RU2205658C1 - Method of correction of biochemical and immunological disturbances at condition of experimental acute toxic hepatopathy accompanying with enhancement of immunological reactivity - Google Patents

Abstract

FIELD: medicine, hepatology, immunology, biochemistry. SUBSTANCE: invention relates to development of method for correction of biochemical and immunological shifts at conditions of acute toxic hepatopathy accompanying with enhancement of immunological reactivity. For this purpose experimental animal is administrated by parenteral (intraperitoneal) route tripeptide H₂N-Gly-L-His-L-Lys-COOH dissolved in sodium chloride solution in the dose 1.5 mcg/kg of body mass, 10 times with interval for 24 h between injections. Method provides arresting hyperactivation of immune system that inhibits the development of autoimmune process. EFFECT: enhanced effectiveness of method. 2 tbl, 1 dwg, 1 ex

Description

 The proposed method relates to medicine, namely to hepatology and immunology, and can be used for the correction and prevention of biochemical and immunological changes in toxic hepatopathies.

 The reactivity of the immune system in various forms of liver pathology plays a significant role in the pathogenesis of the disease, which is due to the direct influence of the body’s immune system on the development of autoimmune complications of toxic and infectious liver lesions, on the transition of the acute form of liver disease to chronic and cirrhosis of the liver (Loginov A.S. Immunological aspects of chronic liver diseases. // Wiss. Beitr. M.- Luther-Univ. Halle-Wittenberg R.-1987.- 99.-P.192-198.), As well as in maintaining the chronic course of the pathological process (Yakhontova OI, Karashurova L. L., Starkova SA Some immunological changes and their diagnostic value in chronic liver diseases. // Doctor. Case. -1988.- 4.- S. 39-42.) Many questions connections of the liver and the immune system are solved experimentally when modeling the pathological process in the liver using various hepatotropic substances (Alekseeva I.N., Bryzgina T.M., Pavlovich S.I., Ilchevich N.V. Liver and immunological reactivity. -Kiev: Science. Dumka, 1991.-C.5). The reaction of the immune system depends not only on the damaging agent, but also on its dose, multiplicity and route of administration. Under the conditions of toxic hepatopathies, one can expect a decrease in immunological reactivity (A. Molotkov, Violations of the physiological mechanisms of the regulation of the immune system in acute poisoning with organophosphorus compounds karbofos: Abstract for the degree of candidate of medical sciences. - Saratov, 2002.- P. 20 ), and its increase (Konoplya A.I. Endogenous immunomodulators as a factor in preserving homeostasis in liver pathology: Abstract of thesis for the degree of Doctor of Medical Sciences. - Kiev, 1989.-S.39). Such dysfunction in the immune system requires appropriate immunocorrective therapy (Alekseeva I.N., Bryzgina T.M., Pavlovich S.I., Ilchevich N.V. Liver and immunological reactivity. -Kiev: Science. Doom-ka, 1991. - S.43-45). It can be effective only if the stage of the disease, the nature and extent of immune disorders are strictly taken into account. Since under the conditions of toxic hepatopathies a different reaction of the immune system can be observed, the effectiveness of treatment in some cases with immunostimulants and in others with immunosuppressants has been shown. Immunostimulants are effective in combating immunodeficiencies observed in some forms of liver pathology, and for activating regenerative processes in the organ. But the use of immunostimulants (thymogen) in conditions of liver pathology namely with increased reactivity of the immune system and high levels of immunoglobulins in the blood often leads to an exacerbation of the pathological process (B.I. Kuznik, V.G. Morozov, V.Kh. Khavinson. Cytomedins 25- summer experience in experimental and clinical research. St. Petersburg .- "Science" .- 1998.-S.197-198.). Immunosuppressants are indicated in order to avoid the damaging effects of immune mechanisms on the liver (Alekseeva I.N., Bryzgina T.M., Pavlovich S.I., Ilchevich N.V. Liver and immunological reactivity. - Kiev: Science. Doctrine , 1991.-S.43), since the activation of the immune response may indicate the launch of autoimmune mechanisms of liver damage (Ibid., P. 77).

