New features of Skulachev ions

Antioxidants against pyelonephritis

Fyodor Galkin, "Biomolecule"More than half a century ago, scientists saw a connection between the process of cellular respiration and aging.

Today it is known as a fact that when organic matter is oxidized, potentially dangerous metabolites are released in the cell, which play an important role in the development of a number of diseases. Mitochondrially targeted antioxidants, also known as Skulachev ions, provide new opportunities in the fight against pathologies such as heart attack and stroke. And in 2013, the laboratory of the structure and functions of mitochondria under the direction of D. B. Zorov from the Research Institute of the Russian Academy of Medical Sciences. Belozersky showed that Skulachev ions can also help patients with acute pyelonephritis.

Antibiotics, or aggressive therapyThe principle of treating bacterial infections has not changed since the discovery of penicillin in the first half of the last century: the patient uses antibiotics in one form or another in the hope that the bacteria will die faster than they will have resistance to the prescribed medication.

At the same time, antibiotics can cause a number of side effects in the patient, such as dysbiosis and digestive problems, and some categories of patients (children, pregnant women, allergies) antibiotics may be generally contraindicated.

Here it is appropriate to draw a parallel between the fight against bacterial infection and martial arts. The philosophy of Aikido, the world–famous Japanese martial art, is based on the idea of opposing force not by force, but by gentleness. Almost any philosophy of combat assumes that it is logical to resist the enemy's power with your own aggression. In Aikido, this method of fighting is considered a dead end: it is necessary to moderate your ardor, because the multiplication of aggression leads only to a violation of harmony, which, in turn, leads to defeat.

Oddly enough, the idea of retaining one's own aggression in battle has also found application in cell biology, and in particular in the development of a new approach to the treatment of acute infectious diseases. This approach is based on the control of cellular respiration in the tissues of the patient, whose role in the fight against infection is extremely important.

The breath of death is likeCellular respiration is the production of energy by controlled oxidation of nutrients in structures called mitochondria.

Sometimes, during respiration, oxygen, for reasons not yet completely clear, can form a number of short−lived, but extremely active compounds: superoxide O2^•-, hydroxyl radical OH^•, hydrogen peroxide H ₂ O ₂. These substances, called reactive oxygen species (ROS), seem to burn out the cell from the inside and can even lead to its death [1]. There are seven sources of ROS in mitochondria, and two of them are arranged in such a way that the formed ROS easily leave the place of synthesis and, like fire, spread to other cellular structures [2]. But even that part of the ROS that remains inside the mitochondria is capable of destroying mitochondrial DNA and other parts of this organelle necessary to control cellular respiration.

In the 50s of the last century, the free radical theory of aging originated under the authorship of Denham Harman. By 1980, it had developed into the mitochondrial theory of aging, according to which it is the ROS produced by these structures that are the cause of degenerative age-related changes [3]. Nowadays, although a correlation between the level of ROS and age has been proven, the accumulated data do not allow us to conclude whether they are the cause or only a consequence of aging [4].

A spoonful of honey in a barrel of tarSo AFC is bad?

Not quite like that, not even like that at all. ROS are involved in many signaling pathways in the cell [5], some of which are aimed at increasing resistance to stressful situations (starvation, hypoxia).

Among other things, peroxide formed by mitochondria or the enzyme NADPH oxidase (NOX, NADPH-Oxydase) [6] is used by immune cells as a weapon in the fight against infection. Such conscious production of ROS in bacterial infections is carried out by a subtype of white blood cells – neutrophils – as well as macrophages. Peroxide can either be produced into the intracellular reservoir – the phagosome that has captured the bacteria – or it can be released into the intercellular space – outwards. In any case, the bacteria cannot withstand oxidative stress, their number decreases, and the infection disappears. At least that's how it should be, but unfortunately, in practice you rarely see such an ideal course of events.

As mentioned above, high concentrations of ROS can cause damage not only to bacteria, but also to the patient's cells. Generally speaking, there are many signaling pathways intertwined in mitochondria, some of which lead to cell survival, and others to its apoptosis, death. And in these ways, the level of ROS often plays a major role. Here we return to the topic of resistance by the force of aggression, which was touched upon at the very beginning of the article. Excessive formation of ROS as a protective reaction to infection often disrupts the balance between life signals and death signals. Learning how to manage them is a cherished dream of scientists.

