CLINICAL-MORPHOLOGICAL STUDY OF LUNG DISEASES IN BASIC TREATMENT WITH THE USE OF ANTIOXIDANTS OF DIFFERENT ORIGIN AND WITHOUT

Annotation. The purpose of the work is to analyze contemporary views on the morphological changes of lung tissue in inflammatory disease in the clinical and experimental conditions and results of correction by using the antioxidants. The analysis is based in a review if foreign articles for 2014-2019, using the scientometric databases PubMed, Web of Science and Google Scholar. According to the latest data from WHO and WORLD HEALTH RANKINGS 5-year mortality rate of such disease as COPD typically ranges from 40% to 70%, depending on disease severity, while the 2-year mortality rate for people with severe COPD is about 50%. We know that almost 90% of COPD deaths occur in lowand middle-income countries such as Ukraine. Thereby mortality rate from lung tissue diseases in Ukraine is 11.11%. These diseases often accompanied by inflammation and oxidative stress. The last can cause mitochondrial dysfunction, dynamic changes and mitophagy impairment, which leads to increase the number of superoxide anions, hydrogen peroxide etc., inflammatory responses and cellular senescence. They all play important roles in the pathogenesis of chronic lung diseases, such as chronic obstructive pulmonary disease, pulmonary fibrosis and bronchopulmonary dysplasia. Many studies in vitro approved the role of antioxidants in the decreasing of the degree of morphological changes: inflammatory cells infiltration and alveolar edema, permeability and inflammation. In vivo disease development is mainly related to the many conditions, but closely to its severity and possible of combination with other diseases. Thereby treatment of such diseases by using e.g. leaves extract of different herbs can be prescribe to ameliorate the level of reactive oxygen species and decrease the possible cell injure made by antiinflammatory medicines.

Frequency of mortality from lung tissue diseases in Ukraine is 11.11 that is our rank 168th according to WORLD HEALTH RANKINGS (2017). Although there are many questions concerning developing lung tissue diseases, it is progressing and relation of impact of oxidative stress on mortality rate and morphological changes. Accordingly these we can highlight the main causes of formation of reactive oxygen, peroxide radicals and others products of cell metabolism.
T. Finkel and co-authors (2015) discovered that mitochondria plays important part as a source of mitochondrial reactive oxygen (mtROS) under the different conditions in humans during oxidative phosphorylation because of the leakage of activated oxygen. Additionally, calcium overload in the mitochondrial matrix enhances the generation of mitochondrial ROS (mtROS) and the release of cytochrome c, resulting in apoptosis and cell death [8]. Physiological processes, which activated as moderators of increased mtROS level, protect against the adverse effects of various cellular stresses and infection [13]. However, their activity depends on the ability of cell to generate ATP, enzyme level and activity. Other authors discovered there is accumulating evidence that the mitochondria has a nonenergetic role in regulating metabolism, apoptosis, innate immunity, inflammatory responses and aging [9]. All of these processes are involved in the pathogenesis of chronic lung diseases, such as chronic obstructive pulmonary disease (COPD), pulmonary fibrosis and bronchopulmonary dysplasia (BPD) [34].
Estimates are that by the year 2030, COPD will rise to the 3rd leading cause of death worldwide unless urgent action isn't taken to reduce tobacco use [32]. In Ukraine major changes in COPD mortality in 1980-2010 were concurrent with changing economic conditions and could only partially be tobacco-related [3]. COPD is characterized by a persistent airflow limitation and lung function decline, where cigarette smoke (CS) is the main etiological factor. CS contains more than 5,000 different chemicals, many of which are oxidants. It is estimated that each puff may contain >1015 free radicals that deplete endogenous antioxidants and tilt the delicate balance in favor of an oxidative stress (OS). The pathogenesis of COPD involves a variety of cellular processes, including oxidative stress, inflammatory responses and cellular senescence [11,34]. Histopathological COPD is characterized by respiratory (smoker's) bronchiolitis, inflammation and fibrosis of terminal and respiratory bronchioles, reduction in terminal bronchioles, squamous metaplasia and so on [4]. Ahmad T. and co-authors discovered the defects of mitochondria by the methods by which the quality of it is controlled -mitophagy, which has been shown to be involved in the pathogenesis of COPD. Authors have shown that cigarette smoke impairs mitophagy, leading to incomplete degradation of damaged mitochondria [1]. These damaged mitochondria may accumulate in the perinuclear region where generating excessive mtROS leads to nuclear DNA damage and subsequent cellular senescence. This is corroborated by the finding that restoration of mitophagy by the overexpression of Parkin reduces cigarette smoke-induced DNA damage and cellular senescence when used in ©Вінницький національний медичний університет ім. М.І. Пирогова combination with an mtROS scavenger [1,34]. Indeed, the levels of Parkin are reduced in lung tissues of COPD patients compared with those of non-smokers and smokers [1,12,34], which further confirms the impairment of mitophagy in COPD. Other authors admitted that severe stress derived from high concentrations of cigarette smoke induces necroptosis (i.e. necrosis and apoptosis), which is mediated by PINK1-induced mitophagy [20,34]. To attenuate the changes of lung tissue after impact of cigarettes smoke and decrease the oxidative stress Ahmad Husari and co-authors (2016) used supplementation of pomegranate juice (PJ) [11]. One month of sublimation of PJ resulted in limited destruction of the normal alveolar architecture and decreased level of TNF-α; third month of sublimation reversed the emphysematous changes noted histologically and attenuated the increase in linear intercept distance [11]. The in vitro study showed that PJ supplementation significantly suppressed CS-induced reactive oxygen species (ROS) [11]. Another study admitted the role of Atorvastatin and Simvastatin in enhancing mouse lung repair after CS induced lung tissue injury. Pinho-Ribeiro V. and coauthors observed that morphologically changes of lung tissue and noted the same pattern of total cell numbers was observed in macrophages, but not in neutrophils, which were higher in CS [25].
