Prospects of Application of Bismuth Drugs in the Treatment of H. pylori- Associated Gastric Ulcer in the Case of Patients Undergoing Cytostatic Therapy

The aim of the study was to determine the efficacy of the bismuth drugs in patients with gastric ulcer, receiving cytotoxic therapy. Peptic ulcer, in patients receiving combination chemotherapy, dyspeptic syndrome is characterized by severe, advanced stage and a relatively low degree of chronic gastritis and H. pylori contamination of the gastric mucosa. In patients receiving combination chemotherapy is determined by a significant level of violation of regeneration in the form of atrophy, intestinal metaplasia, dysplasia of the gastric mucosa associated with serious violations of cell homeostasis, under the control of regulatory molecules PCNA, Bcl2, NF-kB and tachykinins (substance P, neurokinin A). In the subgroup of patients receiving regimens as part of peptic ulcer drugs bismuth less determined violations of the chair (p <0.05) were verified lower rates of expression of factor NF-kB (p<0.05). The rate of healing of ulcers in the subgroup of patients receiving the therapy as part of bismuth preparations did not differ significantly (р˃0.05) occasionally scarring ulcers in the subgroup of those treated without drugs bismuth.


Introduction
Peptic ulcer (PU) is one of the most wide-spread diseases in therapeutic and gastroenterologic practice.It is considered that 7-10% of adult population on Earth suffers from peptic ulcer [1].As a rule, PU is associated with H. pylori, infection rate of the population of Russia with which exceeds 50% [1].There are population groups, morbidity rate of which from the point of vie of PU is higher than average rate of the population.The ulcer genesis may include various factors such as intake of medicines (non-steroid anti-inflammatory drugs, glucocorticosterois, cytostatic drugs) [2], tuberculosis [3], chronic obstructive lungs disease, liver cirrhosis, ischemic cardiac disease, anemia [4], cholecystectomy [5], accompanied by duodenal reflux [5].The mentioned patient groups are, as a rule, infected by H. pylori, the role of which in the ulcer genesis may compete with other factors contributing to the forming of peptic ulcers.Pathogenesis of the ulcers connected with the influence of several pathological factors is poorly studied, which prevents from the development of efficient schemes of this suffering treatment.However, availability of various damaging factors in the pathogenesis of PU presupposes the necessity of applying in treatment schemes of cytoprotective drugs, the most prospective of which is bismuth drug [6].It is shown that in appearance and acute study of PU the special place is taken by dysfunction of diffuse endocrine system and expression of molecules, regulating the process of cellular renovation of epithelial cells of the gastric mucous coat (GMC) (Bcl-2, Ki-67) [7].The role of PCNA, Bcl-2, NF-Kb and tachykinins is determined (substance P, neurokinin А) in pathogenesis of erosive form of gastroesophagal reflux disease in the case of patients receiving polychemotherapy (PCT) [8].At that the value of the mentioned regulatory molecules in pathogenesis of peptic ulcer in the case of patients receiving cytostatic therapy is not known [9].Determination of the mentioned ulcer genesis aspects allows to optimize anti-ulcer therapy and increase the living standard of the patients.
Study goal: determining the efficiency of bismuth drugs application in the case of patients having gastric ulcer (GU), undergoing cytostatic therapy.

