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Volum 10, Issue 3
September 2023
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Article contents

opened journal
Volum 10, Issue 3
September 2023
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Abstract

Introduction

Insects, throughout evolution, have developed a huge arsenal of active compounds, which they use to defend themselves against enemies and diseases, at the same time in recent years insects have shown great interest as a source of food rich in biologically active substances. Research in recent decades has shown that insects produce a variety of proteins and peptides with antibacterial, antifungal, antiviral, immunomodulatory, anti-inflammatory, antioxidant, antitumor, hepatoprotective, antithrombotic, antihypertensive and detoxifying activity during or after contact with the microbial agent or unfavourable factor.  

Materials and methods

The anti-inflammatory effect of imuheptin and imupurin was investigated in a rat model of subacute inflammation induced by subcutaneous implantation of felt discs. The intensity of the exudative and proliferative phase of inflammation, cytokine profile (TNFalpha, IL-6, IL-10), ceruloplasmin and antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and glutathione-S-transferase) in the serum of rats were evaluated.

Results

Imuheptin and imupurin reduced the level of pro-inflammatory cytokines (TNF-alpha, IL-6) and increased that of the anti-inflammatory cytokine (IL-10), as well as ceruloplasmin, glutathione reductase and glutathione peroxidase in subacute inflammation. Additionally, imupurin significantly increased the level of catalase and imuheptin that of glutathione-S-transferase.

Conclusions

Imuheptin and imupurin determined a moderate effect of inhibiting the exudative and proliferative processes, compared to the reference preparation - dexamethasone, but with a favourable effect on the cytokine profile, decreasing the level of pro-inflammatory cytokines (TNF-alpha, IL-6) and increasing the level the anti-inflammatory one (IL-10), as well as the modulation of antioxidant enzyme activity.

Key Messages

What is not yet known about the issue addressed in the submitted manuscript

At the moment there is limited data on the anti-inflammatory properties and the mechanism of achieving the anti-inflammatory potential of the entomological preparations – imuheptin and imupurin.

The research hypothesis

Preparations of entomological origin (imuheptin and imupurin) through the content of biologically active substances will improve the evolution of the inflammatory process and restore the imbalance of the pro- and antioxidant systems in rat model of felt-pellet-induced granuloma formation.

The novelty added by the manuscript to the already published scientific literature

The ability of imuheptin and imupurin to reduce the level of pro-inflammatory cytokines and increase the level of IL-10 with anti-inflammatory functions, as well as to modulate the activity of antioxidant enzymes in subacute inflammation was revealed.

Introduction

The current arsenal of preparations for the treatment of inflammatory processes consists of non-steroidal, steroidal, and disease-modifying antirheumatic drugs (DMARDs) with good efficacy, but safety issues require the research of new substances with anti-inflammatory properties, possibly with different mechanisms and increased safety profiles. Currently, there is a varied and documented basis of methodological recommendations for the in vitro and in vivo study of the anti-inflammatory properties of new substances that allow determining the influence of the investigated substances on inflammatory processes with the elucidation of the mechanisms and peculiarities of action [1-4].

Insects have become an object of research due to their ability to survive in adverse environmental conditions, including infectious factors and those that produce inflammatory processes. Bioactive compounds such as phenols, flavonoids, terpenes, saponins, sugars, alkaloids, glycosides and fatty acids, identified in a wide variety of insects, have demonstrated biological properties including antioxidant, anti-inflammatory, antiproliferative, cytotoxic, analgesic, immunomodulatory, antidiabetic, cardioprotective, antihypertensive, antimicrobial properties. Analysis of literature data demonstrated that a number of extracts, peptides, and synthetic analogues exhibite anti-inflammatory properties [5-8]. 

Previous preclinical and clinical studies of preparations of entomological origin obtained from Lymantria dispar at different stages of development (entoheptin, imuheptin, imupurin, adenoprosine) have shown hepatoprotective, immunomodulatory, anti-inflammatory properties [9, 10]. The purpose of the study was to determine the influence of preparations of entomological origin (imuheptin and imupurin) on the exudative and proliferative processes of subacute inflammation.    

Materials and methods

This experimental study was conducted in the Department of Pharmacology and Clinical Pharmacology and the Biochemistry Scientific Laboratory of Nicolae Testemiţanu State University of Medicine and Pharmacy. Albino rats were purchased from the Animal House of Nicolae Testemiţanu State University of Medicine and Pharmacy. The animals were allowed standard access to food and water. Rats were housed at room temperature under conditions of 12 h of light and 12 h of dark. The experimental procedures involving rats were approved by the Research Ethics Committee of Nicolae Testemitanu State University of Medicine and Pharmacy, Minutes No. 78 from 22.06.2015. The entomological preparations obtained from insects of the order Lepidoptera, the genus Lymantria at the pupal stage (imupurin) and at the egg and pupae stage (imuheptin) were produced by Arena Group SA, Romania. Dexamethasone was purchased from KRKA d.d., Slovenia.

Adult male Wistar rats (180-330 g) were used for the study. They were randomly divided into the following groups: intact (n=8) – no manipulations, only saline (0.9% NaCl) solution intraperitoneally was administered; control (n=6) – felt pellets were implanted, saline (0.9% NaCl) solution intraperitoneally was administered; standard (n=9) - felt pellets were implanted, the steroid anti-inflammatory drug dexamethasone was administered; treatment 1 (n=7) - imuheptin was administered; treatment 2 (n=9) – imupurin was administered. In all animals, except intact group, subacute inflammation was induced by implanting felt pellets, weighing 26±1 mg, in the groin region of the animal's body on the right and left sides (1st day). The intervention was performed in aseptic conditions, under general anaesthesia with sodium thiopental (50 mg/kg intraperitoneally). Substances of entomological origin (imuheptin, imupurin) were administered daily internally for seven days, in doses of 500 mg/kg, dexamethasone (the reference preparation) – in a dose of 2.5 mg/kg intraperitoneally. On the 8th day, under general anaesthesia, the pellets were extracted together with the formed granulation tissue, weighed wet, and then dried at 60°C to constant weight.

The degree of the exudative reaction was assessed calculating the difference between the weight of the wet and the dry granuloma, and the percentage of inhibition of the exudative phase. Proliferative reaction was evaluated calculating the difference between the weight of the dry granuloma formed and the initial weight of the pellet, as well as the percentage of inhibition of the proliferative phase. To calculate the percentage of inhibition of the exudative and proliferative phases, the formula was used:

where: Pi – the percentage of inhibition; 

Mt – wet/dry weight of granuloma in treated group; 

Mm – wet/dry weight of granuloma in control group [1, 3]. 