Mechanical ventilation (MV) as a method of breath support in COPD closely involved in developing ventilator-induced lung injury (VILI). Last is characterized by a disruption of the alveolar-capillary barrier which increases permeability, thus causing edema, inflammatory leukocyte infiltration (mainly neutrophils), and hemorrhage [30]. As follows, the correction of ROS and mtROS level in COPD opens the new ways of increasing patient's life level. Bixin induces the expression of nuclear factor-erythroid-related factor 2 (NRF2) and its downstream targets in lung tissues of Nrf2+/+ mice. More importantly, bixin pretreatment restored normal lung morphology and alleviated MV-induced inflammation and oxidative stress, these effects seem to be dependent on NRF2 signaling since Nrf2-/-mice did not benefit from bixin pretreatment [30]. Although the use of direct antioxidants, like N-acetyl cysteine (NAC), has some degree of beneficial effects but Shasha Tao and co-authors suggested that activating the body's own defensive responses through upregulation of the NRF2 pathway in combination with low tidal ventilator strategies will result in greater benefits for the patients [30,23]. Another histological study revealed that inflammatory cells infiltration and alveolar edema, permeability and inflammation induced by ventilation were significantly severe in VILI as compared to other groups treated by curcumin. In addition, curcumin ameliorates level of tumor necrosis factor (TNF)-α and NF-βB activity which were significantly increased in VILI group [31]. Mairead Hayes and co-authors observed that human mesenchymal stem cells (hMSCs) improved lung compliance, reducing alveolar edema, and restoring lung architecture. hMSCs attenuated lung inflammation, decreasing alveolar cellular infiltration, and decreasing cytokine-induced neutrophil chemoattractant-1 and interleukin-6 while increasing keratinocyte growth factor concentrations, as result level of ROS and mtROS [10].
The outcome of progressing of chronic inflammation of lung tissue, excessive number of mesenchymal cells near inflammatory area, dysfunction of cells will lead to excessive accumulation of macromolecules in extracellular matrix, thereby cause the scar and loss of elasticity in lungs. Both fibroblasts and myofibroblasts are the principal effectors cells of the lung for the generation of extracellular matrix [33]. M. Bueno and co-authors (2015) have highlighted the importance of mitochondrial function and mitophagy in the pathogenesis of lung fibrosis. It has been shown that damaged or dysfunctional mitochondria accumulate in alveolar epithelial cells of patients with idiopathic pulmonary fibrosis [5]. However, it is not clear whether mitochondrial dysfunction and mitophagy impairment occur in (myo) fibroblasts during pulmonary fibrosis. Also further studies are required to determine how mitochondrial dysfunction in lung epithelial cells interacts with (myo)fibroblasts, leading to fibrogenesis [34]. It has been shown that once released mtDNA recruits peripheral blood mononuclear cells and stimulates epithelial cells to generate TGF-β1 [16]. Targeting mtDNA by DNase I protects against paraquat-induced pulmonary fibrosis [16]. Interestingly, TGF-β1 increases the number of mitochondria, mitochondria-specific proteins, voltage-dependent anion channels, adenine nucleotide transporter and mtDNA content, whereas mitochondrial oxidative phosphorylation and mitophagy are impaired during fibroblast differentiation [21,27,34]. Authors proposed that mitochondrial biogenesis is needed for fibroblast differentiation that this effect is further promoted by mitophagy impairment and damaged mitochondria during fibrogenesis. However, it is not clear how TGF-β1 alters mitochondrial homeostasis, inducing the accumulation of damaged mitochondria and subsequent fibrogenesis [34]. M. Bueno et al. (2015) and M. L. Sosulski et al. (2015) admitted in relation aging in developing lung fibrosis. They has shown that it participate in the pathogenesis of lung fibrosis as both mitochondria respiration and mitophagy are compromised in aging, whereas the levels of the mitochondrial biogenesis marker PGC1α, mitochondrial transcription factor A TFAM and mitochondrial gene cytochrome c are comparable between young and old mouse lungs [5,27,34].
