RU2674920C1 - Method for forecasting the development of nephritis in induced model of systemic lupus erythematosus - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to experimental medicine and concerns the prediction of the development of nephritis in an induced model of systemic lupus erythematosus (SLE). Method consists in the fact that healthy female mice-hybrids (C57BL/6xDBA2)F1 are intraperitoneally injected with lipopolysaccharide at a dose of 0.5 mcg/kg. Then, after 24 hours, the SLE model is induced by intravenous injection of spleen cells of mice of the female DBA/2 line. On the 4^th and 8^th week from the moment of induction of SLE, the index of the ratio of neutrophils to blood lymphocytes (N/L) is determined and, with a significant increase in the value of N/L at the 8^th week relative to the 4^th week from the moment of induction of SLE, the development of nephritis is predicted.

EFFECT: method for predicting the development of nephritis is proposed.

1 cl, 1 tbl, 1 ex

Description

The invention relates to a method for predicting systemic inflammatory conditions in healthy animals that can lead to the development of nephritis in an induced model of systemic lupus erythematosus (SLE).

In recent years, research has been actively developing related to the study of the phenotypic heterogeneity of autoimmune, allergic, and oncological diseases to develop approaches to personalized therapy. An example of the phenotypic heterogeneity of systemic lupus erythematosus (hereinafter - SLE) in humans is the presence of nephritis (more than half of patients) or its absence. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease in which a combination of genetic and epigenetic factors gives a heterogeneous clinical picture: from skin manifestations to kidney damage (in 50% of patients). Lupus nephritis is one of the most serious manifestations and one of the strongest predictors of the adverse outcome of SLE. It is relevant to search for early predictors of nephritis when various parameters of inflammation are evaluated in patients with SLE (US 2008187944, C12Q 1/46, 2008; KR 101320694, C12Q 1/37, 2013).

At present, a view is being formed regarding laboratory animals that takes into account the fact that, despite the initial genetic identity, animals undergo onstogenesis under the influence of various environmental factors stable differing characters through epigenetic mechanisms. Phenotypic heterogeneity is known not only in size and weight among inbred (purely linear) litter mice bred under controlled conditions, but they exhibit significant variability in complex behavioral manifestations. Epigenetic modifications (environmental influences) without changing the nucleotide sequence in the genome, make adjustments to the activity of genes, and these changes are preserved throughout the life of the individual and can be inherited. Epigenetic modifications affect the development of the body, the pace of aging, they can provoke autoimmune and other diseases, which today are designated as “lifestyle” diseases. The nature of epigenetic changes largely depends on the external environment.

Actually, such modifications are one of the most powerful levers of environmental influence on the body (Oeu N., Isbel L, Hickey P., Ebaid B., Whitelaw E. Genetic and epigenetic variation among inbred mouse littermates: identification of inter-individual differentially methylated regions Epigenetics & Chromatin (2015) 8:54 DOI 10.1186 / s13072-015-0047-z). Epigenetic changes may be associated with an increase in the level of LPS in the blood of healthy animals.

It is known that the cause of autoimmune diseases is the presence of lipopolysaccharide (hereinafter - LPS) - endotoxin - a bacterial product from the gastrointestinal tract that appears in the blood of patients and determines the picture of inflammation of a low degree of severity (WO 2008029897, A61K 38/00, 2008). To study the mechanisms of such inflammation, various experimental models are currently being tested. Among these models is the experimental model of low-dose endotoxemia, when healthy volunteer people are given intravenous lipopolysaccharide (LPS). The response to the introduction of LPS in some healthy individuals reveals deviations characteristic of patients with cardiovascular diseases. The response to intravenous administration of LPS, which activates innate immunity and causes specific metabolic effects, is regarded as an integral indicator of the complex physiological, molecular and genetic interactions that determine inflammation in each individual (Patel PN, Shah RY, Ferguson JF, Reilly MP Human Experimental Endotoxemia in Modeling the Pathophysiology, Genomics and Therapeutics of Innate Immunity in Complex Cardiometabolic Diseases. Arterioscler Thromb Vase Biol. 2015; 35 (3): 525-534). The introduction of LPS to intravenously healthy people, to assess their response and identify a predisposition to systemic inflammatory processes, is a more complete reaction compared to in vitro conditions.

