RU2780490C2 - Method for use of thrombin and activated protein c as anti-inflammatory remedies - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to the use of thrombin. The use of thrombin at a concentration of 50 NM per 50 thousand cells is proposed for reduction in inflammation with diabetes mellitus on the background of hyperglycemia on an in vitro model.

EFFECT: above-described invention allows for effective reduction in inflammation with diabetes mellitus on the background of hyperglycemia on an in vitro model.

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Description

Technical field

The present invention relates to the field of serine proteases, in particular thrombin and activated protein C (APC), which are protease-activated receptor type 1 (PAR-1) agonists for use in reducing the inflammatory activity of immune cells in the treatment of an inflammatory response. In particular, the invention relates to the treatment of an inflammatory reaction that occurs against the background of complications of diabetes mellitus and other metabolic diseases accompanied by hyperglycemia.

State of the art

This document describes the protective effect of activated protein c (APC) and thrombin in conditions of inflammation that develops against diabetes. Some current approaches to the treatment of diseases accompanied by the development of inflammatory reactions use thrombin inhibitors as concomitant drugs (US patent No. 6362190 B2), which is due to the pro-inflammatory effect of thrombin in high concentrations. In our study, thrombin was used at low (up to 50 nM) concentrations and had the opposite effect, which is the hallmark and basis of the current application.

Under physiological conditions, APC is formed from protein C by proteolytic cleavage, for example, by thrombin. Protein C takes time to activate. Our invention uses already activated protein C, which begins to act immediately after being added to the immune cells under study. The use of the anti-inflammatory and protective action of APS was previously proposed to protect nerve cells from death (CA patent No. 2398929 C). This is applicable for various neurological diseases closely related to inflammatory processes, namely: ischemia, Alzheimer's and Parkinson's diseases, encephalitis, multiple sclerosis, and others. Our invention is aimed at the use of APS to inhibit the inflammatory process specifically in the treatment of diabetes mellitus.

One of the main manifestations of diabetes is a high concentration of glucose in the blood - hyperglycemia. A prolonged increase in glucose levels leads to the emergence and development of pathological processes in the body, such as retinopathy, angiopathy, nephropathy, diabetic foot, etc. [Volozhin, 2006; Fowler, 2008]. These complications, like diabetes itself, are characterized by inflammatory processes, the regulation of which involves different cell types [Wang et al., 2007; Donath et al., 2011]. Patent RU No. 2066550 C1 discusses the use of APS as an agent with analgesic and anti-inflammatory effects. However, diabetes mellitus was not considered by the authors as a condition in which it is possible to use APS as a component of anti-inflammatory therapy. Preservation of the anti-inflammatory effect of APS in diabetes mellitus was demonstrated in our studies.

The therapeutic approach to the treatment of psoriasis, based on the anti-inflammatory and anti-apoptotic properties of APS, is considered in the patent RU No. 2662564 C2. Our proposed approach to the use of APS is also a preventive measure, since the development of skin diseases in pro-inflammatory conditions is one of the comorbidities in diabetes mellitus. Diabetes mellitus can exacerbate existing skin diseases such as psoriasis, eczema [Cohen AD et al., 2007].

The essence of the invention

Thrombin is a key serine protease in blood coagulation. For thrombin, a displaced agonism is shown - a dose-dependent realization of pro- or anti-inflammatory properties. Thrombin in high concentrations has a negative effect on various tissues, leading to cell death. APS, on the contrary, is characterized by anticoagulant activity. APS inactivates factors Va and VIIIa of the blood coagulation system, which leads to inhibition of the propagation phase of blood coagulation. In addition to anticoagulant properties, APS has shown anti-inflammatory and anti-apoptotic properties, realized mainly through PAR-1, as well as participation in the stabilization of the vascular endothelium, stimulation of angiogenesis.