The level of TNF-α, IL-6 and IL-10 was determined in the serum of rats by the ELISA method, using Invitrogen kits, ThermoFisher Scientific Inc, USA. The activity of catalase, superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPO), glutathione-S-transferase (GST) and ceruloplasmin (CP) was determined according to the methods described by Gudumac V. et al. [11, 12]. 

Statistical analysis: The results were statistically processed using the functions of the computer program SPSS (version 25.0) and the basic indicators of descriptive statistics were determined – mean and standard deviation. The differences between the groups were analyzed using One –Way ANOVA, followed by post hoc Bonferonni test. The significance threshold set was for the 95% confidence interval. 

Results

The data presented in table 1 showed that the initial weight of subcutaneously implanted felt pellets was almost identical in all groups. After extracting the pellets with the granuloma formed around them, it was found that their weight increased significantly in all groups, which proves the development of the inflammatory process. Thus, the wet granuloma weight in the control group was 315.1±32.0 mg (increased 11.2 times), in the dexamethasone group 196.1±10.0 mg (increased 7 times), in the imuheptin group 273.1±24.2 mg (increased 9.7 times), and with imupurin 267.5±34.4 mg (increased 9.5 times). Thus, we can state that the implantation of the felt pellets caused a marked exudative inflammatory reaction. In order to assess the influence of the investigated preparations on the exudative phase, the percentage of inhibition was calculated (tab. 1). Dexamethasone caused an inhibition of the exudative process by 38%, imuheptin by 13%, and imupurin by 15%.  The analysis of the weight of the dry granuloma revealed that in the control group, it was 90.4±12.0 mg or 3.2 times higher than the initial weight of implanted pellet, which reveals the presence of a marked proliferative process. In the group treated with dexamethasone, the dry granuloma weight was 49.9±6.1mg or 1.8 times higher than the initial weight, but significantly reduced compared to the control group. In the group treated with imuheptin, the weight of the dry granuloma was 73.4±10.7 mg or 2.6 times higher than the initial one, and in the group with imupurin 73.0±16.7 mg or 2.6 times higher. The intensity of the proliferative process was analyzed, calculating the percentage of inhibition, which for dexamethasone was 45%, for imuheptin 19% and for imupurin 20%. These data confirm that dexamethasone essentially reduced the proliferative inflammation, and preparations of entomological origin showed a moderate effect. Based on the results obtained, we can conclude that dexamethasone effectively inhibited the exudative and proliferative phases in subacute inflammation, and the entomological preparations mainly decreased the proliferative phase. 

Table 1. The effects of imuheptin and imupurin on the exudative and proliferative phase of subacute inflammation in rats

Treatment

Initial weight of pellets

Wet

weight

 

The percentage of inhibition of exudative phase

Dry

weight

 

The percentage of inhibition of  proliferative

Control, saline solution

28.1±0.4

315.1±32.0

 

90.4±12.0

 

Dexamethasone 2.5mg/kg

28.1±0.9

196.1±10.0*

38%

49.9±6.4*

45%

  1. Imuheptin 500 mg/kg

28.1±1.0

273.1±24.2

13%

73.4±10.7

19%

Imupurin 500 mg/kg

28.2±0.9

267.5±34.4

15%

73.0±16.7

20%

Note: Values expressed as mean ± SD, SD – standard deviation; the results were analyzed using Oneway ANOVA followed by Bonferroni multiple comparison test; *- P < 0.05 was used to indicate statistical significance when compared to control

 

At the same time, in the control group with subacute inflammation, the level of TNF-alpha increased compared to the intact group (48.68±10.77 pg/ml) and constituted - 72.67±20.19 pg/ml (P1-2<0.05); increased IL-6 level was observed - 37.57±1.69 pg/ml (P1-2<0.05) compared to the control group (33.75±0.57 pg/ml); as well as the decrease in IL-10 content - 15.28±2.36 pg/ml (P1-2<0.05) compared to the control group, where the level was 32.35±13.39 pg/ml (table 2). Dexamethasone caused a significant decrease in TNF-alpha level and IL-6 level, also slightly increased the IL-10 level. Preparations of entomological origin decreased the level of TNF-alpha and that of IL-6. Imuheptin, and especially imupurin, increased the content of IL-10, a cytokine with anti-inflammatory properties, compared to the control group (table 2). Thus, the steroid anti-inflammatory mainly decreased the level of pro-inflammatory cytokines (TNF-alpha, IL-6), and preparations of entomological origin restored the ratio between pro-inflammatory (TNF-alpha, IL-6) and anti-inflammatory (IL-10) cytokines.

Table 2. The influence of imuheptin and imupurin on cytokines and ceruloplasmin level in rats serum with felt-pellets induced granuloma

Treatment

TNF - alpha,

pg/ml

IL-6,

pg /ml

IL-10,

pg /ml

CP,

mg/L

Intact

(no pellets were implanted)

48.7±10.8

33.7±0.6

32.3±13.4

470.4±87.0

  1. Control, saline solution

72.7±20.2 §§

37.6±1.7§§

15.3±2.4 §§

390.7±78.9

  1. Dexamethasone 2.5 mg/kg

43.3±6.5*

34.2±1.4*

21.2±5.6§§

323.8±42.5§§

  1. Imuheptin 500 mg/kg

46.1±10.9*

35.1±1.5*

23.3±6.6

539.7±68.5*/**

Imupurin 500 mg/kg

46.2±12.9*

34.1±1.0*

27.4±4.2*

501.5±66.4*/**

Note: Values expressed as mean±SD, SD – standard deviation; the results were analyzed using One Way ANOVA followed by Bonferroni multiple comparison test; TNF alpha - tumour necrosis factor alpha ; IL – interleukin; CP - ceruloplasmin; §§ - P<0.05 was used to indicate statistical significance when compared to intact group; * - P<0.05 was used to indicate statistical significance when compared to control group; ** - P<0.05 was used to indicate statistical significance when compared to dexamethasone group.

In felt-pellets-induced granuloma, a decrease in the level of ceruloplasmin was found - from 470.41±87.0 in the intact group to 390.68±78.96 mg/L (P>0.05) in the control group. Dexamethasone caused an even more pronounced reduction in ceruloplasmin levels. Imuheptin and imupurin significantly increased the content of ceruloplasmin compared to the control group with subacute inflammation (table 2). Withal, a tendency to decrease the activity of catalase, SOD and GPO and to increase GR was found in the control group, without essential changes in GST. Dexamethasone virtually restored the activity of catalase and SOD, the level of these enzymes being comparable to that of the intact group, and increased the activity of enzymes of the glutathione system (GR, GPO, GST). Imuheptin, administered to animals with inflammation, reduced SOD activity and restored catalase activity compared with the control group, and increased GR, GPO and GST activity. Compared to the control group, imupurin increased the activity of catalase and decreased that of SOD and GST, simultaneously increasing GR and GPO levels (table 3).

