ГАСТРОЕНТЕРОЛОГIЯGASTROENTEROLOGY Intensity of systemic proteolysis and endotoxicosis in patients with non-alcoholic steatohepatitis associated with obesity and comorbid chronic obstructive pulmonary disease in the dynamics of treatment with hepatoprotectors

. Background. The prevalence of non-alcoholic steatohepatitis (NASH) and chronic obstructive pulmonary disease (COPD) is gaining global significance in the population of economically developed countries with a growing trend in Ukraine. The purpose was the determination of the intensity of systemic proteolysis and endo genous intoxication before treatment and the efficacy of hepatoprotective therapy in patients with NASH against the background of obesity, depending on the comorbidity of COPD. Materials and methods. Seventy-six patients with NASH, grade 1 obesity and COPD 2–3 D were screened and divided into 3 groups according to the treatment received. The control group (C group) consisted of 23 patients receiving basic treatment for NASH (essential fatty acids complex 300 mg 2 capsules 3 times daily) for 30 days and baseline COPD therapy. Twenty-five patients (group 2 — primary, O1), in addition to similar COPD therapy, for the treatment of NASH, instead of essential fatty acids complex, received antral at a dose of 200 mg 3 times a day for 30 days. The third group (basic, O2) involving 28 patients with NASH, grade 1 obesity and COPD 2–3 D, in addition to similar COPD therapy, for the treatment of NASH, instead of essential fatty acids complex, received antral at a dose of 200 mg 3 times daily and, additio nally, policosanol at a dose of 20 mg after the dinner for 30 days. The comparison group consisted of 30 apparently healthy individuals. Results. The proposed therapy with antral reduced the intensity of lysis of azoalbumin, azocasein and azocol in patients of group O2: at day 30, the decrease was 1.3, 1.2 and 1.6 times (p < 0.05), respectively, in patients of the group O1: on day 30, the decrease was 1.2, 1.2 and 1.6 times (p < 0.05), respectively, compared to the pre-treatment values. In the group C, the values decreased less intensively (p < 0.05): only the azocol values were likely to change — 1.3 times (p < 0.05) with the presence of a significant difference with the groups O1 and O2 (p < 0.05). Conclusions. The combined administration of antral for 30 days resulted in a significant correction of proteinase-inhibitory homeostasis in patients with NASH associated with obesity and COPD, which was accompanied by a significant decrease in endotoxicosis (p < 0.05) and damaging effect of systemic proteolysis (p < 0.05).


Introduction
Over the last few years, there has been an explosive interest of the scientific community in non-alcoholic fatty liver disease (NAFLD) and its progressive stage -non-alcoholic steatohepatitis (NASH) due to their widespread among the population of economically developed countries with a growing trend in Ukraine [1][2][3]. In the USA, the number of cases of NAFLD is projected to reach 100.9 million people (35 %) by 2030, and the proportion of NASH among these patients will increase to 27 % [4]. Unfortunately, in Ukraine, there is no systematic data on the prevalence of NASH that makes it difficult to formulate a prognosis, but Gastroenterologìa, ISSN 2308-2097 (print), ISSN 2518-7880 (online) Патологія печінки і жовчовивідної системи / Pathology of Liver and Biliary Excretion System we can definitely speak of a heavy economic burden and an increase in the number of patients with liver cirrhosis, terminal stages of the disease, requiring transplantation, and with hepatocellular carcinoma.
The experience of treating NASH is still small, so the therapy has not yet been developed in detail. The use of drugs with different mechanisms of action is investigated. There are known results from some studies of predominantly uncontrolled or descriptive nature, as well as small-scale randomized controlled (pilot) trials of several drugs, most of which are based on morphological data [5][6][7][8]. Researchers emphasize the reduction (arresting) of laboratory and morphological features of NASH activity [9,10], but the results of pharmacotherapy are ambiguous. Despite the increase in the comorbidity of NASH and chronic obstructive pulmonary disease (COPD), there is a need to conduct studies regarding general mechanisms of development and burden interaction of these nosologies with the development of new correction methods.
According to a number of studies on the use of antral, the remedy promotes the restoration of glycogen and protein-synthesizing function of the liver, improves phospholipid synthesis, activates cytochrome system, stimulates tissue respiration processes, improves energy supply and functioning of hepatocyte monooxygenase system, contri buting to the reduction of endotoxicosis intensity [11,12]. By inhibiting cyclooxygenase and reducing the synthesis of prostaglandins and other mediators of inflammation, antral exerts its anti-inflammatory effect, inhibiting the processes of free radical oxidation and enhancing the processes of neutralization of free radicals, leading to a decrease in the severity of cytolytic syndrome that prevents the destruction of hepatocytes and reduces the intensity of the inflammatory process in the bronchi [13].
In Ukraine, a new phytopreparation has appeared, which improves the lipid spectrum of the blood and has an antioxidant effect protecting low-density lipoprotein cholesterol from destruction by free radicals, reducing hypertriglycemia, suppressing excessive platelet aggregation and improving the effectiveness of statin therapy in combined use -phytostatin, which is a mixture of fatty alcohols extracted from the waxy mass of sugar cane [14].
Purpose. Determination of the intensity of systemic proteolysis and endogenous intoxication before treatment and the efficacy of the use of antral in patients with NASH against the background of obesity, depending on the comorbidity of COPD.