The present invention is based on the discovery of the anti-inflammatory effects of APS (10 nM) and thrombin (50 nM) in conditions of diabetes mellitus, hyperglycemia, and against the background of complications of diabetes mellitus caused by inflammatory reactions. It is known that in diabetic patients there is a significant change in the hemostasis system, a shift in hemostatic balance towards procoagulant factors, which also contribute to the aggravation of inflammation. Our data indicate the possibility of using APS and thrombin at the studied concentrations for therapeutic purposes.

Mast cells (MCs) play an important role in the onset and development of inflammation, including in diabetes. They contain a large number of cytoplasmic granules that store preformed inflammatory mediators. When mast cells are activated by pro-inflammatory stimuli, they degranulate and secrete mediators into the extracellular environment [Silva da et al., 2014]. The level of degranulation serves as a marker of mast cell activation and the intensity of the inflammatory process.

A high glucose level in diabetes potentiates the pro-inflammatory activity of mast cells, causes changes in their ultrastructure, increases their number in the body, and releases pro-inflammatory cytokines from granules [Soma et al., 2002, Donath et al., 2011; Shi et al., 2012]. At the same time, substances secreted by mast cells contribute to the worsening of diabetes and the development of complications [Donath et al., 2011; Nagai et al., 2012]. Thus, there is a loop with positive feedback.

As shown earlier, thrombin and APS are able to regulate mast cell function and, in particular, the secretion of pro-inflammatory mediators. Thrombin and activated protein C hydrolyze a certain peptide bond at the N-terminus of the receptor, which leads to the triggering of various signaling pathways and cellular responses [Mosnier et al., 2012].

Purpose of the invention

Creation of a method that can be used as an effective anti-inflammatory therapy for patients with diabetes mellitus and other metabolic diseases accompanied by a persistent increase in blood glucose levels.

Technical result

In the present study, we compared the contribution of PAR-1 agonists, thrombin and APC, to the regulation of the secretory activity of mast cells and their cultured analogs, RBL-2H3 cells, against the background of the isolated and combined effects of diabetes and inflammation. The level of pro-inflammatory activity of mast cells, which can serve as an indicator of the intensity of the inflammatory process, was assessed by the level of secretion of β-hexosaminidase.

Our study showed that incubation of mast cells derived from animals with 24-hour thioglycolate-induced inflammation with thrombin at a concentration of 50 nM for 1 hour reduced the level of β-hexosaminidase secretion of these cells by 44.4% (Fig. one).

At the same time, incubation of mast cells obtained from animals with 7-day streptozotocin-induced diabetes mellitus with thrombin at a concentration of 50 nM for 1 hour reduces the level of β-hexosaminidase secretion of these cells by 70.2% (Fig. one).

Under the combined action of inflammation and diabetes, the level of cell secretion in the presence of thrombin at the same concentration decreases by 70.0% (Fig. 1).

In a similar experiment on RBL-2H3 cells, activated protein C at a concentration of 10 nM after an hour of incubation with cells also caused a decrease in the level of β-hexosaminidase secretion by cells cultured in a medium with a high (25 nM) glucose concentration for 7 days (hyperglycemia model) , by 10.4% (Fig. 2)

This anti-inflammatory effect of activated protein C was preserved for cells cultured under the same conditions and additionally subjected to calcium-induced activation (Fig. 2).

At the same time, neither activated protein C nor thrombin in our experiments had any statistically significant effects on the level of spontaneous secretion of β-hexosaminidase by mast cells obtained from untreated animals or RBL-2H3 cells cultivated under normal conditions (Fig. 1, Fig. 2).

Thus, serine proteases of hemostasis, thrombin and activated protein C (APC) in concentrations determined by us can be considered as an active ingredient for anti-inflammatory therapy in diabetes mellitus, as well as complications of diabetes mellitus associated with the development and course of inflammatory processes.