Plant antioxidants on the guard of healthIn mitochondria, the energy obtained during the oxidation of nutrients is used to create an electrochemical potential difference of hydrogen ions on the inner membrane of mitochondria.

This potential difference, or voltage, subsequently allows mitochondrial enzymes to store energy in the form of adenosine triphosphate (ATP) molecules.

The formation of ROS by the cell also directly depends on the electrochemical characteristics of mitochondria. Thus, reducing the voltage on the inner membranes of mitochondria by only 10% reduces the formation of ROS tenfold [7]! This is a really impressive figure. There is only one question: what substance is able to accumulate and reduce the generation of ROS in mitochondria?

In the 1970s, academician V. P. Skulachev, now dean of the Faculty of Bioengineering and Bioinformatics of Moscow State University, showed that lipophilic cations with high selectivity penetrate into mitochondria, driven by a potential difference (there is a negative charge inside the mitochondria, and the cations themselves are positively charged). Now such substances are called Skulachev ions at the suggestion of the American biochemist David Green in 1974. By splicing these cations with antioxidants, you can get a powerful tool in the fight against oxidative stress.

In 2005-2006, a project began to search for a new type of drugs based on Skulachev ions, which was registered as the Mitotech company. The first attempts to create such a biotechnological drug were not entirely successful. However, promising results on domestic mitochondrial-targeted antioxidants (MAA) appeared already in 2007: Skulachev ions slow down the development of age-related diseases, reduce the lesion area in heart attacks and strokes, and help in the treatment of certain forms of cancer [8, 9]. Nevertheless, a year later, due to the global economic crisis, the project lost its investor – Oleg Deripaska. But soon Mitotech found new investors: Rostock and RUSNANO. Now research is continuing, and even a full–fledged cure for a whole complex of eye diseases has been created - Visomitin. The plans are to create a systemic drug and enter the global drug market.

Now one of the most promising substances of this class is SkQR1 (see Fig. 1), an antioxidant in which is represented by plastoquinone, an electron carrier from plant chloroplasts. This substance effectively reduces the electrochemical voltage even in very small concentrations. This phenomenon is explained by the fact that SkQR1, due to interaction with mitochondrial proteins, returns to its original form, ready to take on the impact of ROS.

Figure 1. Structure of the mitochondrially targeted antioxidant SkQR1: plastoquinone neutralizes reactive oxygen species, and the lipophilic cation ensures that this drug enters the place of their formation.The last squeak

In July 2013, the leading American scientific publication Proceedings of the National Academy of Sciences published an extremely interesting work "The protective effect of mitochondrially targeted antioxidants in acute bacterial infection" [10], which describes the result of using MAA as the only medicine for acute bacterial diseases on the example of pyelonephritis.

Further on about this work carried out on the basis of the Scientific Research Institute of the FKHB. Belozersky, described in more detail.

Pyelonephritis is an inflammation of the kidneys and upper urinary tract caused by the proliferation of bacteria in them. With this disease, pockets with pus are formed, the kidneys themselves increase in size due to poor outflow of urine and blood. Even after successful treatment, multiple scars remain in them. In the worst case, pyelonephritis can lead to blood poisoning and sepsis – the spread of infection throughout the body. Moreover, acute pyelonephritis develops rapidly (within a few days), which is why it is so important to diagnose it quickly and start treating it.

Traditionally, antibiotics and anti-inflammatory drugs are used for these purposes. However, neither is able to reduce the oxidative stress that the patient's tissues face when releasing ROS by neutrophils. Moreover, in some cases, antibiotics may be ineffective or simply contraindicated to the patient. MAA is a completely different class of substances, on which great hopes are pinned.

Returning to the parallel with Aikido, we can say that the strategy of "softness" – moderate regulation of oxidative stress and normalization of the inflammatory and antibacterial response of the body itself – is capable of bringing greater success in the fight against infectious diseases in the long term. Whereas "strength" – that is, the unrestrained build–up of the arsenal of antibiotics - increasingly turns into defeat, generating more and more strains of resistant bacteria and causing serious side effects in patients.

