Protective effects of Leuzea uniflora (Rhaponticum uniflorum) on the brain mitochondrial function in white rats at hypoxia/reoxygenation

Abstract Protective effects of Leuzea uniflora (L.) Holub (Rhaponticum uniflorum (L.) DC., Asteraceae) on the white rats’ brain mitochondrial function in acute hypobaric hypoxia/reoxygenation were studied. The extract of L. uniflora in the dose of 100 mg/kg was administered to animals for 14 days. The effects of the extract on the brain mitochondria respiration rate as well as on the NADH- and succinate dehydrogenase activities were determined. The extract of L. uniflora increased the oxidative phosphorylation processes coupling in brain mitochondria, namely, it significantly stimulated basal respiration, caused an increase in NADH-oxidase and succinate dehydrogenase complexes activity, increased the ATP content, reduced the lipid peroxidation intensity and improved the antioxidant state. The results obtained indicate the presence of energy-protective and antioxidant activities of L. uniflora, which are due to its ability to reduce the oxidation of biomacromolecules in hypoxia/reoxygenation.


Introduction
The study of molecular-cellular mechanisms of biological processes of regulation in the conditions of stress-factors impact (hypoxia, ischemia, physical exertion, etc.) is an important task of biology and medicine.Particular attention should be given to the problem of the brain cells protection from the impact of chronic or acute hypoxia followed by reoxygenation resulting in imbalance between pro-and antioxidant systems, increased production of active forms of oxygen, intensification of lipid peroxidation of biological structures and, as a consequence, the disturbance of the energy metabolism in the organ cells (Yamada et al. 2020;Pille and Riess 2021).In hypoxia, mitochondria are essential to aggravation of intracellular reactions process due to the increase of O 2 ‾ production, malfunction of electron transport chain and decrease of ATP production; therefore they are target of potential methods of treatment of hypoxic states (Kalogeris et al. 2012;Li et al. 2012).
Leuzea uniflora (L.) Holub (Rhaponticum uniflorum (L.) DC.) is a perennial plant of Asteraceae family, growing in the territory of Siberia and Russian Far East as well as in northern Mongolia.In Tibetan, Chinese and Mongolian traditional medicines, the L. uniflora herb decoction used as a tonic (Olennikov 2022).The plants of Leuzea genus and, in particular, L. uniflora species are reportedly characterized by a high concentration of ecdysteroids: their content in the rhizomes of the latter is 1.19-1.33%and the herb contains from 1.68 to 1.82% of ecdysteroids (Volodina et al. 2012).L. uniflora contains a wide spectrum of phenolic compounds having marked antihypoxic, nootropic, antioxidant and other pharmacological properties (Olennikov 2022).The studies on animals have shown that the extract from the L. uniflora aerial part has antianxiety and anti-depressive effects, stimulates cognitive functions, promotes formation of conditional reflexes and stimulates exploratory activity (Olennikov 2022).Shantanova et al. (2021) have demonstrated that the extracts from the aerial part and roots of L. uniflora have adaptogenic activity in acute hypoxic states of different nature.
The aim of the present study was to estimate the influence of ecdysteroid-containing extract, derived from L. uniflora leaves on the energy metabolism in the white rats brain in the acute hypobaric hypoxia/reoxygenation.
Against the background of acute hypobaric hypoxia/reoxygenation, there was noted the increase in respiration rate of the brain cells mitochondria in the control rats before addition of ADP (state 2) and its 1.3-and 1.5-fold decrease phosphorylation of the ATP (state 3), respectively, as compared to the same indices in the animals of the intact group (Figure S1).In the control group, the increased rate in the state 2 is indicative of the disturbances in the functioning of electron transport chain of mitochondria and uncoupling of oxidative phosphorylation processes due to the decrease in the activity of the respiratory chain enzyme complex: activities of NADH-and succinate-dehydrogenase complexes were 1.4-and 2.0-fold lower, respectively, as compared to the indices in the animals of the intact group.The respiratory control ratio was 2.0-fold decrease in the control group of animals to the intact one (Figure S2).
In the homogenate of control animals' brain, the lactate was significantly accumulated, and lactate/pyruvate ratio was 1:27 vs 1:8 in the intact group of animals.Against the background of hypoxia/reoxygenation, the activity of pyruvate kinase was 60% inhibited as compared to the intact.The abovementioned biochemical changes were followed by the 3.0-fold decrease in the content of ATP as compared to the analogous index in the animals of the intact group that was indicative of the developing energy deficiency in the tested organ (Figure S1).