Brief description of the graphic material

In a graphic that illustrates embodiments of the invention:

in fig. Figure 1 shows a comparison of control-normalized (untreated) levels of beta-hexosaminidase secretion by mast cells, both with and without thrombin at 50 nM, for groups of cells derived from untreated or inflammatory animals. , or with experimental diabetes, or with the combined effect of these factors;

in fig. Figure 2 shows a comparison of levels of beta-hexosaminidase secretion normalized to the level of secretion in the control (without any interventions) by RBL-2H3 mast cells both in the presence of activated protein C at a concentration of 10 nM and without it, for groups of cells without treatment or with caused by activation (inflammation model), or with experimental hyperglycemia, or with the combined action of these factors;

Detailed description of the invention

Research objects.

In experiments in vivo were used male Wistar rats at the age of 3-4 months, weighing 250-300 grams. The animals were kept in the vivarium of the Department of Human and Animal Physiology at a temperature of 23°C, a standard daily cycle of illumination (12 h/12 h), dry food and water were provided to the animals ad libitum. All animal experiments were performed in accordance with the ethical principles and guidelines recommended by the European Science Foundation (ESF) and the Declaration of Humane Treatment of Animals.

In vitro experiments were carried out on RBL-2H3 cells (Rat Basophilic Leukemia cell line), cultured analogues of mast cells.

Model of experimental diabetes induced by streptocin administration.

Experimental type 1 diabetes was induced by a single intraperitoneal administration of streptozotocin (Sigma, USA) at a dose of 60 mg/kg. This method of diabetes induction is used in many studies. The introduction of streptozotocin into the peritoneal cavity causes an increase in blood glucose concentration up to 25 mM after 2 days [Coutinho et al., 2014; Dashtban, Sarir, Omidi, 2016; Zhao et al., 2016] A solution of streptozotocin at a concentration of 30 mg/ml was prepared in 0.1 M citrate buffer, pH 4.2, immediately before administration. Animals were deprived of food 8 hours before the injection of the solution and 4 hours after the injection (total fasting time - 12 hours). Control animals were injected with 0.2 ml of citrate buffer. The blood glucose level was measured one week after the administration of streptozotocin using a glucometer (OK Biotech Co. Ltd, Taiwan). An animal was considered to have entered diabetes if the blood glucose level exceeded 16.7 mM (300 mg/dL).

Model of acute peritonitis caused by the introduction of thioglycolate.

Experimental peritonitis was induced by a single injection of a thioglycolate solution (Sigma-Aldrich, USA) into the animal's peritoneal cavity. The use of thioglycolate as an inflammatory agent has been described in a large number of studies [Dugina et al., 2003; Hecking et al., 2003; Umarova et al., 2006]. A 4% thioglycolate solution was prepared using phosphate buffered saline (PBS, Biorad, USA) of the following composition (in mM): 137 NaCl, 2.7 KCl, 10 Na2HPO4, 1.76 KH2PO4, pH 7.4. Phosphate buffered saline was passed through an antibacterial filter (0.22 µm, GE Water & Process Technologies, USA) before being added to thioglycolate. A solution of thioglycolate was prepared immediately before administration. To induce peritonitis, the animals were injected intraperitoneally with 5 ml, if the animal's weight did not exceed 300 grams, or 10 ml (the animal's weight was more than 300 grams) of a thioglycolate solution. Control animals received phosphate-buffered saline passed through an antibacterial filter, the volume of which was calculated similarly.

Method for the isolation of mast cells from the abdominal cavity of rats.

, Thon, 1959] в модификациях [Babkina и др., 2015; Makarova и др., 2006; Бабкина и др., 2016]. Методика была частично изменена и адаптирована под настоящее исследование.The isolation technique described in [

, Thon, 1959] in modifications [Babkina et al., 2015; Makarova et al., 2006; Babkina et al., 2016]. The methodology was partially modified and adapted for the present study.