Discussion 

The screening of the anti-inflammatory properties in the previous research, namely formaldehyde-induced paw oedema allowed us to find that the drugs of entomological origin (entoheptin, imuheptin, imupurin) do not prevent inflammation but had an anti-inflammatory activity comparable to that of diclofenac. The comparative analysis between the anti-inflammatory potential of entoheptin, imuheptin, imupurin and diclofenac revealed that entoheptin possesses the strongest anti-inflammatory activity, achieving complete healing in 48 hours, followed by diclofenac and imuheptin. Imupurin showed the weakest anti-inflammatory action, but it was more intense than in the group of untreated animals [9, 10].

Table 3. The influence of imuheptin and imupurin on antioxidant enzymes in rats serum with felt-pellets induced granuloma

Treatment

Catalase

µM/s.L

SOD

c/u

GR,

nM/s.L

GPO,

nM/s.L

GST,

nM/s.L

Intact (no pellets were implanted)

19.7±1.7

918.1±45.7

64.8±18.9

430.8±90.3

24.5±12.9

Control, saline solution

15.9±3.2

905.3±49.2

80.4±21.9

380.3±42.3

24.8±10.3

  1. Dexamethasone 2.5 mg/kg

18.3±2.4

944.9±79.3

99.2±33.5

531.7±116.8

50.2±13.4§§/*

Imuheptin 500 mg/kg

20.4±2.9

865.0±96.6

127.6±21.7§§

551.8±96.8*

34.3±8.2

Imupurin 500 mg/kg

31.8±9.5§§/*/**

888.9±135.9

150.8±65.7§§/*

535.5±100.6*

21.1±9.5**

Note: Values expressed as mean±SD, SD – standard deviation; the results were analyzed using One Way ANOVA followed by Bonferroni multiple comparison test; SOD - superoxide dismutase; GR - glutathione reductase; GPO - glutathione peroxidase; GST - glutathione-S-transferase; §§ - P<0.05 was used to indicate statistical significance when compared to intact group; * - P<0.05 was used to indicate statistical significance when compared to control group; ** - P<0.05 was used to indicate statistical significance when compared to dexamethasone group.

 

Insects include the largest number of species and play an important role in the terrestrial ecosystem and have been considered a useful natural resource as food, especially due to their protein and fatty acids. Some studies have shown that insects not only have a high protein content, but micronutrients and bioactive peptides with various pharmacological effects, including anti-inflammatory, antioxidant, antimicrobial and antitumor activity. Wasps (Vespa orientalis) have a major protein content, and the aqueous extract of Vespa affinis has demonstrated antioxidant effects by activating the antioxidant enzymes glutathione-S-transferase (GST) and catalase (CAT) [13-16].

Oxidative and inflammatory processes are closely related, so antioxidants annihilate free radicals that damage cells and lead to inflammation. Several studies have shown that antioxidant and anti-inflammatory peptides have protective effects against reactive oxygen species (ROS) and can contribute to a significant reduction in oxidative stress levels. Tenebrio molitor, Schistocerca gregaria and Grylodes sigillatus have been shown to be a rich source of bioactive peptides with antioxidant and anti-inflammatory properties, which have shown high antiradical activity and an ability to chelate iron ions and inhibit the activity of lipoxygenase and cyclooxygenase-2 [16, 17].

Subacute and chronic inflammation is a response to prolonged stimulation of proinflammatory factors on tissues and is characterized by leukocyte infiltration at the site of inflammation, fibrosis, and granuloma formation. The mechanism of chronic inflammation is attributed, in part, to the release of ROS from activated neutrophils and macrophages, excessive cytokine production, dysregulation of cell signalling, and loss of barrier function. This overproduction causes peroxidation of membrane lipids, which leads to tissue damage by damaging macromolecules. ROS cause or extend inflammation by stimulating the release of cytokines (IL-1beta, TNF-alpha, INF-alpha), which stimulate the recruitment of additional neutrophils and macrophages [18].

Subcutaneous implantation of felt pellets causes the formation of granulomatous tissue. This granulomatous tissue is due to the accumulation of macrophages, neutrophils and lymphocytes around the foreign particles, followed by the proliferation of fibroblast cells. The implanted felt pellets stimulate the immune system to produce interleukins and antibodies that stimulate the proliferation of lymphocytes and the accumulation of cells around them. Initially, exudative processes develop through the transudation of liquid and a marked increase in the weight of wet felt pellets. Steroidal and nonsteroidal anti-inflammatory drugs are shown to reduce granuloma size and transudate by inhibiting the production of proinflammatory mediators (inflammatory cytokines, leukotrienes, and prostaglandins), inhibiting cell (leukocyte) infiltration, and preventing fibroblast proliferation and collagen fibre production and mucopolysaccharide synthesis. A similar effect was demonstrated by dexamethasone in our study. Imupurin showed a lower ability, compared to dexamethasone, to reduce the exudative and proliferative processes. Possibly, unlike the steroid anti-inflammatory, the preparation of entomological origin develops a slower effect due to its immunotropic properties on cellular immunity - modulation of T-lymphocytes [18, 19].

Implantation of felt discs causes an exudative and proliferative reaction and an increase in the level of pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6, products that characterize the function of macrophages (activation, infiltration). The administration of indomethacin, a non-steroidal anti-inflammatory, causes a decrease in the mass of the granuloma and the level of IL-6, with an increase in TNF-alpha, without changing that of IL-1beta [20].

When foreign bodies, such as the implantation of felt discs, penetrate the skin, the production of nitric oxide occurs under the action of nitric oxide synthase. Subsequently, the cascade of proinflammatory mediators and cytokines is activated which includes cyclooxygenase 2, interleukins IL-1β and IL-6, and TNF-alpha. These pro-inflammatory mediators and cytokines cause the activation of the classical inflammatory pathway, nuclear factor NF-κB and mitogen-activated protein kinase (MAPK) triggering an uncontrolled inflammatory response. The use of wasp venom suppressed the production of nitric oxide and reduced the mRNA expression of IL-1β, IL-6 and TNF-α [21].