Thus, the disturbances in the functioning of electron transport chain of brain mitochondrial against the background of hypoxia/reoxygenation correlated with anaerobic glycolysis predominance and inhibition of the Krebs's cycle reactions due to the activation of pro-oxidant processes and increase in oxygen active forms synthesis initiating the oxidation of biomacromolecules and inhibiting the activity of endogenic antioxidant system.In the control group of animals, there was noted the 2.7-fold increase in MDA content, 1.7-and 1.4-fold decrease in the SOD and catalase activities, respectively, as compared to these indices in the intact animals (Table S2).At the same time, the disturbance in the functioning of the glutathione unit was observed: GSH content was decreased by 58% as well as the activity of GP and GR was decreased by 43% and 55%, respectively, as compared to the indices of the intact group.
The preventive introduction of L. uniflora extract to white rats against the background of hypobaric hypoxia with subsequent reoxygenation was followed by the 18% decrease of oxygen uptake rate in the state 2 and its 27% increase when mitochondria were stimulated by ADP addition (state 3) as compared to that of the control animals.Besides, the respiratory control ratio was 1.5-fold increased (Figure S1) as compared to the control index.The acceleration of phosphorylating mitochondrial respiration under the influence of L. uniflora extract was due to the increasing activity of NADH-dehydrogenase and succinate-dehydrogenase complexes by 30% and 37% respectively as compared to the same indices in the control (Figure S2).
The introduction of L. uniflora extract promotes the correlation between glycolytic processes and activation of oxidative phosphorylation in hypoxia/reoxygenation resulting in the decrease of ATP anaerobic resynthesis intensity.In the brain homogenate of experimental animals, there was noted twofold decrease of the lactate concentration, 29% decrease of lactate/pyruvate ratio and 1.4-fold increase of pyruvate kinase activity as compared to the same indices in the control animals (Table S1).
The use of the tested preparation promotes the inhibition of free radical oxidation reactions, increase of the enzymes activity and content of endogen antioxidant system peptides in the white rats brain.Particularly, the MDA concentration was decreased by 49%, SOD and catalase activities were increased by 25% and 15% respectively as compared to the data in control animals.The indices of the glutathione redox system in the experimental group of animals were 1.7-fold increased as compared to the control (Table S2).
Thus, L. uniflora extract influenced the oxidative phosphorylation in mitochondria inducing the efficacy of aerobic glycolysis and oxidative phosphorylation and promoting the antioxidant system activity in the brain structures of white rats in acute hypoxia/reoxygenation.
In the present study, it was found that L. uniflora extract increased activity of enzymes and tripeptide content in the endogenic antioxidant system at hypoxia/reoxygenation that sustained the functional activity of cells in oxidative stress conditions.The tested extract also corrected the intensity of glycolytic processes and reactions of tricarboxylic acid cycle in the brain cells, supported the coupling of oxidation and phosphorylation in mitochondria and increased the respiration rate of mitochondria in the active state and the ATP content.
The revealed pharmacotherapeutic effect of L. uniflora extract is due to the presence of ecdysteroids (20-hydroxyecdysone and ajugasterone C derivatives, rapontisterone R 1 , ecdysone), flavonoids (cosmosiin, apigenin-7-0-glucuronide, vicenin-2, quercitrine etc.), amino acids (alanine, lysine, valine), fatty acids and various structural classes (thiamine, riboflavin, folic, nicotinic and pantothenic acids etc.) (Olennikov 2022).The studies (Cahl ıkov a et al. 2011;Pan et al. 2016) have shown that ecdysterone has a neuroprotective effect in neurodegenerative diseases resulting in preservation of mitochondria functional activity and decrease of apoptotic processes intensity.In the authors' opinion, the given pharmacotherapeutic effect of ecdysterone is due to its capability to decrease the MDA level and to increase the activity of the antioxidant enzyme SOD in the brain structures.The plant polyphenolic compounds are reportedly capable to stimulate the energy metabolism in the cells due to the direct interaction with the breathing chain, particularly, with NADH-dehydrogenase and due to the decrease of free lipid oxidation processes (Moini et al. 1999;Gibellini et al. 2015).

Conclusions
It has been established that the course introduction of ecdysteroid-containing extract of L. uniflora has energy protective and antioxidant effects in acute hypobaric hypoxia/reoxygenation.The extract corrects the functional activity of electron transport chain of the brain mitochondria, efficiency of glycolysis and citric acid cycle reactions.The revealed effect of the plant remedy is provided by its capability to decrease the manifestation of biomacromolecules peroxidation due to the antioxidant activity of chemical substances complex in its composition.The data obtained are very important for explanation of L. uniflora antihypoxic effect mechanism.