Material and Methods
Total amount of the examined patients with GU is 82.The first group -30 personsis composed of patients with GU.The second group is composed of 52 patients having GU, suffering from hemoblastosis in the period of full clinical hematological remission of the disease and receiving supporting PCT (GU PCT ).As a control group of the study 30 practically healthy persons were examined complaining insufficiently for setting the diagnosis of functional dyspepsia and lack of macroscopic and microscopic traits of chronic gastritis.
The group of patients with GU has persons having ulcer anamnesis and at the moment of the present examination having recurrence of the disease being infected with H. pylori at the age from 60 to 74.The first group of patients with GU underwent eradication therapy in accordance with Maastricht treaties IV (2010) [10] at the scheme of amoxicillin in the dosage of 1 000 mg 2 times per day, clarithromycin in the dosage of 500 mg 2 times per day and omeprazole in the dosage of 20 mg 2 times per day during 10 days.
The 2 nd group of patients having GU had 2 subgroups distinguished.The first subgroup (GU PCT1 ) -30 persons had eradication therapy of the first line in accordance with the above scheme.The 2nd subgroup (GU PCT2 ) -22 persons had eradication therapy in accordance with the scheme of amoxicillin 1 000 mg 2 times per day, clarithromycin in the dosage of 500 mg 2 times per day, omeprazole 20 mg 2 times per day and bismuth drugs in the dosage of 120 mg 4 times per day during 10 days.In lack of effect of the carried out eradication all the patients were appointed eradication quadrotherapy.If markers of H. pylori retained after the second therapy line, the third treatment scheme was appointed taking into account sensitivity of H. pylori to antibiotics.In the 1st group and 1st subgroup of patients having GU receiving PCT omeprazole was appointed in the dosage of 20 mg 2 times per day up to statement of the fact of the ulcer scarring.In the 2 nd subgroup omeprazole therapy was carried out in the dosage of 20 mg 2 times per day and bismuth drugs in the dosage of 120 mg 4 times per day up to the fact statement of the ulcer scarring.
All the patients having GU were examined in accordance with a single program prior to the appointment of eradication therapy and after healing of ulcer defects and reaching elimination of H. pylori.Control group persons were examined one time.All the patients underwent fibrogastroduodenoscopy added by chromoendoscopy with methylene blue.The diagnosis of GU was set in the presence of ulcer defect after carrying out general morphological examination.Morphological traits of accompanying chronic gastritis were evaluated in accordance with OLGA classification (2008).For determination of H. pylori 2 methods were used: histological with Romanowsky-Giemsa stain, as well as fast urease test.Eradication control was carried out after 4-6 weeks following the intake of antibiotics and no less than in 2 weeks after the last consumption of FAP (First-Aid Package).In accordance with existing recommendations, when ulcers are not scarred during 6 weeks, they are recognized as scarred with difficulty [11].
Evaluation of sizes of ulcer defects was carried out in accordance with the classification of A.L. Grebnev and A.A. Sheptulin (1995).The degree of expression of dyspeptic syndrome was evaluated in accordance with 10-score scale with the help of VAS (Visual Analogue Scale) questionnaire.
Morphological and immunohistochemical studies were carried out in a scientific research institute of obstetrics, gynecology and reproduction named after D.O.Ott in the laboratory of pathomorphology under the guidance of Doctor of Science Emeritus of the Russian Federation, Professor I.M. Kvetnoy.Biopsy materials were taken from mucous coat of antral section and from body of stomach.
The biopsy material was fixed in 10% neutral buffered formalin (рН 7.2), the subsequent treatment was carried out with the help of automatic material conducting station Leica TP1020 in accordance with standard methods producing paraffin blocks.For morphological study the microscopic sections were coloured with hematoxylin and eosin in accordance with standard methods.For As negative control immunohistochemical reaction was carried out without use of initial antibodies.The specificity of antibodies was proved in control experiments.
The studies were carried out on dewaxed and dehydrated microscopic sections with the use of avidin-biotin immunoperoxidase method.Temperature disclosure of antigens was carried out with the use of 0.01М citrate buffer рН=6.0 under pressure.
For the purpose of blocking endogenous peroxidase laboratory glasses were placed in 3% solution of hydrogen peroxide for 10 minutes.For washing tris-NaCl-buffer was used with рН comprising 7.6.As secondary antibodies those conjugated with polymer were used, marked with peroxidase (universal set DAKO EnVision TM + System).
Stain visualization was carried out with the use of DAB+ complex and substrate buffer.
The evaluation of immunohistochemical dye results was carried out with the help of morphometric study using the system of computer analysis of microscopic images applied.The results were considered statistically significant when р was ˃0.05.For more objective evaluation of the results we carried out dynamic comparison of indicators of the 1 st patient group and 1 st subgroup of the 2 nd group, as they were of equal size.We also compared indicators of two subgroups of patients in the dynamics: in acute condition and remission of GU.Accordingly, they represented patients with GU PCT1 and with GU PCT2 .