Glucocorticoids (GCs) play an important role in the regulation of the inflammatory and immune response, acting on most types of immune cells. Glucocorticoids can: regulate the phenotype, survival and functions of monocytes and macrophages; exhibits anti-apoptotic effects that promote the survival of anti-inflammatory macrophages; improve the phagocytic activity of macrophages; stimulates the clearance of neutrophils; inhibits the release of pro-inflammatory mediators (cytokines, chemokines, etc.) and reactive oxygen species; regulate the maturation, survival and migration to lymph nodes and the functionality of dendritic cells. Glucocorticoids inhibit transcription factors that control the synthesis of proinflammatory mediators and cells, including macrophages, eosinophils, lymphocytes, mast cells, and dendritic cells. Another important effect is the inhibition of phospholipase A2, responsible for the production of pro-inflammatory mediators. Glucocorticoids inhibit the genes responsible for the expression of cyclooxygenase-2, iNOS and proinflammatory cytokines. Concomitantly, GCs produce an increase in lipocortin and annexin A1, with subsequent reduction in the synthesis of prostaglandins and leukotrienes [22-24].

The plasma level of ceruloplasmin, considered an acute-phase inflammatory plasma protein, produced predominantly by hepatocytes and activated monocytes and macrophages, increases in response to inflammation, trauma, or infection. Ceruloplasmin production by myeloid cells is induced by interferon-γ (IFN-γ) and tumour necrosis factor-alpha (TNFα). The ferroxidase activity of ceruloplasmin inhibits the ferrous ion-mediated production of reactive oxygen species with the manifestation of antioxidant activity. Ceruloplasmin also exhibits ferroxidase-dependent bactericidal activity. The increase in the plasma level of ceruloplasmin during the acute phase reaction suggests a possible anti-inflammatory function of the antioxidant, bactericidal and ferroxidase activity. Ceruloplasmin, due to its antioxidant activity, prevents the carbonylation of proteins by reactive oxygen species in inflammatory diseases. The anti-inflammatory action of ceruloplasmin, most likely, is determined by its synthesis by infiltrated macrophages at the site of inflammation and, less so, by the modulation of the T-cell response. Thus, the prevention of oxidation and tissue damage can be considered the basic mechanism of ceruloplasmin, generated by macrophages recruited to the site of inflammation [25].

Most organisms use aerobic cellular respiration to produce energy for their functioning, but this process is also accompanied by side effects caused by metabolic products in the form of free radicals. Living organisms use exogenous and endogenous antioxidants to defend themselves against the harmful effects of free radicals, and studies on the antioxidant activity of substances of plant, animal, entomological, or biological origin have captured the interest of researchers for many years. [26]. Antioxidants are compounds capable of counteracting the effects of oxidative processes in cells or exogenous systems, reacting in particular with reactive oxygen or nitrogen species or with other free radicals or unstable molecules generated during normal metabolic oxidative reactions. Antioxidant systems include enzymes (SOD, catalase, GP, GR, GST) and non-enzymatic substrates (glutathione, coenzyme Q, ascorbic acid, retinols, tocopherols, flavonoids, carotenoids, etc.). Antioxidants are found in products of vegetable, animal, or entomological origin, in food supplements. Antioxidant capacity is the general ability of organisms or compounds to interact with free radicals and prevent their harmful effect. The antioxidant effect includes the protection of cells and cellular structures against the effect of free radicals, especially oxygen and nitrogen [26-28].

The enzymes of glutathione metabolism - glutathione reductase (GR), glutathione peroxidase (GPO) and glutathione-S-transferase (GST) constitute a group of antioxidants that ensure the protection of cells against ROS and RNS action, also against lipid peroxidation products. The main role in the degradation of hydroperoxides belongs to the GPO/GR enzyme system. GR has a variable distribution in organs and intracellular organelles and ensures the maintenance of the optimal level of glutathione (GSH) by reducing oxidized GSH (GSSG). The enzyme reduces the need for the new synthesis of GSH from amino acids. GR function is in constant correlation with GPO and GST, enzymes that oxidize GSH in peroxide reduction processes [29, 30].

Glutathione peroxidase catalyzes the cleavage of hydrogen peroxide and organic peroxides by using GSH and converting it to GSSG. This enzyme is in competitive relations, due to its different intracellular localization, with catalase and SOD in the neutralization of excess hydrogen peroxide and organic peroxides, which ensures the efficient functioning of these enzymes. Glutathione peroxidase in mitochondria and peroxisomes works in tandem with catalase, and in the cytoplasm with SOD, which ensures, together with non-enzymatic antioxidants, the protection of subcellular structures and the modulation of the oxygen activation process by deregulating the formation of the hydroxyl radical (OH•). Glutathione-S-transferase catalyzes the conjugation of GSH with electrophilic organic compounds, an important detoxification reaction of exogenous products and the neutralization of endogenous substances within the physiological processes of metabolic waste elimination [29, 30].

Insect antioxidant systems are of crucial importance in defence mechanisms against xenobiotics that produce endogenous reactive oxygen species (ROS) in insects. Increased levels of radicals from xenobiotics, such as plant secondary metabolites, are associated with oxidative stress in the midgut tissues of lepidopteran larvae. Xenobiotics (prooxidant substances, heavy metals, pesticides) and their metabolism are associated with the production of free radicals, which react with various biomolecules and affect cellular functions. These radicals are removed by innate antioxidant defence systems, including antioxidant enzymes and various antioxidant compounds. Deficiency of the antioxidant defence system leads to increased ROS, which interacts with many cellular biomolecules, including proteins, lipids, enzymes, carbohydrates, and DNA with their damage. Insects, in order to overcome the toxic effects of SRO, have developed a complex antioxidant mechanism consisting mainly of the enzymatic action of glutathione peroxidase (GPX), catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase, and glutathione transferases (GST). In insects, GSTs are involved in the transformation of many insecticides, and their overexpression is responsible for the development of resistance against those insecticides. Glutathione-S-transferases present selenium-independent glutathione peroxidase activity and can remove highly reactive electrophilic components, lipid hydroperoxides (DAM, trans-4-hydroxy-2-nonenal), generated by ROS-initiated lipid peroxidation. After exposure to xenobiotics, increased levels of DAM have been correlated with a variety of tissue and cell membrane damage in animals [15, 31, 32].

The anti-inflammatory properties of edible insects have been evaluated in vivo and in cellular models. In vivo, studies have revealed a reduction in circulating cytokine levels elevated by various stressors after administration of different insect extracts. An increase in cytokine levels was found only at high doses of Hermetia illucens administered to healthy fish, without being confirmed by inflammatory events on histological analysis. In studies of healthy subjects, circulating levels of TNF-α have been shown to be reduced, data that must be reviewed because they may have had a reduced level of inflammation [32]. Levels of NF-κB, the transcription factor regulatory genes involved in inflammatory responses, were decreased in cell and animal models. At the same time, the levels of TLR4, whose stimulation leads to the activation of NF-kB, were not affected. Some studies have shown activity in reducing the production of NO in macrophages, a radical involved in the modulation of inflammation and immunity. In conclusion, evidence from cellular and animal models supports an effect on reducing inflammatory cytokines by modulating NF-kB levels, without affecting immunoglobulins [33, 34].

