EffEct of β-amyloid pEptidE 42 on thE dynamics of expression and formation of Аβ 40 , il-1 β , tnf α , il-6 , il-10 by pEriphEral blood mononuclEar cElls in vitro and its corrEction by curcumin

The toxic effect of Аβ-oligomers accompanies chronic inflammation, with cytokines as main mediators. Therefore, the cytokine link of inflammation becomes a new target on the way to restrain amyloidosis. The aim of the study was the effect of aggregated Аβ42 on the dynamics of expression and formation of endogenous Аβ40 and cytokines (IL-1β, TNFα, IL-6, IL-10) by peripheral blood mononuclear cells in vitro and its correction by curcumin. A suspension of mononuclear cells isolated ex tempore using ficoll-urografin gradient from venous blood samples of healthy volunteers were used to study the effects of Аβ42 (15 nM), curcumin (54 pM), and their combined action (at similar concentrations) in time dynamics: 0, 1, 3, 6 and 24 h incubation at 37 °C. Polymerase chain reaction with appropriate primers was used to determine the relative expression of mRNA for AβPP, TNFα, IL-1β, IL-6, IL-10 and enzyme-linked immunosorbent assay – to determine the content of Аβ40 and cytokines in mononuclear suspension during all periods of incubation. The individual dynamics AβPP and cytokine expression was shown under the action of the Aβ42, which had influence on the content of Aβ40, TNFα, IL-1β, IL-6 and IL-10 in mononuclear suspension. Curcumin displayed the inhibitory effect on gene expression of AβPP, TNFα and IL6, which resulted in the decrease of the level of these two cytokines and Aβ40. Thus, the dynamics of anti-inflammatory effect of curcumin in vitro for transcriptional and translational levels of cytokine’s formation by mononuclear cells was shown in the work. Direct inhibitory effect of curcumin on the concentration of endogenous Aβ40 during the 24 h incubation in conditions of toxic action of Aβ42 aggregates was established.

UDC 616.894-092:615.3The toxic effect of Аβ-oligomers accompanies chronic inflammation, with cytokines as main mediators.Therefore, the cytokine link of inflammation becomes a new target on the way to restrain amyloidosis.The aim of the study was the effect of aggregated Аβ 42 on the dynamics of expression and formation of endogenous Аβ 40 and cytokines (IL-1β, TNFα, IL-6, IL-10) by peripheral blood mononuclear cells in vitro and its correction by curcumin.A suspension of mononuclear cells isolated ex tempore using ficoll-urografin gradient from venous blood samples of healthy volunteers were used to study the effects of Аβ 42 (15 nM), curcumin (54 pM), and their combined action (at similar concentrations) in time dynamics: 0, 1, 3, 6 and 24 h incubation at 37 °C.Polymerase chain reaction with appropriate primers was used to determine the relative expression of mRNA for AβPP, TNFα, IL-1β, IL-6, IL-10 and enzyme-linked immunosorbent assay -to determine the content of Аβ 40 and cytokines in mononuclear suspension during all periods of incubation.The individual dynamics AβPP and cytokine expression was shown under the action of the Aβ 42 , which had influence on the content of Aβ 40 , TNFα, IL-1β, IL-6 and IL-10 in mononuclear suspension.Curcumin displayed the inhibitory effect on gene expression of AβPP, TNFα and IL6, which resulted in the decrease of the level of these two cytokines and Aβ 40 .Thus, the dynamics of anti-inflammatory effect of curcumin in vitro for transcriptional and translational levels of cytokine's formation by mononuclear cells was shown in the work.Direct inhibitory effect of curcumin on the concentration of endogenous Aβ 40 during the 24 h incubation in conditions of toxic action of Aβ 42 aggregates was established.K e y w o r d s: curcumin, β-amyloid peptides 40 and 42, cytokines, mRNA, human peripheral blood mononuclear cells.