Results of the Study
Analysis of the carried out studies testifies of similarity of GU clinical evidence in the case of patients receiving PCT (GU PCT1 ), and in the case of the patients with isolated course of GU.Nevertheless, patients with GU receiving PCT reliably more frequently (р˃0.05)complained of epigastric burning, nausea, vomiting, reduction of appetite, getting thinner, disorders of stool.The patients receiving PCT reliably more frequently (р˃0.05)showed diarrheal syndrome and less frequently -constipation (р˃0.05).At that the aggregate level of dyspepsia syndrome in accordance with VAS scale in the case of patients suffering from GU PCT1 was reliably higher (р˃0.05) in comparison with patients having GU.It is necessary to remark that in the case of patients having GU PCT1 reducing of clinical symptoms took place significantly slower (Table 1).At that 13 (43.3%)patients having GU PCT1 showed hardly cicatrizing ulcers compared to the group of patients having GU with the same phenomenon typical only of 4 (13.3%)patients.In the subgroup having GU PCT2 6 (27.3%) persons showed hardly cicatrizing ulcers which was of no significant difference (р˃0.05) from the occurrence of hardly cicatrizing ulcers in the subgroup of GU PCT1 .
In the period of GU remission the group of patients receiving PCT (GU PCT1 ) reliably more frequently (р˃0.05)showed discomfort in epigastric region, belch, epigastric burning, nausea, reduction of appetite, diarrheal syndrome and complaints of weight reduction explained by the effect of the prescribed medicines.The aggregate level of dyspepsia syndrome in the case of patients having GU PCT1 was significantly different (р˃0.05) from that in the case of GU (Table 1).Comparison of clinical traits of the disease in the subgroups of patients having GU PCT1 and GU PCT2 revealed no statistically significant difference (р˃0.05).When reaching remission the group of patients having GU PCT2 significantly less frequently (р˃0.05)showed stool disorder in the form of diarrheal syndrome.Endoscopic examination showed in the case of 9 (30%) patients having GU small ulcer defects, in the case of 13 (43.3%)patientsmedium defects, in the case of 7 patients (23.3%)large defects and in the case of 1 patient (3.3%)huge defects.At that average size of ulcer defect comprised 15.6±0.28mm.Small ulcers were determined in the case of 5 (16.7%) patients having GUpct, medium onesin the case of 14 (46.7%)patients, large onesin the case of 9 (30%) patients and huge onesin the case of 2 (6.7%) patients.Average size of ulcer defect in the case of patients suffering from GU PCT1 comprised 18.3±0.29 mm, which was of no significant difference (р˃0.05) from the size of ulcers in the case of patients receiving no PCT.
Morphological examination of GMC in the case of patients having GU PCT1 reliable more frequently (р˃0.05)revealed atrophic changes of the body of stomach, at that the degree of expression of inflammatory changes was reliably lower (р˃0.05).Thus, patients with GU PCT more frequently (р<0.05)verified II and III stages of chronic gastritis, at the same time I stage was less frequent (р<0.05).The patients with GU PCT significantly more frequently (р<0.05)showed II stage of chronic gastritis and reliably less frequently (р<0.05)-IV stage.Also patients having GU PCT verified more frequently (р<0.05)I stage of H. pylori seeding and reliably more frequently (р<0.05)-II stage of it.In the period of GU remission the frequency of determination of GMC dysregeneratory disorders (atrophy, intestinal metaplasia, dysplasia) in the patient groups having GU PCT1 and GU was not differentiated (р˃0.05) from that in the acute disease period (Table 2).When endoscopic and morphological traits of the disease were compared in the subgroups of patients in the acute phase and remission stage of GU PCT1 and GU PCT2 no significant difference was observed (р˃0.05).Note: "A" letter denotes traits in the period of GU acute stage, "R" letterin the period of remission.Mark "*" denotes actual difference (р˃0.05) between the analyzed symptoms in the acute period and period of the disease remission.Mark "**" denotes acute difference (р˃0.05) between corresponding symptoms in the acute stage of GU and GU PCT1 .Mark "***" denotes acute difference (р˃0.05) between the analyzed symptoms in the period of GU and GU PCT1 remission, accordingly.In the groups of GU PCT1 and GU PCT2 no actual difference was revealed in the analyzed indicators.
Patients having GU PCT1 showed reliably lower (р<0.05)indicators of expression of PCNA, Р substance and neurokinin А and reliably higher (р<0.05)indicators of expression of Bcl-2 and Nf-kB factor in comparison with the group of patients having GU (Fig. 1,2).In the period of GU remission patients of both groups had the tendency of all analyzed immunohistochemical indicators to get normalized.Nevertheless, their values in all cases were reliably different from those of practically healthy persons (Table 3).When comparing the results of immunohistochemical studies in the subgroups of GU PCT1 and GU PCT2 no reliable difference was discovered (р˃0.05) in no trait.When   shows actual difference (р˃0.05) between groups of patients having GU PCT1 and GU PCT2 in the remission phase.