A well-known mouse model is C57BL / 6, which transfects the lymphoproliferation gene - Ipr. The introduction of the Ipr lymphoproliferation gene inevitably causes the development of polyclonal activation of B cells, an increase in the titer of anti-DNA antibodies, i.e. the development of systemic lupus erythematosus. The model is used as a method of testing therapeutic agents for the treatment of systemic lupus erythematosus by measuring life expectancy, changes in tissues, organs, cells, changes in serum anti-DNA antibody titer (US 2005066378, A61K 67/027, 2005).

The described methods do not allow predicting a predisposition to systemic inflammatory diseases in potentially healthy organisms.

There is a method for creating an SLE model in a semi-allogeneic model: transfer of spleen cells from female DBA / 2 mice to> female (C57BL / 6 x DBA2) F1 hybrids. After model induction, after 12 weeks in the population of genetically homogeneous F1 hybrids, all mice showed signs of SLE: splenomegaly, hypergammaglobulinemia, production of autoantibodies to DNA, while some mice with more severe symptoms of SLE develop nephritis (SLE ^{nephritis +} ), and in mice with less symptomatic SLE nephritis absent - borehole ^nefrit- (Kolesnikov OP, Kudaeva OT, Wolski NN Gaumann EV, Gavrilova ED, Perminova OM, Demchenko E. N., Kozlov VA An experimental model of the autoimmune process: the role of epigenetic variation spine in mice hybrids population Bulletin of Medical Sciences 2015; 70 (2). 152-158). Assessment of the development of nephritis is made 12 weeks after the induction of the model according to the level of protein in the urine. It can be assumed that the separation of mice into groups of SLE ^nephritis and SLE ^{nephritis +} , equal to approximately 50/50 upon induction of the SLE model, is caused in some mice by epigenetic changes in the innate immunity system, which determine a stronger inflammatory response, which will subsequently lead to the formation of a group SLE ^{jade +} .

The disadvantage of this method is the long time to determine the development of jade with SLE by the level of protein in the urine.

The objective of the invention is to provide a method for earlier predicting the development of jade in an induced model of systemic lupus erythematosus in vivo.

The problem is solved in that in a method for predicting the development of jade in an induced model of systemic lupus erythematosus (SLE), healthy female hybrid mice (C57BL / 6 x DBA2) F1 are injected with a lipopolysaccharide at a dose of 0.5 μg / kg, then after 24 hours SLE model is induced by intravenous injection of spleen cells of mice of DBA / 2 females; at the 4th and 8th week from the time of SLE induction, the neutrophil to blood lymphocyte ratio index (N / L) is determined and with a significant increase in the N / L value by 8 week relative to the 4th week from the moment of SLE products predict the development of jade.

Invention, in our opinion, is new and has an inventive step.

The method is as follows.

1) to 27 healthy female mice - hybrids (C57BL / 6 x DBA / 2) F1 we administer LPS at a dose of 0.5 μg / kg intraperitoneally;

2) after 24 hours, these mice are injected intravenously with spleen cells of mice of the DBA / 2 female line, thus inducing an SLE model;

3) on the 4th and 8th week from the moment of induction of the model, we determine the N / L index (ratio of neutrophils to blood lymphocytes); we estimate the index change at 8 weeks relative to 4 weeks for each mouse individually.

4) in some of the mice, an increase in the N / L index by 25% was detected, and in the other part of the mice, no changes in the index were found (the increase was 1.5% - within the error);

5) 12 weeks after the induction of the model, we measure urine protein in all 27 mice to establish the development of nephritis;

6) it was found that 14 mice have nephritis (protein is greater than or equal to 3 mg / ml), while 13 mice have no nephritis (protein is less than 3 mg / ml);

It is important that an increase in the N / L index reveals a subgroup of SLE ^{nephritis +} mice before detecting proteinuria, which begins to be detected in mice of this group at 12 weeks from the moment of induction of the model. Experience shows that after 8 weeks it is possible to predict the development of nephritis in an induced model of systemic lupus erythematosus in vivo, and not after 12 weeks - as with the known methods.