EffEct of β-amyloid pEptidE 42 on thE dynamics of expression and formation of Аβ
T he family of β-amyloid peptides (Аβ) includes peptides with 38-43 amino acid residues (a.r.) being a product of amyloidogenic processing of the precursor protein of β-amyloid peptide (АβРР).Soluble Аβ 40 in standard conditions performs a trophic function in neurons [1] and only with manifestation of protein conformation disease (amyloidosis) its aggregates acquire toxic properties [2].The Аβ isoform with 2 additional a. r. at C-end of this peptide (Аβ42) is more aggregation aggressive; the above acid residues increase its hydrophobicity and thus determine high bias towards formation of insoluble fibrils [3].β-Amyloid pep-tides acquire toxic properties owing to their own oligomerization.Rather soluble Аβ oligomers than their monomers or insoluble amyloid fibrils are responsible for neurotoxicity and synaptic dysfunction under amyloidosis [4].
The toxic effect of Аβ-system which produces the excessive number of inflammation cytokinesinterleukins-1β, -6, -8 (IL-1β, -6, -8), tumor necrosis factor α (TNFα), etc. [5], accompanies the course of chronic inflammatory process, cytokines being its main mediators.Hyperactivity of mononuclearphagocytic link of the immune system appears as independent motive force of amyloidosis process.It has doi: http://dx.doi.org/10.15407/ubj88.01.109 been found out that cytokines affect metabolism of AβPP, activating its synthesis and processing in amyloidogenic way with formation of Aβ.IL-1 and IL-6 can regulate АβРР synthesis both transcriptiona lly and translationally, depending on the cell type [6][7].Certain combinations of cytokines display additive effect on AβPP metabolism: combinations of TNFα and interferon γ (IFNγ) or IL-1β + IFNγ increase essentially Aβ secretion, while used separately they have no effect [8].Thus, the cytokine link of inflammatory process becomes a new target on the way of amyloidosis suppression.
Polyphenols draw attention among natural substances with anti-inflammatory properties [9].Curcumin [1,7-bis(hydroxy-3-methoxyphenyl)-1,6heptadiene-3,5-dion] is one of the most studied representatives of this class compounds.The system bioavailability of curcumin is very low that is explained by its insolubility in water and quick metabolism in the liver, kidneys and gastric wall.Despite that, curcumin is considered one of the most promising candidates of natural origin having antiinflammatory influence without side effects [10].It has been established that the mechanism of anti-inflammatory activity of curcumin includes: • activation inhibition of the transcription factor NFκВ which regulates expression of pro-inflammatory gene products [11]; • down-regulation of expression of cyclooxygenase-2 (СОХ-2) [12]; • expression inhibition of inflammatory cytokines, including TNFα, IL-1β, IL-6, IL-8, chemokines [13].
The inhibiting effect of curcumin on the nuclear factor κB (NFκB)-signal path occupies a central place in ensuring its anti-inflammatory properties.Curcumin blocks IκB-mediated phosphorylation and degradation of inhibitory protein IκBα, as a result NFκB remains in the complex with IκBα in cytoplasm and cannot enter a nucleus to activate the transcription.Investigations of the suppression of NFκB activity have demonstrated further down-regulation of СОХ-2 and inducible NO-synthase, as well as a decrease of synthesis of inflammatory markers [14].
Microglia is a derivative of bone marrow monocytes.It consists of a network of immunoprotective cells which respond to impulse activity of neurons and mediate neuro-immune interaction [16].That is why the modeling of the effects of β-amyloid peptide 42 and curcumin on monocyte suspension in vitro corresponds to the effect of these factors in the brain microglia in vivo.
The work aim was to investigate the effect of aggregated Аβ 42 on the expression dynamics and formation of endogenous Аβ 40 and cytokines (IL-1β, TNFα, IL-6, IL-10) by mononuclears of the peripheral blood in vitro and correction of such effect by curcumin.