Figure 2. Mitochondria under stressful conditions emit excessive amounts of reactive oxygen species (ROS), which damage healthy tissues. Mitochondrial antioxidants are able to neutralize their negative effect.The effectiveness of MAA was tested by a variety of parameters on two models: in vivo (in rats) and in vitro ("in vitro").

In the first case, a culture of bacteria diluted on a nutrient medium was injected into the bladder of rats, and four injections of SkQR1 were given to some experimental subjects for two days. By the end of a certain period (two days or a week), their kidney tissues were taken for analysis. In the second case, the culture of renal cells (RTC – Renal Tubular Cells) was cultured for two days together with leukocytes isolated from the blood on a nutrient medium containing bacterial endotoxin lipopolysaccharide (LPS) or the shells of the bacteria themselves. These additives are recognized by immune cells as living pathogens and trigger the corresponding reactions (the Nobel Prize in Physiology and Medicine was awarded in 2011 for the discovery of mechanisms for the recognition of foreign agents by immune cells [11, 12]). Various MAAS were also added to the nutrient medium.

Effectiveness of SkQR1 in the fight against pyelonephritisIt is proved that the activation of leukocytes leads to a five-fold jump in the level of ROS and a significant increase in indirect indicators of oxidative stress.

However, the mere presence of contact between non-activated leukocytes and RTC also leads to an increase in ROS, but two times less than activated ones.

In addition, the contact of RTC and leukocytes leads to an increase in TNFalfa, a protein that provokes apoptosis and is a key element in the process of inflammation. To test the relationship between TNFalfa and ROS, the RTC culture was grown in an environment rich in this protein. The level of reactive oxygen species increased in direct proportion to the content of TNFalfa, which was released by the culture cells themselves (mainly leukocytes). The effect of TNFalfa on the kidney can provoke an avalanche-like process in which the released ROS create favorable conditions for the formation of new ROS molecules [13].

In addition to TNFalfa, many other proteins are involved in the processes of inflammation, including Bcl-2 and pGSK-3beta. These proteins are responsible for cell survival and its resistance to ROS. Usually, with pyelonephritis, the content of these proteins decreases, but the MAA to some extent restores their amount.

Thus, MAA – and SkQR1 in particular – with timely application directly reduce the concentration of ROS, restrain their avalanche-like release and ensure the predominance of survival signals (see Fig. 3). All this is achieved only by restoring the normal functioning of mitochondria in RTC and leukocytes.

Figure 3. SkQR1 reduces the level of oxidative stress in inflamed kidney cells. Inflammation in the kidneys was caused by the addition of leukocytes activated by bacterial lysate or lipopolysaccharide (a component of bacterial cell walls) to the cell culture. The indicator of inflammation is the level of ROS, which drops almost to the level of healthy kidneys in the presence of SkQR1. Designations: RTC – healthy kidney cells. RTC+Lk+LPS – grown together RTC and leukocytes in the presence of LPS. RTC+Lk+Bac – grown together RTC and leukocytes in the presence of bacterial shells. RTC+Lk+Bac+SkQR1 – the same, with the addition of SkQR1. Picture from [10].

How effective is SkQR1, not in an in vitro model far from life, but in the treatment of live rats? First of all, it should be said that this drug, not being an antibiotic, lowered the level of pathogenic microorganisms in the urine of rats. But what is much more exciting: the mortality rate of rats injected with SkQR1 has sharply decreased (Fig. 4)!

Figure 4. The use of SkQR1 significantly reduces the mortality of rats with pyelonephritis. There were twelve rats in all three groups. Picture from [10].Such results make one think: mitochondria appeared in cells more than a billion years ago and in the course of evolution should have turned into an infallible mechanism.

Nevertheless, the studies conducted with MAA indicate monstrous malfunctions in this seemingly ideal element of the cellular structure. Did the scientists manage to correct the mistakes of Mother nature, or does their myopia prevent them from noticing some blemish in their calculations?

According to the theory of symbiogenesis, mitochondria originated from bacteria that began to live inside other unicellular organisms. Maybe such inconsistency of actions is a remnant of the autonomy of bacterial ancestors?

Be that as it may, preparations based on Skulachev ions provide hope that in the near future humanity will be ready to oppose bacterial infections and cardiovascular diseases with something more effective than what they have now.

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