ratio in fetuses in the groups with the combined administration of the investigated drugs, showed an improvement in this indicator up to the level of the intact animals (Table 2).

 

Table 2 – The effect of the combined use of Preductal^® MB and Detralex^®, as well as their combined use with Dopegit^®, on the density of the cell pool in the fetal and maternal parts of the placenta; the diameter of the chorionic villi and the growth-weight index of the fetuses in the correction of ADMA-like preeclampsia (M±m)

Indicator/Index Group	Density of decidual cells, 0,008 mm2	Cell density in fetal part of placenta, 0,008 mm2	Diameter of villi, х10-3mkm	Growth-weight index, mm/g
Int.	118.3±2.14*	235.8±2.75*	32.40±0.41*	14.78±0.22*
L-NAME	23.1±0.33#	80.7±2.57#	17.19±0.26#	15.79±0.23#
L-NAME+ Dopegyt® 86 mg/kg	55.6±0.45*#	98.9±1.73*#	18.78±0.17*#	15.62±0.15#
L-NAME+ Preductal® MR 6 mg/kg	102.7±0.77*#	150.5±1.71*#	29.93±0.17*#	15.36±0.22*#
L-NAME+Detralex® 260 mg/kg	104.8±0.87*#	151.3±1.69*#	29.90±0.16*#	15.31±0.58
L-NAME+ Preductal® MR + Detralex®	132.7±1.92*#y	179.3±1.60*#y	34.89±0.16*#y	14.42±0.63*
L-NAME+ Preductal® MR + Dopegyt®	141.3±2.21*#y	177.6±1.59*#y	31.79±0.14*y	14.86±0.30*
L-NAME+ Detralex®+ Dopegyt®	138.8±2.29*#y	181.0±1.69*#y	26.85±0.15*#y	14.53±0.69*

Note: # – p <0.05 in comparison with the group of intact animals; * – p <0.05 in comparison with the L-NAME group; y – p <0.05 in comparison with the groups in monotherapy.

 

Thus, the combined use of Preductal^® MB and Detralex^®, as well as their combined use with Dopegit^®, led to a significantly positive effect in the correction of ADMA-like preeclampsia compared with the use of the same drugs in monotherapy. This was reflected in a more pronounced decrease in blood pressure. The microcirculation level reached the target values. There was a pronounced positive effect on the size of the fetal and maternal parts of the placenta, the concentration of the cell pool in the maternal and fetal parts of the placenta increased, the diameter of the chorionic villi was restored. There was also a significant improvement in the morphometric parameters of the fetus.

DISCUSSION

The most pronounced positive effects in the correction of morphofunctional disorders arising under the conditions of experimental preeclampsia when using combinations of the studied drugs in comparison with the use of the same drugs in monotherapy, can be explained by the possibility of influencing various points of pathogenesis. This is ensured by the fact that each drug has its own, different from the others, mechanism for the implementation of effects.

The positive effects of trimetazidine, are explained by its capacity to improve the energy metabolism of tissues under ischemic conditions. During the oxygen starvation, under the influence of trimetazidine, the cells activate the oxidation of pyruvate for the synthesis of ATP. This leads to a decrease in oxygen deficiency by 10-12% compared to the oxidation of fatty acids, which makes it possible for the cells to use oxygen more efficiently under the conditions of the oxygen deficiency [33, 34].

In addition, trimetazidine prevents the accumulation of insufficiently oxidized fatty acid products in the mitochondria of cells, and increases the stability of cell membranes due to the inclusion of fatty acids in phospholipids [35, 36]. This leads to a decrease in the severity of the oxidative stress and, as a result of a decrease in the synthesis of reactive oxygen species by mitochondria, its negative effect [37, 38]. In addition, the endothelioprotective properties of trimetazidine due to an increase in the amount of eNOS and the synthesis of nitric oxide as one of the most important factors of vasorelaxation, also explain the effectiveness of its use [39, 40]. The capacity of trimetazidine to reduce the formation of pro-inflammatory cytokines [41, 42], can promote both a decrease in the systemic content of markers of the oxidative stress, and a decrease in the eNOS activity. In addition, the endothelioprotective properties of trimetazidine may lie in its capacity to protect the endothelium from the direct damaging action of free radicals [43] and to reduce the inactivation of nitric oxide by inactivating lipid peroxidation processes [44, 45]. The result of the realization of direct and indirect effects of trimetazidine on the endothelium, is an improvement in endothelium-dependent vascular relaxation not only in this experiment, but also in patients with chronic heart failure (CHF) [46, 47].

The effective use of Detralex is explained by the presence of its several protective properties. One of them is a pronounced anti-inflammatory and antioxidant effect [48- 51]. The anti-inflammatory effect is associated with the capacity of diosmin to reduce the production of pro-inflammatory cytokines: IL-6, IL-1β, TNF-α, etc. [52–54]. In addition, diosmin is can reduce the induced production of NO by inhibiting eNOS [48, 53]. The antioxidant activity includes the capacity of the studied drug to increase the activity of glutathione peroxidase, superoxide dismutase, catalase [53, 55, 56], and to prevent lipid peroxidation with an increase in the activity of the antioxidant system [56, 57]. A decrease in the formation of proinflammatory cytokines and markers of the oxidative stress, leads not only to a decrease in the injury of the placental tissues, but also to the improvement in the endothelial function.