The work has been made in accordance with
Universal declaration on Bioethics and human Rights (UN, 1997).Mononuclear cells of human peripheral blood were isolated ex tempera with the help of ficoll-urografin gradient from the samples of venous blood of three healthy volunteers, separately.After triple washing of cell suspension with sterile physiologic solution of room temperature the mononuclears were resuspended in RPMI medium to concentration of 2×10 6 cells/ml.The RNAse inhibitor was added to RPMI to prevent mRNA degradation.Thus the obtained samples of suspensions of mononuclear cells were used to investigate the effect of β-amyloid peptide 42 (in a dose of 15 nM), curcumin (in a dose of 54 nM) separately and their united action (at analogous concentrations).The results were standardi zed by the corresponding indices of cell suspension which was added only 0.9% NaCl.The ratio of volu mes of mononuclear suspension and corresponding impurities (curcumin, Аβ 42 , 0.9% NaCl) was 100:1.
Аβ42_Human (Human Amyloid β Protein Fragment 1-42, Sigma-Aldrich), dissolved in bidistilled water, was aggregated during 24 h at 37 °С.Big coarse conglomerates of Аβ42_Human were dispersed with ultrasound and sterilized directly before adding.Since curcumin is low-soluble in water, the concentrated output curcumin solution was first prepared in 96% ethanol, diluted by bidistilled water about 0.7 g/l (directly before adding) to mononuclear suspension.
The dynamics of Аβ 42 and 0.9% NaCl effect on mononuclears was studied at the 0, 1, 3, 6 and 24 th hours of their incubation at 37 °С and stirring rate 600 rpm.The dynamics of curcumin effect and joint action of Аβ 42 + curcumin was studied at the 3, 6 and 24 th hours of incubation, thereat curcumin was added after 1 h incubation of mononuclear suspension with Аβ 42 or with 0.9% NaCl.In correspon ding terms of incubation the aliquots of suspensions were taken and cells were ruined under three-minute action of ultrasound with frequency 2.64 MHz and intensity 0.25 W/cm 3 in the device MUSSON-1.After that the samples were centrifuged at 6 000 rpm during 20 min, and supernatant was used for further measurements.
Relative expression at mRNA level for АβРР, TNFα, IL-1β, Il-6 and Il-10 genes was determined in mononuclears by the method of polymerase chain reaction (PCR) with the help of corresponding primers in all terms of incubation.RNA was isolated from cells using a set "RIBO-sorb" "AmpliSens " (RF).Gene expression analysis was performed by PCR method in real time.Complementary DNA (cDNA), obtained in the course of the reverse transcription reaction using a set "AmpliSens" (RF), were used as a matrix.Rotor-Gene Q (QIAGEN) (Germany) and specific primers produced by Sintol (RF) were used for amplification: The amplification reactions were conducted as triplets for each gene in the following conditions: 60 s at 95 °С, 40 cycles: 30 s at 94 °С, 30 s at 60 °С, 30 s at 72 °С using SYBR Green mix (Amplisence, RF).To the reaction mixtures were added the following concentrations of MgCl 2 (AmpliSence, RF): 1 mM for amplification of gene AβPP, 2 mМ for amplification of genes IL-1β, Il-6, Il-10 and aCTB, 4 mМ for amplification of gene TNFα.The expression level was determined by Ct delta method.
The data obtained were standardized by the expression of referent gene aCTB (for β-actin) in the form of the relation of the number of cDNA copies of the determined factor to the number of cDNA copies of aCTB [17] and were expressed in conventional expression units (c.e.u.).
ELISA determined the concentration of cytokines in supernatants of mononuclear suspension in correspondence to the protocol of Vector-Brest, RF for IL-1β, IL-6, IL-10 and TNFα, and also Аβ 40 by the set ELISA Kit Human Аβ40, Invitrogen Corporation.Optical density was read by microplate analyzer GBG Stat FAX 2100 (USA) at 450 nm with wavelength correction at 630 nm.ELISA data were computed for total protein (ng/g of protein).Results were presented on Figures in percentage of the indices of mononuclear suspensions, which had been incubated with 0.9% NaCl.Total protein concentration was determined by the Lowry method [18].
The results were processed statistically, mean values and standard deviations for mononuclear suspension indices were calculated.Statistical analysis of differences was carried out using Student's t-test.The value P < 0.05 was considered significant.