The animals were anesthetized with chloroform, decapitated, and bled. 10 ml (24 hours after inflammation induction) of 145 mM NaCl solution containing 10 mM HEPES (pH 7.4) was injected into the abdominal cavity, the abdomen was gently massaged for 1–2 min. The peritoneal fluid was collected, the cells were placed on ice for the duration of the work, and then centrifuged for 5 min at 157 G, after taking 10 µl for staining and cell counting. The supernatant was discarded, the pellet was suspended in 2 ml of buffer. Mast cells of animals without inflammation were purified on a two-stage ficoll gradient (35% and 25%) by centrifugation at 4°C and 250 G for 10 minutes. To isolate mast cells from animals with induced peritonitis, a ficoll gradient (28.6% and 21.8%) was used, centrifuged at 4°C and 294 G for 17 minutes. A two-step ficoll gradient was prepared as follows: a layer of 35% ficoll (3 ml) was covered with 2 ml of 25% ficoll dissolved in Na-HEPES buffer (pH 7.4) containing 145 mM NaCl, serum albumin (1 mg/ml) and glucose ( 2 mg/ml). Ficoll gradient 28.6% and 21.8% were prepared similarly. The pool of mast cells during centrifugation is concentrated at the interface of ficoll solutions. Cells were carefully collected with a 25% Ficoll pipette, transferred to a balanced solution tube (5 ml) and washed. A balanced solution (pH 7.4) was used with the following composition (in mM): NaCl 145, HEPES 10, KCl 5, CaCl2 1, MgCl2 1, glucose 5, 0.1% serum albumin. After each washing, the cells were centrifuged for 10 min at 250, 210, and 174 G, respectively. The mast cell pellet from the last wash was resuspended in a small volume of balanced solution (0.5 ml). Mast cells were counted in a Goryaev chamber, cells were preliminarily stained with toluidine blue. The dye was mixed with the cell suspension in a ratio of 1:1. After 10 minutes, the number of cells in five large squares was counted. Using the formula n(cell/ml)=N*50*2*1000, where N is the number of cells in five large squares, the concentration of cells in the resulting suspension was calculated. After counting, the cell suspension was divided into samples and diluted with a balanced solution based on the required number of cells in the experiment.

experimental impact.

After calculating the concentration of isolated mast cells, they were distributed into separate samples, 50 thousand cells per sample (sample volume was 250 μl). To assess the effect of PAR-1 agonists on the functioning of mast cells, they were incubated for 1 hour at 37°C in the presence of APS (10 nM) and thrombin (50 nM and 100 nM). After adding the agonists, the samples were mixed. No agonists were added to the control sample. After incubation with PAR-1 agonists, the samples were placed on ice for 10 minutes, then centrifuged at 157 G for 5 minutes in order to pellet the cells. The supernatant was taken for subsequent evaluation of released β-hexosaminidase and histamine. The selected samples were placed on ice prior to analysis or frozen at -20°C if the analysis was performed on another day. To destroy the cells, they were resuspended in a 0.1% solution of Triton X-100 prepared in a balanced solution (see method for isolation of mast cells). Cells were incubated with Triton for 15 minutes at 37°C and then centrifuged at 1576 G for 10 minutes. The supernatant was taken to evaluate the remaining β-hexosaminidase and histamine in the cells. Selected samples were frozen at -20°C if the analysis was performed on a different day.

Method for determination of β-hexosaminidase secreted by peritoneal mast cells and RBL-2H3 cell line.

To determine β-hexosaminidase, we used the method proposed by [Schwartz, Austen, Wasserman, 1979], in our modification. The degree of secretion of peritoneal mast cells and RBL-2H3 cells was determined by the cleavage of a specific chromogenic substrate - 4-nitrophenyl-N-acetyl-β-D-glucosaminide. The substrate at a concentration of 4 mM, dissolved in 0.04 M citrate buffer (pH 4.5) containing 0.9% NaCl, was added in a ratio of 1:1 to the obtained samples. Incubation with the substrate was carried out in a 96-well plate for 2 hours at a temperature of 40°C. The reaction was stopped by adding an equal volume of 0.2 M Glycine-NaOH (pH 10.7) buffer. After the addition of the stop reagent, the solution acquired a bright yellow color, the intensity of which was proportional to the amount of β-hexosaminidase contained in it. The optical density of the samples was measured on a spectrophotometrically measured immunoassay analyzer "Uniplan" AIFR-01 (CJSC "Pikon", Russia) at λ=405 nm.