 Conclusions 

Imuheptin and imupurin showed a moderate inhibitory effect, predominantly of proliferative processes compared to dexamethasone, which essentially diminished inflammation's exudative and proliferative phases. Imuheptin and imupurin reduced the level of pro-inflammatory cytokines (TNF-alpha, IL-6) and increased that of anti-inflammatory cytokines (IL-10). The studied entomological preparations increased the ceruloplasmin level and restored the activity of catalase and glutathione peroxidase with the increase of glutathione reductase activity in subacute inflammation. Due to the effects mentioned earlier, the researched entomological preparations – imupurin and imuheptin have an anti-inflammatory potential, which requires a more in-depth study to determine the mechanisms of anti-inflammatory action and the pathological conditions where these effects would be beneficial. 

Competing interests 

None declared. 

Authors’ contribution

IG conceived and participated in the study design, performed the experiments and statistical analysis, and drafted the manuscript. NB participated in the study design and helped drafted the manuscript. VG had contribution to acquisition and interpretation of data, and helped drafted the manuscript. All the authors reviewed the work critically and approved the final version of the manuscript.

Ethical Statement

This study was carried out in accordance with the European Convention for the Protection of Vertabrate Animals Used for Experimental and Other Scientific Purposes and approved by the Research Ethics Committee of Nicolae Testemiţanu State University of Medicine and Pharmacy, Minutes No. 78 from 22.06.2015. 

