Preservation of the physiological function of the mammalian uterus is a critical condition for ensuring the reproductive capacity of the organism, an important part of which is the spontaneous contractile activity of smooth muscle cells (SMCs) of the myometrium. One of the factors that disrupts the function of the myometrium is lipopolysaccharide, a structural component of the wall of gram-negative bacteria. This endotoxin triggers a cascade of inflammatory reactions through Toll-like receptors (TLRs), leading to oxidative stress, mitochondrial dysfunction, and changes in the signalling pathways of regulatory molecules, such as oxytocin. Since oxytocin is a key regulator of labour, studying changes in uterine sensitivity to it under conditions of endotoxemia is critically important for understanding the pathogenesis of preterm labour and developing new therapeutic strategies.
The work aimed to investigate the sensitivity of the rat uterine myometrium to oxytocin in terms of contractile activity and oxytocin receptor expression under conditions of lipopolysaccharide-induced experimental endotoxemia, and to assess the effectiveness of pharmacological approaches to prevent and correct the identified disorders. The work used a comprehensive approach using physiological methods (strain-based measurement of contractions of isolated myometrial strips ex vivo), molecular genetic (Real-time PCR and reverse transcription for gene expression analysis), biochemical (determination of oxidative stress markers, NO synthase activity and hydrogen sulfide content), as well as statistical processing of the results using Excel and Origin 7.0.
In ex vivo experiments, it was shown that the direct effect of LPS on myometrial tissue leads to an increase in the amplitude and duration of spontaneous contractions, but reduces their frequency and disrupts the contraction-relaxation phases. It was first established that systemic endotoxemia (administration of LPS at a dose of 3 mg/kg) causes hypersensitivity of the myometrium to oxytocin. This is manifested in a significant increase in the amplitude of contractions (by 3.1 times) and an increase in basal tone. The explanation for this fact is a significant increase of 4.6 times in the expression of oxytocin receptor mRNA (OXTR). Along with this, an intensification of oxidative stress was detected: the rate of formation of superoxide radicals increased by 2.5 times, and the content of lipid peroxidation products (diene conjugates) by 4 times. An imbalance in the nitric oxide system was detected: inhibition of constitutive NO synthesis against the background of significant activation of inducible NO synthase (iNOS by 3.7 times). An interesting fact is the detection of the body's response to the toxic effects of lipopolysaccharide: an increase in the expression of the antioxidant enzyme catalase, the H2S-synthesizing enzyme cystathionine-γ-lyase, and the Kir6.1 subunit of ATP-sensitive potassium channels. However, these resources were insufficient to restrain the pathogenic effect of lipopolysaccharide. The following approaches were used in the work to prevent and restore myometrial contractility. The introduction of exogenous glutathione (52 mg/kg) before and after lipopolysaccharide injection made it possible to almost completely eliminate the negative effect of endotoxin. Glutathione normalized the expression of the OXTR gene, restored the balance of NO synthases, and stabilized the indicators of contractile activity to control values. The use of the ATP-sensitive potassium channel activator floccalin against the background of already developed endotoxemia demonstrated an effective relaxing effect. Flocalin (10 μmol/L) inhibited oxytocin-induced hyperactivity in rats with experimental endotoxemia, reducing the amplitude of contractions by 122% and the frequency by 47%.
Scientific novelty and practical significance. For the first time, the mechanism by which bacterial endotoxin increases the sensitivity of the uterus to oxytocin through the modulation of gene expression and oxidative stress is comprehensively described. The role of glutathione and ATP-sensitive potassium channels as protective links in the uterine myometrium is proven. The results of the work indicate the prospects for the use of antioxidants and activators of ATP-sensitive potassium channels for the prevention of reproductive disorders caused by infectious agents. The materials of the dissertation can be integrated into courses in physiology, biochemistry and obstetrics, as well as become a basis for the development of new pharmacological drugs.