results and discussion
Table 1 data illustrate the absence of dynamics of the basal level of mRNA expression of АβРР and cytokines (IL-1β, TNFα, Il-6, Il-10) in mononuclears under the effect of 0.9% NaCl, except for inconsiderable, in view of the research objective, varia tion of mRNA TNFα , since the latter is connected with nonspecific circadian fluctuations of gene TNFα expression in mononuclear suspension in vitro [19,20].
In contrast to basal expression level of mRNA under study, concentrations of Аβ 40 and cytokines (IL-1β, TNFα, IL-6, IL-10) in the mononuclear suspension under the effect of 0.9% NaCl changed in different directions during 24 h (Table 2).
The increase of content of endogenous Аβ 40 for the 6 th hour of incubation with the absence of АβРР expression activation may be explained by intensification of amyloidogenic processing of already existing molecules of the precursor protein of β-amyloid peptide.As to dynamics of the basal level of cytokines (spontaneous production) in mononuclear suspension (Table 2), a probable decrease of IL-1β concentrations (for the 1 and 24 th h), IL-6 (1-3 th h) and IL-10 (1 st h) explains the degradation of those peptides with a short half-life (˂0.5 h) [21], while the increase of TNFα content on the background of the corresponding inhibition of expression (Table 1) probably owes to enzymatic activity of TNFα-converting enzyme (ADAM17) [22].
β-Amyloid peptide 42 in aggregated form did no effect on the expression of mRNA TNFα in the suspension of mononuclears in vitro and caused an increase of concentration of the tumor necrosis factor α itself for the 3 rd hour of incubation (Fig. 1).
Curcumin solution added to the suspension of mononuclear cells of human peripheral blood after 1 hour of toxic effect of Аβ 42 inhibited the expression of mRNA TNFα (for the 3 rd hour of incubation) and decreased considerably TNFα concentration.However, the inhibiting effect of curcumin proved short-term and, already for the 6-24 th hour of incubation the expression level of mRNA TNFα was renewed (Fig. 1, a), while TNFα content was doubled (Fig. 1, B).The modulating effect of curcumin itself on the dynamics of mRNA TNFα and TNFα content in the mononuclear suspension coincided with this polyphenol effect on the background of toxic effect of Аβ 42 (Fig. 1).
In our research the changes of mRNA IL-1β expression by mononuclears were not clarified for any

T a b l e 1. Basal level of mRNA expression of АβРР and cytokines (IL-1β, TNFα, IL-6, IL-10) in mononuclears under the effect of 0.9% NaCl during 24 h
* P ˂ 0.05 compared to preincubation (0 h). Conventional  factor under study (Fig. 2, a), but the content of this pro-inflammatory interleukin varied essentially with adding curcumin (Fig. 2, B).Such non-apparent result may be explained only by curcumin ability to accelerate creation of the active form of IL-1β from its precursor under the catalytic action of caspase-1 [23].
The induction in the interval of Аβ 42 effect of 3-6 h and simultaneous essential expression inhibition with curcumin were established for gene IL-6 (Fig. 3, a).The inhibiting effect of curcumin at the transcription level in mononuclear suspension was preserved for the day of incubation as well.At the translation level the increase of mRNA IL-6 under the effect of Аβ 42 resulted in the corresponding enrichment of the mononuclear suspension with IL-6 for the 3 th h and further decrease of this interleukin concentration at time interval of 6-24 h (Fig. 3, B).The content of IL-6 proved to be lower in 6-24 h of curcumin effect itself compared to Аβ 42 effect in that period.Thus the effect of curcumin on the course of inflammatory process in mononuclears proved to be direct and dominating, though short-term, compared to Аβ 42 effect.