The closest correction method in conditions of acute toxic liver damage CCL ₄ (carbon tetrachloride (TCM), another name for carbon tetrachloride (ChCU), is the use of a polysaccharide complex extracted from the roots and rhizomes of the six-petaled meadowsweet (PSK LS), which has immunosuppressive activity (Mansimova O. V. Abstract of dissertation for the degree of Candidate of Medical Sciences (Kursk, 1999, pp. 12-16). The polysaccharide complex of rhizomes and roots of meadowsweet contains six glucose - 13.6%, arabinose - 8.5%, xylose - %, galactose -%, mannose - 3.5%, rhamnose - 7.1%. Under conditions of increased immunological reactivity caused by the introduction of CCL ₄ , LSC UCS shows a significant immunocorrective effect, that is, reduces the increased immunological reactivity to the level of healthy animals. normalization of immunological reactivity is accompanied by a decrease in the severity of biochemical symptoms of liver damage: cytolysis syndromes (activity of alanine and aspartate aminotransferases), cholestasis (concentration of bilirubin, cholesterol) and hepatosuppressive s ndroma (fibrinogen concentration), which indicates an improvement in the liver. Thus, in this correction method, hyperactivation of the immune system is limited, which can lead to the development of autoimmune complications, chronicity of the process and deterioration of the liver, but immunodeficiency does not occur, since the reactivity of the immune system decreases to the level of healthy animals, but not lower than it.

 The disadvantage of this correction method is the complexity of obtaining PSK LSH, the difficulty in standardizing it, since the percentage of active substances in PSK LSh may depend on the quality of the initial raw materials and the conditions of selection, in addition, it is not clear what is the main active substance in this complex, from which its effects depend on the exact percentage of monosaccharides or their sequence in polysaccharides.

 The objective of the invention is to develop a method for the correction of biochemical and immunological changes in experimental acute toxic hepatopathies, accompanied by an increase in immunological reactivity.

The objective of the invention is achieved in that an experimental animal (rat) with toxic hepatopathy, accompanied by increased immunological reactivity, is administered parenterally (intraperitoneally) dissolved in physiological saline (RF) sodium chloride, the suppressing immune response tripeptide H ₂ N-Gly-L-His-L- Lys-COOH (chemical name is glycyl-L-histidyl-L-lysine) (hereinafter referred to as Gly-His-Lys) at a dose of 1.5 μg / kg body weight ten times, with an interval of 24 hours between injections.

 In our correction method, we use a tripeptide that has a weak immunosuppressive effect, and such a dose and a frequency of its administration are selected that, without causing immunodeficiency, it limits the hyperactivation of the immune system. It can be argued that such immunocorrection positively affects the condition of the liver, since the severity of the biochemical symptoms of its defeat (inflammation syndrome and partially cholestasis) decreases.

This peptide is isolated from blood plasma and its stimulating effect on hair growth, collagen synthesis by fibroblasts, and the accumulation of intercellular substance of connective tissue is known, which largely explains the accelerated healing of wounds and ulcers under its influence and on the basis of which there are suggestions about the physiological role of this peptide, such as on a wound healing stimulator (Maquart FX, Pickart L., Laurent M., Gillery P., Monboisse JC, Borel JP. Stimulation of collagen synthesis in fibroblast cultures by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu ^{2 +} . // FEBS Lett. 1988. Vol. 238. N2. P.343-346; Trachy RE, Fors TD, Pickart L., Uno H. The hair follicle-stimulating properties of peptide copper complexes. Results in C3H mice. // Ann. NY Acad. Sci. 1991. Vol. 642. P.468-469.) Gly-His-Lys binds bivalent copper and can thus be involved in the activation of copper-containing enzymes thereby providing antioxidant processes, cell bioenergetics, synthesis of elastin, collagen, catecholamines (Maquart FX, Bellon G., Chaqour B., Wegrowski J, Patt LM, Trachy RE, Monboisse JC, Chastang F, Birembaut P, Gillery P. In vivo stimulation of connective tissue accumulation by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu ²⁺ . // J. Clin. Invest. 1993. Vol 92. N5. P. 2368-2376; Linder MC Biochemistry of Copper. E. Freiden, Ed. Plenum Press. 1991).

 At the moment, since the chemical formula of this peptide is precisely known, it can be synthesized in any laboratory for the synthesis of peptides or purchased from companies engaged in the supply of biologically active compounds (Sigma, ICN, Merk, Bachim, etc.). We used a preparation synthesized at the Research Institute of Chemistry of St. Petersburg State University.

 The drawing shows a histogram showing the immunocorrective effect of the Gly-His-Lys peptide under toxic liver damage with carbon tetrachloride.