Authors’ ORCID IDs

Ina Gutu – https://orcid.org/0000-0002-7839-5415

Nicolae Bacinschi – https://orcid.org/0000-0003-4854-5715

Valentin Gudumac – https://orcid.org/0000-0001-9773-1878

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Oxygen-ozone therapy stands as a medically endorsed practice confirmed by numerous international clinical studies. Various authors have illustrated the beneficial clinical outcomes of ozone therapy in terms of its capacity to regulate redox balance, cellular inflammatory responses, and adaptation to ischemia/reperfusion processes. Ozone therapy extends to encompass a range of viral infections, inflammatory disorders, and degenerative ailments, used as both monotherapy and as an adjunct to unified conventional therapies.Introduction Ozone (O3), a gas discovered in the mid-19th century and composed of three oxygen atoms, represents a highly reactive allotropic form of oxygen. It exhibits high solubility in plasma, extracellular fluids, and water (approximately 10 times more soluble in water than conventional oxygen). At room temperature, it is unstable, causing rapid decomposition into ordinary diatomic oxygen. Notably, its half-life measures 25 minutes at 30°C, 40 minutes at 20°C, and 140 minutes at 0°C [1-10]. Medical ozone is a blend of oxygen and ozone derived from medical-grade oxygen through the utilization of a medical ozone generator. This medical ozone contains a concentration of 1-5% ozone and 90-95% pure medical oxygen, or 10-80 μg/mL (0.21-1.68 μmol/ml) of ozone per milliliter of blood. Ozone therapy stands as a current and significant avenue of research in contemporary medicine [1, 3-5, 7, 9, 10-15]. Oxygen-ozone therapy is a medically validated practice supported by numerous international clinical studies. Nowadays, many clinical trials have shown its beneficial effects on the modulation of the oxidoreduction balance, cellular inflammation state, and adaptation to ischemia/reperfusion processes. Ozonotherapy is an effective, safe, feasible, and easy-to-perform technique, which finds applications in various inflammatory, infectious, degenerative diseases, as well as in rehabilitation following acute cardiac and cerebral ischemic events. It demonstrates good efficacy both as an independent treatment and, notably, as an adjunct to conventional therapies [3-5, 7, 11, 16-22]. By incorporating this medical practice, patients can attain significant clinical benefits. When combined with standard therapies, it often leads to reduced medication dosages, complication rates, treatment duration, medication toxicity, and medical expenses. It also addresses the issue of bacterial resistance to antibiotics [2, 4, 18, 19, 21, 23]. In the context of the aforementioned, the purpose of this article is to present a synthesis of the most recent findings regarding ozone's mechanisms of action. Materials and methods To achieve the outlined purpose, an initial search of specialized scientific publications was conducted. These were identified through the Google Search engine, namely, PubMed, Hinari, SpringerLink, the National Center of Biotechnology Information, and Medline. The article selection criteria encompassed contemporary data regarding the mechanisms of action of ozone therapy, utilizing the following keywords: “ozone”, “ozone therapy”, “ozone mechanisms of action”, “biological effects of ozone”, “antioxidant effect”, “anti-inflammatory effect”, and “immunomodulatory effect.” These keywords were employed in various combinations to optimize search efficiency. For the advanced selection of bibliographic sources, the following filters were used: full-text articles, articles in English, articles published between 1990 and 2022. 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To minimize the potential systematic errors (bias) in the study, a meticulous search was conducted within databases to identify a maximum number of relevant publications for the study's purpose. Only studies that satisfy validity criteria were evaluated, rigorous exclusion criteria for articles under consideration were applied, and a comprehensive review was conducted of both positive outcome studies and those that did not highlight the treatment's benefits. If necessary, additional sources of information were consulted to clarify some concepts. Duplicate publications and articles that did not meet the purpose of the article and were not available for full viewing were excluded from the list of publications generated by the search engine. 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Publications, the content of which did not reflect the relevant topic, despite being selected by the search program, as well as articles that were not accessible for open viewing through the HINARI (Health Internet Work Access to Research Initiative) database or available in the scientific medical library of the Nicolae Testemițanu State University of Medicine and Pharmacy, were subsequently removed from the list. Although ozone is the most potent natural oxidant, capable of oxidizing a wide range of organic and inorganic substances, and has the potential for cytotoxicity, researchers believe that under controlled conditions, it possesses numerous therapeutic effects. Moreover, the reactivity to ozone can be effectively mitigated by the blood and cellular antioxidant system [1, 2, 15, 24-28]. Initially used as an empirical approach, oxygen-ozone therapy has now evolved to a stage where the majority of ozone's biological mechanisms of action have been extensively studied and elucidated, these findings being found within the fields of biochemistry, physiology, and pharmacology [18, 22, 25, 27-29]. Ozone is not a pharmaceutical medicine but rather a regulatory molecule capable of generating bioactive mediators. The effects of ozone have been demonstrated to be consistent, safe, and associated with minimal preventable side effects [2, 30]. Chronic oxidative stress, chronic inflammatory processes, and immune overactivation are present and highly detrimental in a wide variety of diseases. The effectiveness of ozone therapy is determined by moderate oxidative stress, resulting from the interaction of ozone with the biological components of the body, triggering an endogenous cascade of biochemical reactions [31]. Ozone can function as an oxidant either directly, when it dissolves in plasma and other biological fluids, immediately reacting with polyunsaturated fatty acids, antioxidants, cysteine-rich proteins, and carbohydrates; or indirectly, by generating reactive oxygen species (ROS) and lipid oxidation products (LOP) [25, 28, 32-37]. At the onset of ozone therapy, an endogenous cascade is triggered, releasing bioactive substances in response to transient and moderately induced oxidative stress by ozone ('oxidative eustress'). Ozone can easily induce this oxidative stress due to its plasma solubility. Reacting with polyunsaturated fatty acids and water, ozone forms ROS in human fluids and tissues. The main molecule among ROS is hydrogen peroxide (H2O2) – a non-radical oxidant. Concurrently, ozone also gives rise to LOP – the lipoperoxide radical, hydroperoxides, malondialdehyde, isoprostanes, ozoneides, alkenes, and predominantly, 4-hydroxynonenal. ROS and LOP are the effector molecules responsible for modulating several biological and therapeutic effects in the body following ozone therapy [3, 8, 13, 27, 34, 37-39]. Having reacted with a number of biomolecules, ozone disappears, and hydrogen peroxide, the main molecule of ROS, and other mediators rapidly diffuse into cells, activating various metabolic pathways with numerous biological and therapeutic effects [3, 5, 27, 28, 35, 40]. Therefore, ROS and LOP are “biological messengers of ozone” and are responsible for the biological and therapeutic effects of ozone. ROSs are short-acting early messengers and are responsible for immediate biological effects, while LOPs are important late and long-term messengers [3, 5, 10, 13, 14, 17, 28, 36]. The formation of ROS in plasma occurs extremely quickly (less than a minute), accompanied by a moderate and transient decrease in the antioxidant capacity of the blood (from 5% to 25%). However, this antioxidant capacity returns to normal within 15-20 minutes [3, 9, 17, 28, 31, 40, 41]. Discussion Although not fully comprehended, the present article will delve into the mechanisms underlying the antioxidant, anti-inflammatory, immunomodulatory, antimicrobial, antiviral, and analgesic effects of ozone. The antioxidative capacity is considered one of the key impacts of ozone therapy. Moderate oxidative stress induced by ozone within the therapeutic range (10-80 μg/mL), most commonly 30-45 μg/mL (the 'physiological' dose of ozone), physiologically effective and recommended levels for systemic application, elicits controlled low doses of ROS acting primarily as signaling molecules, thereby stimulating the formation of LOP. ROS triggers the activation of nuclear erythroid factor 2 (Nrf2), well known as a pivotal regulator of manifold cytoprotective responses, responsible for upregulating antioxidant enzyme activity. In response to transient, moderate oxidative stress, the levels of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, glutathione S-transferase, catalase, and