This can be confirmed by the results of the studies by other authors. Endothelioprotective effects are manifested both in the treatment for varicose veins [58, 59] and in the correction of arterial pathology [60–62]. Special attention should be paid to the data on the protective effects of diosmin in ischemia-reperfusion injuries, since this is comparable with the pathogenetic features of preeclampsia [63, 64], especially at the capillary level [65]. The molecular mechanisms by which endothelioprotective effects are realized, include: suppression of the synthesis of proinflammatory humoral factors, a decrease in the production of cell adhesion molecules, a modulating effect on the permeability of the vascular wall, a favorable effect on the ratio of prooxidant and antioxidant factors [66].

In the protective effects of flavonoids, an important role is played by their capacity to improve the drainage function of tissues [60, 67–69]. Since edema increases in ischemia or inflammatory phenomena, this disrupts tissue trophism, and an improvement in the drainage function causes the opposite effect.

Methyldopa is a prodrug by its nature, and belongs to the group of centrally acting antihypertensive drugs. Passing through the blood-brain barrier (BBB), methyldopa turns into alpha-methylnorepinephrine, which depletes the resources of norepinephrine, displacing it from the granules (which brings this drug closer to sympatholytics), excites the central α2-adrenergic receptors of the vasomotor center, causes a decrease in its rhenium, and inhibits the formation of angiotensin [70]. A decreased peripheral vascular tone is an important addition to the mechanisms of the studied drugs’ action. In addition, there is evidence of the endothelioprotective properties of this drug. In an in vitro study, it was established that the incubation of endothelial cell culture with methyldopa, promoted the leveling of the inhibitory TNF-α effect on the endothelial NO-synthase, and also led to an increase in the content of the vascular growth factor VEGF [71]. Herewith, in the absence of TNF-α, this drug had no effect on the eNOS expression [72].

CONCLUSION

The combined use of drugs in the treatment of a lot of diseases, is the most urgent direction. The results of this study witness the fact that the use of trimetazidine and a purified micronized flavonoid fraction in the complex therapy of preeclampsia, is a promising direction necessary to continue the research in.

FUNDING

This study did not receive any financial support from outside organizations.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

AUTHORS’ CONTRIBUTION

O.E. Antsiferova – administration of drugs to the animals, modeling of ADMA-like preeclampsia, functional tests and other studies;

M.P. Teleschenko – administration of drugs to the animals, modeling of ADMA-like preeclampsia, functional tests and other studies;

Yu.M. Tsuverkalova – administration of drugs to the animals, modeling of ADMA-like preeclampsia, carrying out functional tests and other studies;

M.V. Pokrovsky – idea, research planning, consultations on the implementation of individual stages of the experimental work;

V.V. Gureev – article writing, development of the research design;

M.A. Zatolokina – preparing samples for histological examination, morphological description of placenta sections;

A.V. Gureeva – article writing, preparing samples for histological examination, formalization of the bibliography, working with graphic materials.

About the authors

Oksana E. Antsiferova

Belgorod State National Research University

Author for correspondence.
Email: AnciferovaO@ya.ru
ORCID iD: 0000-0002-9456-9569

postgraduate student of the Department of Pharmacology and Clinical Pharmacology

Russian Federation, 85, Pobeda St., Belgorod, 308015

Maria P. Teleschenko

Belgorod State National Research University

Email: marijastreltsowa@yandex.ru
ORCID iD: 0000-0003-0849-4351

postgraduate student of the Department of Pharmacology and Clinical Pharmacology

Russian Federation, 85, Pobeda St., Belgorod, 308015

Yulia M. Tsuverkalova

Belgorod State National Research University

Email: cvd404@mail.ru
ORCID iD: 0000-0001-8489-247X

post-graduate student of the Department of Pharmacology and Clinical Pharmacology

Russian Federation, 85, Pobeda St., Belgorod, 308015

Mikhail V. Pokrovsky

Belgorod State National Research University

Email: mpokrovsky@yandex.ru
ORCID iD: 0000-0002-2761-6249

Doctor of Sciences (Medicine), Professor, Professor of the Department of Pharmacology and Clinical Pharmacology, the Head of the Research Institute of Pharmacology of Living Systems

Russian Federation, 85, Pobeda St., Belgorod, 308015

Vladimir V. Gureev

Belgorod State National Research University

Email: produmen@yandex.ru
ORCID iD: 0000-0003-1433-1225

Doctor of Sciences (Medicine), Associate Professor, Professor of the Department of Pharmacology and Clinical Pharmacology

Russian Federation, 85, Pobeda St., Belgorod, 308015

Maria A. Zatolokina

Kursk State Medical University

Email: marika1212@mail.ru
ORCID iD: 0000-0002-9553-1597

Doctor of Sciences (Medicine), Professor, Professor of the Department of Histology, Cytology, Embryology

Russian Federation, 3, Karl Marx St., Kursk, 305041

Anastasia V. Gureeva

Kursk State Medical University

Email: nastasyi.207@gmail.com
ORCID iD: 0000-0003-1719-7316

4^th year student of Medical Department

Russian Federation, 3, Karl Marx St., Kursk, 305041

References

Supplementary files