Material and methods
Seventy-six patients with NASH, grade 1 obesity and COPD 2-3 D were screened and divided into 3 groups according to the treatment received. Before treatment, the ave rage activity of cytolytic syndrome (the activity of alanine aminotransferase) was (1.440 ± 0.011) μmol/h • l). The control group (group C) consisted of 23 patients receiving basic treatment for NASH (essential fatty acids complex 300 mg 2 capsules 3 times daily) for 30 days and baseline COPD therapy (budesonide 160 μg/d + formoterol fumarate 4.5 μg/d) by inhalation 2 times a day for 30 days; ipratropium/fenoterol (250/500 μg/ml) by inhalation with a nebulizer twice a day, azithromycin 500 mg once per day for 10 days. Twenty-five patients (group 2primary, O1), in addition to similar COPD therapy, for the treatment of NASH, instead of essential fatty acids complex, received antral at a dose of 200 mg 3 times a day for 30 days. The third group (basic, O2) involving 28 patients with NASH, grade 1 obesity and COPD 2-3 D, in addition to similar baseline COPD therapy, for the treatment of NASH, instead of essential fatty acids complex, received antral at a dose of 200 mg 3 times daily and, additionally, policosanol at a dose of 20 mg after the dinner for 30 days. The mean age of the patients was (55.70 ± 3.22) years. The comparison group consisted of 30 apparently healthy individuals (AHI).
The diagnosis of NASH was made according to the unified clinical protocol approved by the order of the Ministry of Health of Ukraine No. 826 of 06.11.2014, in the pre sence of criteria for the exclusion of chronic diffuse liver disease of viral, hereditary, autoimmune or drug genesis as the cause of cytolytic, cholestatic, mesenchymal-inflammatory syndromes, and the results of ultrasonography with shear wave elastography, SteatoTest.
The diagnosis was established and the treatment for COPD was conducted following the clinical guidelines (MOH Order No. 555 of June 27, 2013, taking into account the recommendations of GOLD, 2019).
The obesity was diagnosed according to the classification of the WHO International Obesity Working Group (1997). The patients were subjected to measurements of height and body weight, their body mass index (BMI) was calculated according to the Kettle formula (1):
On admission to the hospital, the functional state of the liver was determined according to the approval list of activity of enzymes, markers of pigment and nitrogen metabolism, proteinogram, lipidogram, ionogram, and calculation of the de Ritis ratio. All the patients underwent anthropome try to determine BMI, waist circumference (WC), hip circumference (HC), and waist/hip index (WHI = WC/HC).
The intensity of endotoxicosis was studied by the content of medium molecular weight peptides (MMWP) by the method of N.I. Gabrielyan at 254 and 280 nm. The proteolytic activity of blood plasma was studied using azoalbumin (AA) (lysis of low molecular weight proteins), azocasein (AC) (lysis of high molecular weight proteins), azocol (CLA) (collagen lysis) using standard reagent kits (Simko Ltd, Lviv).
Statistical analysis of the results was performed according to the type of study conducted and the types of numerical data obtained. The normality of the distribution was checked using the Lilliefors, Shapiro-Wilk tests and the method of direct visual evaluation of the histograms of the distribution of eigenvalues. Quantitative indicators that had a normal distribution are presented as mean (M) ± standard deviation (S). For comparisons of data that had a normal www.gastro.org.ua, http://gastro.zaslavsky.com.ua
The analysis of the effect of antral complex therapy on the state of endotoxicosis and the proteinase-inhibitory system (Table 1) indicates a higher degree of efficacy in the groups of patients O1 and O2 since only these groups developed a significant decrease in the intensity of lysis of AA, AС, СLA (p < 0.05).