Fig. 1. dynamics of mRNa TNFα expression (А) and TNFα content (B) in the suspension of mononuclears under the effect of Аβ 42 , curcumin and their joint action (in % of the basal level with 0.9% NaCl, taken as 100%).
Here and in Fig. 2-5 * P ˂ 0.05 compared with Аβ 42 effect Fig. 2. dynamics of mRNa IL-1β  The results obtained conform to recently published data on dose-dependent inhibiting effect of curcumin on the content of IL-1β, TNFα, IL-6 and their mRNA in Аβ 42 -activated microglia [24].The work authors associate curcumin effect with phosphorylation of ERK1/2 and p38 and thus blocking of just these intracellular signal paths.Our data also partially coincide with results of the work by Jian Jiao with co-authors [25], where they show the in-crease of TNFα and IL-1β concentrations in microglia culture when adding monomers, oligomers or fibrils of Аβ 42 in concentration range of 0.625-2.5 μM with maximum for the 12 th h of incubation.The authors suppose the NFκВ-dependent induction of proinflammatory cytokines by various forms of Аβ 42 .

expression (А) and IL-1β content (B) in mononuclear suspension under the effect of Аβ 42 , curcumin and their joint action (in % of the basal level with 0.9% NaCl, taken as 100%)
The effect of Аβ 42 on the expression of antiinflammatory IL-10 was not established (Fig. 4, a), but curcumin raised mRNA IL-10 content for the 6 th h, while simultaneously with Аβ 42 -only for the 24 th h of incubation.Beginning from the 1 st h of incubation with Аβ 42 the authors observed a sharp drop of IL-10 level in mononuclear suspension, which could be stopped by curcumin from the 6 th h only (Fig. 4,  B).Some authors supposed that high level of IL-10 might be a good prediction sign in the dynamics of inflammatory process in general and under amyloidosis in particular [26,27].But there appeared researches which results evidence for the effect of Fig. 3. dynamics of mRNa Il-6 expression (А) and IL-6 content (B) in mononuclear suspension under the effect of Аβ 42 , curcumin and their joint action (in % of the basal level with 0.9% NaCl, taken as 100%) Fig. 4. dynamics of mRNa Il-10

expression (А) and IL-10 content (B) in mononuclear suspension under the effect of Аβ 42 , curcumin and their joint action (in % of the basal level with 0.9% NaCl, taken as 100%)
IL-10 on the level of apolipoprotein E and clearance of β-amyloid peptide that led to formation of amyloid platelets [28].
We have studied the dynamics of concentration of endogenous Аβ 40 in mononuclear suspension under the effect of exogenous Аβ 42 (Fig. 5, B).We can see two maxima of this peptide formation: for the 1 st and 24 th h of incubation.In view of the absence of AβPP expression activation for the 1 st h (Fig. 5, a) such a quick and intensive response to the action of Аβ 42 , compared with the cytokine system, owes to activation of processing of β-amyloid peptide precursor following the amyloidogenous scenery with formation of endogenous Аβ 40 .The second peak of Аβ 40 concentration (for the 24 th h) is the result of induction of AβPP expression and increase of its mRNA АβРР in this time interval (Fig. 5, a, B).
The inhibiting effect of curcumin has been shown both on the expression and synthesis of AβPP and formation of Аβ 40 .The mRNA АβРР expression minimum was noted for the 3 rd h of incubation, and the level of Аβ 40 in the presence of curcumin did not essentially differ from the initial one (Fig. 5, a).The data obtained may evidence for curcumin efficiency in normalization of β-amyloid peptide metabolism even in pro-inflammatory condition, determined by the local excess of Аβ 42 aggregates.Curcumin proper ties in inhibiting the formation and destabilization of Aβ-oligomers as well as in ruining a senile platelet are well known now [29][30].
Thus the dynamics of anti-inflammatory effect of curcumin in vitro at transcriptional and translational levels of cytokines formation by mononuclears has been cleared, as well as its direct inhibiting effect on the level of endogenous Аβ 40 during the 24-h incubation under the toxic effect of Аβ 42 aggregates has been established in this work.К л ю ч е в ы е с л о ва: куркумин, β-амилоидные пептиды 40 и 42, цитокины, мРНК, мононуклеары периферической крови человека.