heme oxygenase-1 increase. Thus, moderate, transient, and repetitive oxidative stress causes an intense modulation of antioxidants in the body. A multitude of cells across various organs upregulate the synthesis of antioxidants, which are capable of significantly countering excessive ROS, thereby alleviating chronic oxidative stress, which is present and extremely harmful in a variety of diseases Consequently, ozone, either through oxidative preconditioning or adaptation to chronic oxidative stress, safeguards tissue integrity against ROS-induced damage, fostering a balance between antioxidant and pro-oxidant factors while preserving cellular redox balance [27, 30, 41-45]. Nrf2 and nuclear factor kappa Β (NF-κB) represent the primary signaling pathways through which ozone exerts its effects. Nrf2 activation regulates cell defense and maintains cellular homeostasis [36]. Furthermore, ozone therapy fosters adaptation to oxidative stress by gently triggering the immune system, releasing growth factors, and/or activating metabolic pathways that contribute to maintaining redox balance [38]. By activating Nrf2, LOP induces oxidative stress proteins, including heme-oxygenase-1 (HO-1), another inhibitor of the NF-κB pathway and one of the most crucial antioxidant defense enzymes. Through inhibiting the high expression level of hypoxia-inducible factor-1α (HIF-1α), it contributes to reducing the production of proinflammatory cytokines, directly activating anti-inflammatory cytokines, enhancing antioxidant protection, and consequently, safeguarding cellular integrity [8, 28, 30, 44-46]. Thus, ozone mimics acute oxidative stress which, when properly balanced, is not harmful, but can trigger several beneficial biochemical mechanisms. It can reactivate the intra- and extracellular antioxidant system, thereby reversing chronic oxidative stress in various inflammatory, degenerative processes, etc. During ozone treatment, cells throughout the body receive gradual and subtle impulses of LOP, significant long-term messengers that play a crucial role in up-regulating antioxidant enzymes in multiple cell types while rebalancing the oxidant/antioxidant system [46, 47]. Vascular and Hematological Modulation. Ozone serves as a catalyst for transmembrane oxygen flow. The increase in cellular oxygen levels resulting from ozone therapy enhances the efficiency of the mitochondrial respiratory chain. Moreover, ozone amplifies the production of prostacyclin, a widely acknowledged vasodilator [2, 6, 8, 26]. The effects of ozone on oxygen metabolism can be explained by promoting (1) changes in the rheological properties of blood (reversal of erythrocyte aggregation, increased flexibility and elasticity of red blood cells, favoring the transport and delivery of tissue oxygen), leading to enhanced blood flow in microcirculation; (2) increasing the speed of glycolysis in erythrocytes; and (3) the release of substances (adenosine triphosphate, nitric oxide, and prostaglandins) that may contribute to reducing peripheral vascular resistance and increasing oxygen supply to tissues [18, 25, 35, 37, 40, 48-50]. Hydrogen peroxide (H2O2) diffuses from the plasma into the cellular cytoplasm and serves as the triggering stimulus. Depending on the cell type, various biochemical pathways can be simultaneously activated in red blood cells, white blood cells, and platelets, leading to a multitude of biological effects [10, 28, 32]. The Impact of Ozone on Erythrocytes. Erythrocytes are the focus of ROS. During erythropoiesis, submicromolar concentrations of LOP positively regulate the synthesis of antioxidant enzymes. Consequently, ozone therapy increases the glycolytic rate by enhancing intracellular adenosine triphosphate production. This approach intensifies erythrocyte generation, yielding metabolically enhanced erythrocytes (super-endowed erythrocytes) capable of more effectively transporting and delivering oxygen to tissues, including ischemic tissues, thereby correcting hypoxia in vascular diseases [7, 12, 25, 27, 28, 39, 48]. Coupled with increased nitric oxide synthase activity, there is a significant increase in nitric oxide, an essential element in maintaining optimal levels of vasodilation and blood perfusion [1, 6, 8, 40]. Ozone therapy, through careful regulation of ozone dosage, stimulates the production of antioxidant enzymes within the system (catalase, glutathione peroxidase, and superoxide dismutase) while mitigating excessive formation of ROS, thereby reducing chronic oxidative stress [1, 6, 12, 14, 39, 43, 49]. The impact of ozone on leukocytes. Ozone acts as a mild cytokine and serves as a cytokine inducer by lymphocytes and monocytes, thereby enhancing the immune system's activity. This stimulation fosters intercellular matrix synthesis and contributes to the healing process [1, 12, 32, 35, 37, 39]. The Impact of ozone on platelets. Hydrogen Peroxide (H2O2) and other ROS generated through blood ozonation initiate a cascade of enzymatic reactions. These reactions gradually elevate intracellular Ca levels and trigger the release of prostaglandins (F2a and E2), leading to irreversible platelet aggregation. Increased levels of growth factors released from platelets, mobilization of endogenous stem cells, and stimulation of neoangiogenesis promote tissue regeneration, as well as healing of injuries and wounds [27, 49, 51]. Thus, the impact of ozone on oxygen metabolism is explained by how it alters the blood's rheological properties (reversing red blood cell clumping, enhancing the flexibility and elasticity of red blood cells, promoting the transport and delivery of oxygen to tissues). This, in turn, facilitates blood flow in the microcirculation, increases glycolysis in red blood cells, and triggers the production of substances (such as adenosine triphosphate, nitric oxide, and prostaglandins) that help reduce peripheral vascular resistance. Activation of the immune system. Ozone promotes an increase in the production of interferon-γ (IFN-γ) and some cytokines, with interleukin-2 (IL-2) being the primary one, subsequently triggering a whole cascade of immunological reactions [1, 2]. It has been shown that ROS, including H2O2 and LOP generated by ozone therapy, can easily diffuse into plasma cells and activate NF-κB, inducing the production of immunoactive cytokines in normal cells (IL-2, tumor necrosis factor alpha - TNF-a, IL-6 and IFN-γ), thereby enhancing the immune response [9, 13, 14, 26, 32, 40, 44]. Ozone indirectly activates the innate (non-specific) immune system by enhancing phagocytosis and promoting the synthesis of cytokines and interleukins in neutrophils and leukocytes. It also triggers the components of both cellular and humoral immunity [8, 26, 33, 39]. Within mononuclear cells, ozone stimulates immune responses by modulating the NF-kB transcription factor, thereby reactivating the suppressed immune system [27, 28]. Furthermore, ROSs trigger the activation of the immune system, which acts through monocytes and lymphocytes, promoting the production of a variety of cytokines (IL-1, IL-2, IL-6, IFN-β, IFN-γ, TNFα) [6, 49]. Thus, ozone induces mild immune system activation by stimulating neutrophils and initiating the synthesis of certain cytokines that trigger a whole cascade of immunological responses. Bactericidal, virucidal and fungicidal action of ozone. Ozone used in vitro acts directly on the membrane of bacterial cells (direct oxidative effect), disrupting and damaging the integrity of bacterial cell membranes, oxidizing phospholipids and lipoproteins, thereby impeding their enzymatic function. Additionally, ozone damages the viral capsids, disturbing their structure and interfering with the virus-cell interaction, leading to disruption in the reproductive cycle. When it comes to fungi, ozone inhibits cell growth by perturbing intracellular homeostasis, resulting from the compromised barrier properties of the plasma membrane [2, 6, 12, 16, 26, 44, 48, 50]. Although ozone is one of the most potent disinfectants, used in various ways, it cannot deactivate any pathogens (bacteria, viruses, and fungi) in vivo. This is because pathogens are well protected, especially within cells, by the cell's powerful antioxidant system. Consequently, ozone acts as a gentle enhancer of the immune system by activating neutrophils and stimulating the synthesis of certain cytokines [1, 10, 19, 22, 28, 39, 46]. The anti-inflammatory effect is revealed in ozone's ability to influence the inflammatory cascade by oxidizing biologically active substances (arachidonic acid and its derivatives - prostaglandins), which participate in the development and sustenance of the inflammatory process. Additionally, ozone significantly reduces the levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α) without any signs of toxicity or recorded side effects [8, 26, 30, 31]. These cytokines induce the prostaglandin E2 pathway, which causes pain or increases the sensitivity of nerve roots to other algogenic substances (such as bradykinin) [31]. Severe oxidative stress, triggered by high concentrations of ozone, along with proinflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α), activate NF-κB, a key regulator of the inflammatory response and muscle atrophy. This contributes