Discussion
Detoxification function of the liver requires intensive aerobic metabolism to produce sufficient ATP in the mitochondria of hepatocytes, which leads to the constant formation of reactive oxygen species (ROS) [15]. It should be noted that damage to hepatocytes with the presence of in-

Notes: group С (control) -patients with NASH who received essential fatty acids complex; group O1 (basic)patients with NASH who received antral; group O2 -patients with NASH who received antral and phytostatin; * -the difference is probable compared with the AHI group (p < 0.05); ** -probable difference compared with the values before treatment (p < 0.05); # -probable difference compared with the values after treatment in patients of group C (p < 0.05); *** -probable difference compared with the values after treatment in patients of group O1 (p < 0.05) with the values in patients of group
Патологія печінки і жовчовивідної системи / Pathology of Liver and Biliary Excretion System flammation can significantly increase the intensity of free radical processes, both in each hepatocyte and in the organ as a whole. These processes further intensify under the conditions of hypoxia caused by lung damage [16]. Oxidative stress leads to an increase in the production of ROS and, as a consequence, the oxidative modification of proteins (OMP): their aggregation and fragmentation, which is accompanied by irreversible changes in the physicochemical and biological properties of the protein molecule. It is known from the existing literature on OMP processes that proteins in which their oxidative modification takes place are much easier to proteolysis [17].
During the study, we found a significant increase in proteolysis (lysis intensity of AC, AA, CLA) in the groups before treatment compared to the AHI group (p < 0.05). In our opinion, the activation of some proteolytic enzymes and their involvement in the processes of degradation of protein components are affected by lysosomal proteinases, which after being exposed to free radical oxidation of lipids and oxidative modification of proteins into cells are released from them upon their destruction. Peptide hydrolase released from cells catalyzes specific reactions of limited proteolysis and, in our view, activates inactive precursors of enzymes and isolates specific inhibitors of proteolysis [18][19][20].
Therefore, in case of NASH associated with obesity and comorbid COPD, the development of an imbalance of the protease-inhibitory system is caused by the uncontrolled ele vation in the proteolytic activity of blood plasma.
The degree of endogenous intoxication is to depend not only on the severity and nature of inflammation in the lungs but also on the functional state of the hepatic detoxification system [21,22], which coincides with the data of our study. A significant level of endotoxicosis, which we established for blood levels of MMWP 254 and MMWP 280, was observed in patients with comorbid NASH, obesity and COPD compared with the AHI group (p < 0.05). There is no room for doubt the fact of the involvement of membrane destructive phenomena in hepatocytes, that makes it possible to correct this type of comorbidity using the drugs with a membrane-stabilizing effect. Antral exhibits such properties [23].
In the groups of patients who received antral for a hepatoprotective therapy (O1 and O2), the degree of endogenous intoxication decreased, there was a significant reduction in the intensity of lysis of AA, AC, and collagen (p < 0.05). Due to stimulation of β-oxidation of fatty acids and influence on the synthesis of phospholipids, antral improves energy processes in cells and arranges the functio ning of monooxygenase systems, as a consequence of which the hepatocytes reduce the accumulation of fat, the degree of damage to hepatic cells decreases. There are also data on the effect of antral on immune regulation, namely the effect on T-helper cells and the decrease in the level of circulating immune complexes that contributes to the reduction of the expression of metabolic toxicosis [24]. Regarding the combination of antral with policosanol, we registered the best corrective effect on the proteolysis system, namely a pro bable decrease in the intensity of azoalbumin lysis (p < 0.05).
Prospects for further research in this direction is a further study of the effect of antral and policosanol on the dynamics of clinical, biochemical syndromes of NASH with comorbid COPD on day 60, 90 and 120 of administration, as well as to study the long-term effects of the course of treatment.

Conclusions
1. Patients with non-alcoholic steatohepatitis, obesity and COPD comorbidity showed a significant increase in the intensity of endogenous intoxication with accumulation of medium molecular weight peptides 254 and 280 in the blood (1.8 and 2.0 times, respectively; p < 0.05), which was accompanied by activation of proteolysis (1.4-2.0 times; p < 0.05).
2. Administration of hepatoprotector antral within 30 days led to a significant correction of the damaging effect on the proteolysis system in patients with NASH, obesity and COPD with a significant decrease in the blood content of azoalbumin, azocasein and collagenolytic activity (p < 0.05), accompanied by a significant decrease in endotoxicosis (p < 0.05).
3. Improvements in the effectiveness of NASH and COPD treatment with combined administration of antral and policosanol on the state of proteolysis and endogenous intoxication have been registered by the intensity of azoalbumin lysis (p < 0.05).