Evaluation of the effectiveness of the proposed therapy on energy metabolism parameters in patients with chronic liver lesions on the background of secondary immunodeficiency state

Introduction

Currently, due to the pathology of the digestive organs, in particular, the liver and intestines, the morbidity of the population of Donbass has increased significantly for a variety of reasons [1]. This is primarily due to the high level of environmental pollution with xenobiotics and other chemically hazardous substances or radionuclides, as well as the ongoing special military operation on the territory of the Luhansk People's Republic, which negatively affect the state of the immune system and the chromosomal apparatus of cells and cause the formation of immunodeficiency and cytogenetic disorders. Chronic liver disease (CLD) and non-alcoholic steatohepatitis (NASH) are problems, currently the most common in hepatology, as they occur mainly in people who do not abuse alcohol, suffer from type II diabetes mellitus, and obesity [2][3]. Liver damage in patients with NASH is usually characterized by a combination of multiple changes, such as a chronic inflammatory process in the liver parenchyma, which is often manifested by the presence of necrotic alterations, fatty degeneration of hepatocytes, etc. Currently, this pathology is considered an independent disease characterized by a combination of fatty degeneration of liver cells and chronic hepatitis, i.e. the inflammatory and possibly necrotic process in the liver parenchyma [4]. The prevalence of CLD has increased significantly in recent decades. It is considered to be the most common disease among non-alcohol abusers [5][6]. It is known that patients with CLD often have changes in the intestinal microbiota leading to intestinal dysbiosis [7][8]. The presence of intestinal dysbiosis at the same time negatively affects not only the state of hepatic parenchyma and the functional state of the liver as a whole but also the state of the immune system, which leads to the development of secondary immunodeficiency state (SID) [10]. Thus, the development of pathogenetically justified methods of treatment for patients with CLD associated with SID has practical significance.

To date, essential phospholipids affecting the energy metabolism parameters have been used for the treatment of patients with CLD, but the authors’ attention was attracted by a new drug – human placenta hydrolysate (HPH) [11][12]. In addition, the authors proposed to use a modern immunoactive drug azoximer bromide (AB) in the treatment of CLD and SID, because it exerts a significant detoxification and antioxidant activity and has expressed immunotropic effect [13]. Thus, the authors believe that it is reasonable and promising to use a combination of HPH and AB drugs in the treatment of patients with CLD associated with SID. HPH reduces lipid and cholesterol deposition in hepatic cells, increases the activity of tissue respiration, and stimulates hepatocyte regeneration, which is manifested by detoxification properties [14].

The aim of the study was to investigate the effect of the combination of AB and HPH on metabolic processes in patients with chronic liver lesions associated with SID.

Materials and Methods

Two groups of patients with CLD associated with SID, randomized by age, sex, and duration of the disease, who were treated under dynamic observation in the therapeutic department of the 4th City Clinical Hospital of Luhansk in the Luhansk People’s Republic were examined. The patients’ age ranged from 35 to 56 years (mean age – 45±0.18). Patients were divided into two groups: Group I (n=45) – main, and Group II (n=45) – comparison. The criterion for inclusion in the study was the presence of CLD associated with SID with concomitant diagnosis of “Intestinal Dysbiosis”. Patients who abused alcohol, had autoimmune hepatitis, as well as those who had markers of viral hepatitis B or C detected during the ELISA examination, were excluded from the study. The results of the fecal microbiological examination were analyzed [15]. Changes in intestinal microbiota were detected in patients. Almost all patients were found to have grade II dysbacteriosis [16], characterized by a significant increase (p < 0.05) in the number of staphylococci, Candida yeast-like fungi (>10⁵ CFU/g), Proteus spp. (⩾10¹⁰ CFU/g), and E. coli (⩾10¹⁰ CFU/g). All patients were treated according to current clinical guidelines depending on the manifestations of the pathologic process (unstable remission or exacerbation). In addition to conventional therapy, patients of Group I received an additional combination of AB and HPH drugs. Treatment with HPH was administered intravenously by 10 ml (560 mg) of the drug (5 ampoules) dissolved in 250–500 ml of a 5% dextrose solution or physiologic saline solution and administered through the ulnar vein for <1.5–2 hours. Injections were performed three times a week with an interval of two days. The course of treatment included 10 injections. AB was administered in tablets or suppositories 12 mg for 10 consecutive days. Then, it was continued to be administered 10 mg every other day. After that, the regimen was switched to 10 mg twice a week for 2–3 months. Group II patients received milk thistle preparations as a hepatoprotector and did not use any immunoactive drugs. In addition, bifiform forte was mainly used to correct intestinal dysbacteriosis in both groups of patients.

After general clinical and bacteriological examinations, the level of macroergic compounds in the hemolysate of washed erythrocyte suspension was determined in all patients by thin-layer chromatography [17][18]. In particular, the authors revealed the content of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in μmol/L with the simultaneous calculation of erythron energy charge (EEC) as the ratio of ATP: (ADP+AMP). Metabolic processes and their key enzymes of anaerobic glycolysis lactate dehydrogenase (LDG), anaerobic glycolysis and isoenzyme spectrum (LDG_1-5), were studied in a horizontal electrophoresis chamber on a PEF device. Accordingly, the levels of anodic “aerobic” fractions of LDG₁₊₂, intermediate (LDG₃), and cathodic “anaerobic” fractions of LDG₄₊₅ isoenzymes were calculated. All patients were examined twice – at admission to the hospital and before discharge. Statistical data processing was performed using the Statistica 10.0 and Microsoft Excel 2010 software.