The amount of secreted β-hexosaminidase was expressed as a percentage of the enzyme in a given aliquot of cells and was calculated by the formula:

SV=X/(X+Y)*100%, where SV is the level of secretion, X is the optical density of the sample containing the enzyme released during cell incubation with agonists, Y is the optical density of the sample containing the enzyme released after cell destruction.

Maintenance of the RBL-2H3 cell line.

We used RBL-2H3 cells, cultured analogs of mast cells. This cell line was derived from Wistar rats with chemically induced basophilic leukemia. Cells have the ability to degranulate, i.e. release of a number of substances associated with immune reactions (histamine, β-hexosaminidase) and are used as cultured analogs of mast cells [Baumgartner et al., 1994; Wolfe et al., 1996].

Cells were grown on culture mats 25 cm2 ⁱⁿ α-MEM medium (PaneCo, Russia) containing 0.5 mM L-glutamine, 10% inactivated fetal bovine serum (HI-FBS), and 100 U/mL penicillin/streptomycin (Gibco ", USA). The cells were passaged once every three days, the multiplicity of sieving was 1:4-1:8. Cell reseeding was performed as follows: the culture medium was completely drained, replacing it with Versen's solution (PaneKo, Russia). After washing the cells twice, the solution was completely drained and filled with 1 ml of 0.05% trypsin-EDTA (Sigma) and left in an incubator at 37°C for 7 minutes. Next, up to 5 ml of α-MEM medium was added, suspended, washing the cells from the mattress wall. After that, the cell suspension was completely taken away and 1/4 of the suspension was returned back to the mattress. The rest of the cells were scattered on 96- and 48-well plates and on glasses (glassbottom) for further experiments. Subsequently, RBL-2H3 cells were grown in an incubator at 37°С and 5% CO2; The medium was changed every 2-3 days. 24 hours before the experiment, the medium in the plates was replaced with serum-free medium. After 15-18 passages, the ability to degranulate (secretion of histamine) decreases.

At the end of all experiments, the cells were subjected to cryopreservation in a growth medium containing 5-8% DMSO, 2×106 cells per ampoule. Viability after cryopreservation 80% (0 passage stained with trypan blue).

Experimental effect on RBL-2H3 cells.

Modeling of experimental hyperglycemia was carried out by growing a cell culture in a medium containing glucose at a concentration of 4.5 g/L (25 mM) during the entire duration of the experiment (>4 days). The control group of cells was kept in a medium with a glucose concentration of 1 g/l (5.5 mM). Inflammation was modeled by incubating cells with 50 nM phorbol ester (PMA) and 50 nM calcium ionophore (A23187) for 24 hours at 37°C. To assess the effect of APS and thrombin on the level of secretion of RBL-2H3 cells for an hour at 37°C, they were incubated with the test substances. After incubation, the cells were placed on ice to stop the reaction. Subsequent analysis of the release of β-hexosaminidase was performed similarly to the protocol described above for mast cells.

Statistical data processing.

Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA). The exclusion of outliers was performed based on the ROUT test (Q=1%). Distributions were tested for normality using the D'Agostino & Pearson normality test and Shapiro-Wilk normality test. If the number of obtained values was not sufficient to test for normality, the distribution was considered normal. There is a large amount of data in the literature indicating that, in large samples, the amount of secretion by mast cells of both histamine and β-hexosaminidase has a normal distribution. [Umarova et al., 2011; Tang, 2012; Fukuishi et al., 2014] Data from the current study were presented as mean ± standard error of the mean. The number n in each group is the number of independent experiments. Differences between groups were calculated using a T-test for unrelated samples, using Welch's correction for inequality of sample variances, or one-way ANOVA with Dunnett's correction for multiple comparisons. Differences were considered significant at p<0.1.

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Claims (1)

The use of thrombin at a concentration of 50 nM per 50 thousand cells to reduce inflammation in diabetes mellitus against the background of hyperglycemia in an in vitro model.

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