Reducing the effects of increased production of oxidative stress components that enhance the development of pulmonary fibrosis remains it is actuality. The treatment of Phyllanthus emblica leaves (PELE) to CCl4 exposed rat demonstrated strong repairing ability as manifested by the elevation in activity level of catalase, superoxide dismutase, glutathione peroxidase and GSH in the pulmonary samples of rat. PELE was also able to ameliorate the oxidative injuries induced with CCl4 and decreased the elevated level of TBARS, H2O2 and nitric oxide in lung samples of rat. The repairing abilities of PELE on the histopathology of lungs showed normal alveoli with explicit alveolar spaces and bronchioles having slight cell degeneration, intraalveolar septa thickening were observed in most lung regions [29].
In the modeling experimental condition that will cause the development of pulmonary fibrosis mainly associated with bleomycin. It was found that Nrf2 and its downstream antioxidant factors are involved in the pathogenesis of IPF [18,26]. Nrf2 agonist attenuated pulmonary fibrosis induced by bleomycin (BLM) via the oxide level in lung tissue [15]. Y. Liu and co-authors (2017) demonstrated that Bach1 knockout inhibited the progression of BLM-induced pulmonary fibrosis by regulating the expressions of Nrf2 and its downstream anti-oxidant factors [17]. The changes in lung tissue induced by bleomycin characterized by alveolar edema, a significant increase in septum width and increased inflammatory cells infiltration on 14 days and the alveolus collapsed or disappeared, the structure was markedly damaged, and a large number of inflammatory cells and fibroblasts were infiltrated on 42 days respectively. Administration of pirfenidone (PED) for 4 weeks ameliorated the inflammatory infiltration, the damaged structure in lung tissue and fibrosis as compared to that of the BLM group [18]. Other study demonstrated that celastrol also reduces inflammation in BLM-induced rats as evidenced by decrease in the expressions of mast cells, Tumor necrosis factor-alpha (TNF-α) and matrix metalloproteinases (MMPs) 2 and 9 [7]. O. Khazri and coauthors (2016) admitted the role of grape seed and skin extract properties that could find potential application in the protection against bleomycin-induced lung fibrosis [14].
Another experimental model that initiate fibrosis close linked to lipopolysaccharide (LPS). Toshio Suzuki and coauthors (2017) presented hypothesis in which that stated that LPS exposure leads to pulmonary fibrosis via endothelialto-mesenchymal transition (EndMT). Given that one of the main initial targets of endotoxins is vascular endothelial cells especially in case of acute respiratory distress syndrome from extra-pulmonary origin [6,22,28], EndMT could thus be closely involved in the pathogenesis of pulmonary fibrosis after systemic endotoxemic injury [28]. Morphological examination following LPS induction revealed that several histopathological alterations, including cell structure destruction, neutrophil infiltration, alveolar wall thickening and lung edema, had occurred in the lung tissue In other study Naif O. Al-Harbi and co-authors (2015) discovered the role of riboflavin in comparing to dexamethasone as a method of attenuation lipopolysaccharide-induced lung injury. Cellular LPSinduced changes including interstitial edema, hemorrhage, infiltration of PMNs, etc., which were reversed by riboflavin (100 mg/kg, p.o.) administration, which showed similar protective effects as dexamethasone (1 mg/kg, p.o.) [2].
C. Luo and co-authors (2015) evaluated the role of intestinal ischemia-reperfusion (IIR) that can resulted in severe damage to the lungs, with collapse of the alveoli, interstitial edema, haemorrhage in the alveoli and mesenchyme, neutrophil infiltration and atelectasis. But they discovered that pretreatment with sevoflurane SEV and apocynin (AP) significantly prevented the lung damage induced by IIR in the way of: protecting type II alveolar epithelial cells from the injury induced by IIR and mast cell degranulation, inhibiting inflammatory responses and decreasing the level of ROS [19].

Conclusions and prospects for future development
1. Overall we can state the widespread prevalence of pulmonary tissue diseases and their possible complications caused by oxidative stress and inflammation most studies in vitro approved the role of antioxidants in the decreasing of the degree of morphological changes: inflammatory cells infiltration and alveolar edema, permeability and inflammation. In vivo the role of antioxidants, compared to anti-inflammatory medicines have the same results and can be prescribe to the patients to ameliorate the pathognomonic signs or prevent them.
Therefore importance of including in treatment of these diseases antioxidants remains an important recommendation.