 The method is as follows. As a model of acute toxic hepatopathy (OTG), accompanied by stimulation of immunological reactivity, we selected a model of hepatopathy caused by tetrachloromethane poisoning (TCM), since this model has a large similarity between the mechanisms of development of liver damage with clinical forms of acute, chronic hepatopathy and liver cirrhosis of various etiology in humans (Blyuger A.F., Major A.Ya., Gorshtein E.S. et al. Characterization of violations of the integrity of the membranes of liver cells in some types of organ damage. // Success and hepatology. / Under the editorship of A.F. Blueger.-Riga.-1981.-Iss. 9.-C.5-24.). Experimental animals (rats) were intramuscularly injected with a 50% solution of TXM in vegetable oil at a dose of 3 ml / kg body weight for 5 consecutive days. Simultaneously with the intake of hepatotropic poison, before and after it, ten times (once a day), 0.1 ml of a solution containing Gly-His-Lys peptide at a dose of 1.5 μg / kg of mass dissolved in a sterile isotonic chloride solution is injected intraperitoneally sodium. With this multiplicity of administration, the peptide has a corrective effect on a number of biochemical parameters, such as the level of beta-lipoproteins and the severity of the thymol test, as well as a weak immunosuppressive effect, which, without significantly weakening the function of the immune system, prevents a further increase in immunological reactivity, which can lead to hyperactivation and the possible development of autoimmune complications. With the introduction of the peptide in the same dose, but five times normalizing biochemical shifts, the effect is less pronounced, and the immunological reactivity increased under conditions of TCM poisoning practically does not decrease. Exceeding the recommended dosage by more than 10 times (doses of 50 and 150 μg / kg) is accompanied by a more pronounced suppressive effect of the peptide and a simultaneous increase in biochemical disorders, such as the activity of alanine and aspartate aminotransferases, the concentration of bilirubin, and beta-lipoproteins in the blood, as well as the severity thymol test, which indicates a deterioration in the functional activity of hepatocytes.

Concrete example
Wistar rats were divided into groups of 8-10 rats each. The groups of animals are presented in Table 1. The Gly-His-Lys peptide was administered to animals ten times (once a day) intraperitoneally at a dose of 1.5 μg / kg of body weight and a 100 times higher dose of 150 μg / kg. OTG was caused by five-fold (once a day) intramuscular injection of a 50% solution of TCM in vegetable oil at a dose of 3 ml / kg of body weight, alternating between the paws. The peptide was administered before and simultaneously with the administration of TXM. The day after the last administration of TCM, rats were sacrificed by bleeding under ether anesthesia and blood serum was obtained, which was used for biochemical studies. Under conditions of OTG, an increase in the activity of alanine and aspartate aminotransferases, bilirubin levels, beta-lipoproteins and a thymol test is observed, which indicates liver damage and the development of cytolysis, cholestasis and inflammation syndromes. Under these conditions, the corrective effect of the peptide at a dose of 1.5 μg / kg of mass on the concentration of beta-lipoproteins and the level of thymol sample was manifested, as well as a tendency to normalization with respect to other biochemical parameters, while the use of the peptide at a dose of 150 μg / kg mass led to increased biochemical disturbances (table 1).

The same groups of animals were taken to study the state of immunological reactivity. The intensity of the humoral immune response (GMO) was judged by the number of antibody-forming (AOK) and rosette-forming (ROCK) cells in the spleen on the 5th day after immunization of experimental animals (rats) (K. Malberg, E. Siegl. Immunological methods. / Ed. G. Frimel; Translated from German by A.P. Tarasov. - M., 1987-p. 273-282.). Sheep erythrocytes (EB) were used as antigen. The peptide was administered both before and after immunization according to the scheme in table 2
The results of the experiment are presented in the drawing. The introduction of TCM is accompanied by the development of OTG and the stimulation of GMOs on T-dependent antigen-EB. The introduction of the peptide under these conditions reduces immunological reactivity, which can be used for immunological correction.

 Thus, the use of the proposed tripeptide at a dose of 1.5 μg / kg ten times intraperitoneally in experimental animals (rats, mice) significantly reduces the increased immunological reactivity, thereby preventing its hyperactivation and the development of autoimmune complications, and also normalizes a number of biochemical parameters of blood serum, which indicates the corrective effect of the peptide on the functional activity of hepatocytes. Thus, the Gly-His-Lys tripeptide with this method of administration has a simultaneous corrective effect both in relation to the functional activity of hepatocytes and in relation to immunological reactivity.

Claims (1)

A method for the correction of biochemical and immunological disorders in experimental acute toxic hepatopathy accompanied by an increase in immunological reactivity, characterized in that the suppressive immune response tripeptide H ₂ N-Gly-L-His-L-Lys is administered parenterally once a day for ten days in a row -COOH at a dose of 1.5 μg / kg of body weight.

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