to an increased inflammatory response and tissue damage, including the release of other inflammatory factors that enhance the migration of eosinophils and neutrophils [9, 13, 17, 47, 49]. On the contrary, mild oxidative stress induced by precise and small doses of ozone activates Nrf2. The latter indirectly inhibits the pro-inflammatory mechanism driven by the NF-kB pathway. As a result, there is a reduction in NF-κB activity along with a modification in the expression of inflammatory cytokines associated with NF-kB activity. This triggers an anti-inflammatory effect, leading to a decrease in IL-1, IL-2, IL-6, IL-7, and TNFα, as well as an increase in interleukins such as IL-4, IL-10, IL-13, and the transforming growth factor beta – TGF-β [11, 13, 19, 38, 43, 49, 50]. Nrf2 also plays an important role in intracellular inflammatory signaling pathways. Triggering the Nrf2-antioxidant signal can dampen NF-kB activity, leading to the downregulation of the inflammatory response by suppressing essential inflammatory mediators and cytokines (IL-6, IL-8, and TNF-a) [31, 38, 42, 50]. Moreover, a small amount of H2O2 stimulates the NF-kB pathway, which is typically balanced out by the Nrf2's blocking action, resulting in an immunomodulatory effect [11]. The analgesic effect of ozone is ensured by the oxidation of the byproducts of albuminolysis, known as algopeptides, which act on the nerve endings in the damaged tissue and determine the intensity of the pain response. Additionally, the analgesic effect is attributed to the restoration of the antioxidant system and, subsequently, the reduction of harmful molecular byproducts from lipid peroxidation [26]. Recent preclinical studies have elucidated the role of ROS in hyperalgesia by activating N-methyl-D-aspartate receptors [11]. Following ozone therapy, there has been a demonstrated increase in antioxidant molecules (serotonin and endogenous opioids), which induce pain relief by stimulating antinociceptive pathways [31, 39]. Data from scientific research acknowledge that the mechanisms of action of ozone are due to: (1) a decrease in the production of inflammatory mediators; (2) oxidation (inactivation) of metabolic mediators of pain; (3) improvement of local blood microcirculation leading to improved oxygen delivery to tissues; (4) elimination of toxins and resolution of physiological disorders that generate pain [42, 52]. Therefore, ozone exhibits pleiotropic properties, extending beyond its exclusive role as an antioxidant, anti-inflammatory, or immunomodulatory one. It also encompasses the capacity to employ ROS as a signaling molecule rather than merely as intracellular toxic substances. In existing experimental models and clinical studies, the anti-inflammatory, antioxidant, regenerative and immunomodulatory effects of ozone therapy have been associated with several molecular mechanisms, the main ones being the NF-kB/Nrf2 balance and IL-6 and IL-1β expression. NF-kB and Nrf2 are the most studied and important transcription factors and regulatory proteins that control the expression of a wide range of genes, encoding proteins involved in a multitude of vital biological functions, including those associated with redox status, immunity, and inflammatory responses. Additionally, indirectly through these pathways, LOP initiates the HIF-1α, HO-1, and NO/iNOS pathways. The main pharmacological effects of medical ozone through ozone-produced peroxides are as follows: (1) increased oxygen release by erythrocytes due to activated metabolism; (2) immunomodulation due to leukocyte activation; and (3) regulation of cellular antioxidants via Nrf2 signaling. Ozone therapy can elicit the following biological reactions: (a) improved blood circulation and oxygen delivery to ischemic tissue; (b) optimization of overall metabolism by improving oxygen delivery; (c) regulation of cellular antioxidant enzymes and induction of HO-1; (d) triggering a mild immune system activation and intensified release of growth factors; (e) providing a state of well-being in most patients, probably due to stimulation of the neuroendocrine system. Conclusions 1. Ozone induces both mild and moderate oxidative stress. When appropriately balanced, this stress poses no harm; instead, it can initiate several beneficial biochemical mechanisms. These mechanisms, in turn, reactivate the intracellular and extracellular antioxidant systems, effectively countering long-term oxidative stress in various inflammatory and degenerative processes, etc. Cells throughout the body receive small and gradual bursts of lipid oxidation products, important late and long-term messengers that are responsible for activating antioxidant enzymes in many cell types to rebalance the oxidant/antioxidant system. 2. The impact of ozone on oxygen metabolism is explained by changes in the blood's rheological properties. This involves reversing red blood cell aggregation, enhancing the flexibility and elasticity of hemoglobin, and promoting the efficient transport and delivery of oxygen to tissues. This process also facilitates blood flow within microcirculation, speeds up glycolysis within red blood cells, and triggers the release of substances like adenosine triphosphate, nitric oxide, and prostaglandins, which help to reduce peripheral vascular resistance. 3. Ozone triggers a slight activation of the immune system by up-regulating and activating neutrophils and promoting the synthesis of cytokines (IL-2, TNF-a, IL-6, and IFN-γ), setting off a chain reaction of immune responses. 4. Ozone therapy induces the following biological responses: enhanced blood circulation and oxygen delivery to ischemic tissue, regulation of cellular antioxidant enzymes, mild immune system activation, and intensified release of growth factors. 5. Ozone is an inherently toxic gas that should never be inhaled, cannot be stored, and must be handled with caution. Generally, no toxic effects were reported, and only the respiratory tract was found to be highly sensitive to inhaled ozone since the respiratory mucosal cells contain a minimal amount of antioxidants and are extremely susceptible to oxidation. 6. Although ozone ranks among the most potent disinfectants, being employed in various ways, it cannot neutralize any pathogens (bacteria, viruses, and fungi) in vivo, since pathogens are effectively shielded by the strong blood and cellular antioxidant system. Furthermore, elevated ozone concentrations induce severe oxidative stress, prompting increased inflammatory responses and tissue damage. Competing interests None declared. Authors’ contribution NC and RB conceived the study, participated in the study design and assisted in drafting the manuscript. SȘ and IC performed the analysis and data interpretation. IG drafted the manuscript. SC conceived the significant revision of the manuscript and provided significant intellectual involvement. 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Review The role of the lateral pterygoid muscle in temporomandibular disorders
Vitalie Pântea1*, Felicia Tabără1, Mariana Ceban1, Veronica Burduja1, Lilian Nistor2, Olga Ursu3
https://doi.org/10.52645/MJHS.2023.3.09
The clinical concept that would argue that the activity of the lateral pterygoid muscle, being disturbed, would play an important role as an etiological factor in temporomandibular joint dysfunctions is still widely accepted, being also a decisive factor in the correct choice of the treatment plan. However, because of the fact that very few research and clear evidence were conducted and presented to support completely that concept, it continues to remain a very controversial one.
Case study Laser ureteroscopic endopyelotomy efficacy in pyeloureteral junction stenosis
Vladimir Caraion1*, Eduard Pleșca2, Andrei Mezu2, Corneliu Maximciuc2
https://doi.org/10.52645/MJHS.2023.3.10
Pyeloureteral junction stenosis (PUJS) is a condition that affects urinary drainage at level of the renal pelvis and upper ureter. It is found in approximately 1 in 500 newborns, with a higher prevalence in males (2:1 ratio). PUJS is the main cause of congenital hydronephrosis and can also be caused by other specific pathologies. Endoscopic management is the primary treatment for PUJS, particularly in cases of aperistaltic and <2cm intrinsic ureteral stenosis without aberrant vessels.
Case study Treatment of deep carious lesions with mineral trioxide aggregate: clinical case report
Diana Trifan*, Diana Uncuța
https://doi.org/10.52645/MJHS.2023.3.11
Deep carious lesions are a dental disease widely spread among population of all ages. From clinical point of view, they have little symptoms and go unnoticed by the patients a long time, until they provoke dental pulp inflammations. If diagnosed and treated properly, the tooth can be treated conservatively with certain techniques of pulp vitality preservation. An important role in this process plays the innate capacity of regeneration of the pulp-dentine complex and the enhanced stimulating properties of new biomaterials used in dentistry. The aim of this clinical case report is to describe the clinical manifestations and the diagnostic algorithm used in deep caries and to establish a clinical guideline of treatment of deep carious lesion with a calcium silicate hydraulic cement.