Discussion
Our data testifies of more expressed dyspepsia syndrome of the patients having GU PCT1 , explained by the ability of PCT to have emetogenic effect.The obtained results testify of the fact that in the case of all elderly patients with GU the disease development was pathogenetically connected with the preceding H. pylori-associated atrophic gastritis.It is necessary to remark that patients having GU PCT1 verified a deeper stage (р<0.05)and lower degree (р<0.05) of chronic gastritis against the background of lower degree of GMC seeding (р<0.05) with H. pylori.This testifies of the fact that the basis of chronic gastritis progression of the patients having GU PCT1 is formed not only by an inflammatory process connected with the activity of H. pyloriinfection, but also by chemotherapy cytostatic effect disturbing the processes of cell immunohistochemical study the following initial antibodies were used: Rabbit polyclonal Anti-NF-kB p65 antibody "Abcam" (dilution 1:100, incubation time 1 hour at room temperature); Rabbit monoclonal Anti-Substance P antibody "Abcam" (dilution 1:50, incubation time 1 hour at room temperature); Rabbit polyclonal Anti-Neurokinin A antibody "Abcam" (dilution 1:200, incubation time 1 hour at room temperature); Monoclonal Mouse Anti-Proliferating Cell Nuclear Antigen (PCNA) "DACO" (dilution 1:200, incubation time 30 minutes at room temperature); Monoclonal Mouse Anti-Human BCL2 Oncoprotein "DACO" (dilution 1:100, incubation time 1 hour at room temperature).
consisting of Nikon Eclipse E400 microscope, Nikon DXM1200 digital camera, PC based on Intel Pentium 4 and Vidеotest Morphology 5.2 software.In each case five fields of view were analyzed under magnification of 200х and 400х.Measurement of two parameters was carried outoptical density and the expression area.Optical density was revealed in standard units (s.u.).The area of expression was calculated as relation of the area occupied by immunopositive cells to total area of cells in the FOV expressed in %.FGDS (Fiberoptic Gastroduodenoscopy), morphological and immunohistochemical studies were carried out prior to the beginning of treatment and further on after reaching clinical endoscopic remission of the disease.Data Processing.The obtained data was processed in Microsoft Excel 5,0 for Windows program and SPSS 21.0 for Windows program of Operating System Windows 2010.Average values were determined, as well as standard deviation, arithmetic average error.Normal distribution of the studied indicators was determined based on the criteria of Kolmogorov-Smirnov and Shapiro-Wilk.In most cases close to normal distribution of the studied indicator traits was discovered.When average indicators were compared in the studied groups tcriterion of Student was stating remission in the case of patients having GU PCT2 significantly lower indicators of expression of factor NF-kB were expressed in comparison with those of the patients in GU PCT1 subgroup . .The rest studied immunohistochemical indicators in the remission phase of GU of the persons receiving bismuth drugs in the course of therapy and of the patients receiving no bismuth drugs in the course of therapy were of no statistic difference (р˃0.05).
Mark "*" shows actual difference (р˃0.05) between the group of practically healthy persons and patients having GU; mark "**" shows actual difference (р˃0.05) between the period preceding treatment appointment and the period after reaching the disease remission and elimination of H. pylori; mark "***" shows acute difference (р˃0.05) between groups of patients having GU and GU PCT1 in the acute stage of the disease; mark "****" shows actual difference (р˃0.05) between the groups of patients having GU and GU PCT1 in the remission phase; mark "*****" homeostasis of GMC epithelial cells.This point of view is supported by the discovery of lower values (р<0.05) of proliferative potential of GMC epithelial cells connected with PCNA molecule expression in the case of patients having GU PCT1 ; higher (р<0.05)expression indicators of Bcl-2 and NF-kB, testifying of higher degree of dysregeneratory changes of GMC and relatively low indicators of tachykinins expression (р<0.05),explaining lower degree of expression of inflammatory reaction in the case of such patients.Higher degree of expression disorders of the studied group in regulatory molecules in the case of patients having GU PCT1 is accompanied by a frequent (р<0.05)discovery of deeply aggravated stages of chronic gastritis, intestinal metaplasia and dysplasia of GMC epithelium slowed down by evolution of clinical and endoscopic pattern of GU PCT1 , frequent (р<0.05)occurrence of hardly cicatrizing ulcers.The discovered disorders of cell renewal process are capable of making it harder for GMC to restore even after reaching H. pylori elimination[12].The carried out studies testify that all patients having GU after healing of ulcer defects and reaching H. pylori elimination have the analyzed immunohistochemical indicators which remain changed to a significant extent.At that expression of tachykinins (substance Р, neurokinin А) may keep existing which is described in the literature as the so-called "ex-helicobacter gastritis" capable of long-term persistence, and the disturbed expression of Bcl-2, PCNA, NF-kB progression of chronic gastritis even in the absence of H. pylori[13,14].In the subgroup of patients having GU PCT2 the treatment scheme of whom included bismuth drugs stool disorders occurred reliably less frequently in the form of diarrheal syndrome (р<0.05), which was explained by the ability of this group of drugs to absorb bacterial toxins, inhibit the growth of pathological microflora and reduce the production of anti-inflammatory prostaglandins enhancing intestinal peristalsis[15].The patients receiving bismuth drugs in the treatment scheme reliable lower indicators (р<0.05) of factor NF-kB expression were distinguished, which testified of positive influence of this drug group on cell homeostasis of GMC.At that no reliable difference in evolution of endoscopic and morphological traits of GU in different subgroups of patients having GU was distinguished from the point of view of evolution of endoscopic and morphological evidence of GU in different subgroups of GU patients receiving PCT (р˃0.05).

Table 1
Clinical Symptoms of Patients Having Gastric Ulcer in the Disease Dynamics

Table 2
Morphological Characteristics of Gastric Mucous Coat in the Case of Patients Having

Table 3
Immunohistochemical Indicators of Patients Having Gastric Ulcer in the Disease Dynamics