Example. The dose of LPS is 0.5 μg / kg and the N / L index as an early marker of nephritis.

A retrospective analysis of the average values of leukocytes, neutrophils, lymphocytes in the group of SLE ^nephritis and SLE ^nephritis at 4 and 8 weeks from the moment of induction of the model does not reveal any significant differences. At the same time, the average values and individual fluctuations of the N / L index reveal a tendency to increase in the SLE ^{nephrite +} group compared to the SLE ^nephrite group at 8 weeks compared to 4 weeks from the moment of induction of the model. To normalize the fluctuations of the N / L index in each individual animal, the change (increase / decrease) in the N / L index by the eighth week relative to the fourth week was calculated and it was found that changes in the N / L index in the SLE groups of ^jade _and SLE ^{nephritis +} significantly differ : in SLE ^nephritis mice ^{+ a} significant and significant increase in the N / L index was detected - it averaged 25.1%, in SLE ^nephritis mice ^{there were} no significant changes in the index (an increase of 1.5% - within the measurement error). These changes in groups also relate to frequency characteristics: an increase in the N / L index is detected in 10 out of 14 SLE ^{nephrite +} mice and only 4 out of 13 SLE ^nephrite mice (the level of statistical significance of differences in the index with the presence of proteinuria according to Pearson's criterion x ² corresponds to p < 0.05).

Notes to table 1: n - the number of mice in the group;

in the numerator - the number of mice with proteinuria in the denominator - the number of mice in the group

We believe that a significant increase in the N / L index is associated with the onset of nephritis in the CRD ^{nephrite +} mouse group, which is consistent with human data [Li L, Xia Y., Chen C, Cheng P., Peng C. Neutrophil-lymphocyte ratio in systemic lupus erythematosus disease: a retrospective study. Int J Clin Exp Med. 2015; 8 (7): 11026-31]. The ability to identify a group of CRD ^{nephrite +} mice for 8 weeks by increasing the N / L index relative to 4 weeks seems to be important, because no proteinuria was detected in mice during this period, on the basis of which we divide mice into phenotypically heterogeneous groups of SLE ^{nephritis +} and SLE ^nephritis .

It is important that the administration of LPS to healthy mice at a dose of 0.5 μg / kg does not provoke the development of nephritis, as It is known that the administration of LPS to mice with genetically determined SLE accelerates and aggravates the development of nephritis. Example 2. It was found that after administration of LPS at an dose of 0.5 μg / kg to intact recipients a day before the induction of the model, after 12 weeks, 20 of 38 mice developed jade, 18 mice did not have jade, i.e. the percentage of mice SLE ^{jade +} / SLE ^nephritis was 53/47.

The introduction of LPS in a dose of 0.5 μg / kg a day before the induction of the process does not change the frequency of SLE ^{nephritis +} / SLE ^nephritis , which, under standard experimental conditions, is also approximately 50/50 on average. Thus, after 8 weeks, it is possible to predict the development of nephritis in the induced model of systemic lupus erythematosus in vivo, and not after 12 weeks - as with the known methods.

Claims (2)

A method for predicting the development of nephritis in the induced systemic lupus erythematosus (SLE) model, which consists in injecting 0.5 μg / kg lipopolysaccharide intraperitoneally into healthy female hybrid mice (C57BL / 6 x DBA2) F1, then model is induced after 24 hours SLE by intravenous injection of spleen cells of mice of the female DBA / 2 line, at the 4th and 8th week from the time of SLE induction, the neutrophil to blood lymphocyte ratio index (N / L) is determined and with a significant increase in the N / L value on the 8th week relative to the 4th week from the moment of induction of SLE prog They are responsible for the development of jade. ^- .