Results

Patients from both groups with CLD before treatment complained of general weakness, malaise, poor appetite, decreased efficiency, increased fatigue, negative emotional mood, heaviness or painful sensations in the right subcostal region, and often, also bitterness in the mouth. Most patients had subicteric sclerae, often also bluish (positive Wysokowicz’s symptom) during an objective examination. At palpation, the liver protruded 2–4 cm below the rib edge and was of increased density, the edge was blunt and sensitive. In addition, stool disorders in the form of constipation alternating with diarrhea and flatulence were often noted. At palpation, there was also noted painfulness of the large intestine, which corresponds with the data of Yushchuk et al. [19].

Metabolic homeostasis parameters were studied in all patients with CLD associated with SID before treatment at admission to the hospital (Table 1).

The obtained data showed significant shifts on the part of energy metabolism indicators, first of all, the adenyl system before the beginning of treatment. It is important to note a significant decrease in the ATP content in the blood of the examined patients with a disorder of the adenyl system. Still, in most cases, the levels of ADP and AMP were compensatory elevated, which led to the formation of an imbalance of different adenyl nucleotides. ADP levels were significantly elevated before the beginning of therapy in patients of both groups. A two-fold and higher increase in AMP levels in both groups (p<0.01) indicates significant disturbances of metabolic homeostasis. As a consequence, there was a decrease in the EEC index, indicating a decrease in the energy supply of the patients’ organisms.

Before the beginning of treatment (Table 2), there was an increase in total LDG activity, which averaged 2.58±0.14 μmol/l-sec in Group I and 2.24±0.16 μmol/l-sec in Group II (with a norm of 2.02±0.05 μmol/l-sec), i.e. it was elevated by 1.3 and 1.1 times in different groups (P<0.05). This was accompanied by certain changes in its isoenzyme spectrum, mainly by a decrease in the concentration of “aerobic” fractions of LDG₁₊₂ (in Group I – by 1.7 times, in Group II – by 2.1 times lower than normal) with the growth of “anaerobic” (hepatic) fractions LDH₄₊₅ (in Group I – up to 15.4±0.15%, i.e. 2.5 times higher than normal, in Group II – up to 15.8±0.15%, i.e. 2.6 times higher than normal). A more significant decrease in the ATP level led to the maximum increase in the activity of “anaerobic” LDG fractions, which was shown by individual analysis, and there was a separation of oxidative phosphorylation and switching of energy metabolism to a less efficient pathway of anaerobic glycolysis. In Group I, the relative amount of the sum of LDH₄₊₅ isoenzymes was elevated by 2.5 times (p<0.01), in Group II – by 2.6 times (p<0.01) relative to the norm. In both groups, the absolute content of the sum of “hepatic” LDH₄₊₅ isoenzymes was elevated by 3.25 times (p<0.01) relative to the norm before the treatment. Significant differences between the studied indicators of metabolic homeostasis in patients of both groups were not found (p>0.1).

It should be noted that after treatment completion, there was a clear tendency toward the improvement of adenyl system parameters, first of all, an increase in the ATP level in the blood associated with a decrease in AMP and ADP content. As a result, the EEC value increased, indicating the restoration of the energy supply (Table 3).

In all patients of Group II who received only traditional treatment, after the course of treatment, a repeated biochemical examination showed an increase in the activity of total LDG, as well as its fractions, associated with a progressive decrease in ATP content. At the same time, there was a clear trend toward the improvement of the biochemical parameters studied by the authors in Group I patients who received an additional combination of AB and HPH drugs for treatment.

Similar changes were observed in LDG activity and its isoenzyme spectrum: there was a decrease in the total enzyme activity associated with a clear tendency toward the normalization of its isoenzyme spectrum due to a decrease in the concentration of “anaerobic” fractions with an increase in LDG₁₊₂.

In Group I patients, when studying the state of metabolic disorders parameters, almost complete normalization of the studied parameters after treatment completion was established, which cannot be said about Group II patients who received only conventional drugs in average therapeutic doses in the treatment complex (Table 4).

Discussion

The given data indicate the positive influence of AB and HPH combination on the state of metabolic homeostasis indicators in the studied cohort of patients. There was a tendency toward the normalization of the studied biochemical indices in Group II, but there were differences of the indices both from the norm and from the similar indices in Group I. It is significant that in clinical terms, the detoxication, antioxidant, and immunomodulatory effects of the combination of AB and HPH drugs were accompanied by the improvement of the general condition and well-being of the patients, which was characterized by increased efficiency and appetite, reduction of general weakness and malaise, and normalization of sleep and improvement of mood. As a result of treatment in Group I, heaviness in the right subcostal area, bitterness in the mouth, and subictericity of the sclerae disappeared, and stool normalized. Significantly less pronounced, but still positive dynamics of clinical parameters were also observed in Group II. However, some patients still had complaints about slight heaviness in the right subcostal area, sometimes – about bitterness in the mouth, and in some cases, subicteric sclera was observed. It should be noted that the inclusion of the immunomodulatory drug AB and hepatoprotector HPH into the complex of CLD treatment associated with SID is pathogenetically justified and clinically effective, because such combination promotes normalization of metabolic homeostasis parameters and clinical manifestations of the disease. Consequently, this combination of drugs can be recommended for the treatment of CLD patients with SID.

Conclusion

The inclusion of AB in combination with HPH in the treatment complex of patients with CLD associated with SID eliminates a number of clinical manifestations and metabolic homeostasis disorders, which testifies to the effectiveness of the proposed scheme of therapy.