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10Results of curcuminoids in an experimental model of systemic amyloidosis in rats
Kushnariov V.A, Lapin D.S, Lebedev SV, Vasilyev A.V, Borodin E.A AmurStateMedicalAcademy, Russia
Summary: We studied the effect of curcuminoids from the rhizomes of Curcuma longa on 48 male albino rats with experimentally simulated systemic amyloidosis. The influence of the substance on the protein level of serum, levels of proinflammatory cytokines IL-6 and IL-8 as well as the degree and amount of amyloid in biopsy of animals. According to a study shows the positive effect of a pharmaceutical composition to the level of serum proteins, the reduction of proinflammatory cytokines and regression of amyloid in biopsies of experimental animals.
Key words: amyloidosis, amyloid, curcumin, cytokines
Introduction:
At present, the secondary systemic amyloidosis is attracting more attention as accompanies many chronic inflammatory disease caused by infectious or non-infectious factors. Such diseases include rheumatoid arthritis, tuberculosis, Crohn's disease, ulcerative colitis, and many others, in which amyloidosis promotes dysfunction of multiple organs and systems [7], complicating the course of the underlying disease. The most commonly affects the kidneys, which is manifested nephrotic syndrome in end-stage leading to chronic renal failure [11,15].
Number of detections of systemic amyloidosis in patients with rheumatoid arthritis range from 6 to 23% according to various authors [4,6]in patients with tuberculosisuntil 19% [5]. Development of amyloidosis and the appearance of amyloid associated with genetic, immune, metabolic disorders. Amyloid is biochemically variable structure, representing a glycoprotein. The main component of the glycoprotein are fibrous proteins, of which there are 23 species [3]. Found that amyloid contains albumin, globulin, fibrinogen, fibrin, plasma proteins of predominant gamma globulin [1]. Amyloid accumulates in organs and tissues in large amounts, which leads to the atrophy and sclerosis functional organ failure.
In this regard, prevention and treatment of this disease is in the first place, since there is still no effective treatment, and existing traditional treatment with cytostatics and steroids fail to achieve sustained remission, and making it difficult for the many side effects of the disease [3,7]. It is necessary to create and search for new effective molecules acting purposefully to amyloid fibrils and have a preventive effect. Some of these substances are polyphenol curcumin and its derivatives [2], which are widespread spices derived from plants of the genus Curcuma, have great therapeutic potential [9].
According to some of curcumin and its derivatives are capable of preventing the aggregation of amyloid lesions and promote lysis amyloid in Alzheimer's disease and other neurodegenerative diseases [2]. In the Laboratory of Experimental Pharmacology of the Youth Innovation Centre, we have developed a pharmacological composition based on curcuminoids from the rhizomes of Curcuma longa.
The aim: to prove anti-amyloidogenic properties developed by us on the basis of the drug composition of curcuminoids Curcuma longa in the treatment of experimental systemic amyloidosis induced in male albino rats. Materials and Methods:
Biological models:The study was conducted on 48 male albino rats kept under standard vivarium conditions Amur State Medical Academy, weighing 230-280 g Model of systemic amyloidosis:
Systemic amyloidosis modeled within 30 days from the introduction of amyloidogenic substance in a dose of 1 ml intraperitoneally projection axillary and inguinal lymph nodes by day for 15 days. Amyloidogenic substance consists of native ovalbumin modified with complete Freund's adjuvant. Modified Freund include glycerin PC-94 pharmaceutical grade (MiniMed), 10% sterile gelatin strain Mycobacterium tuberculosis. The ratio between the components of the amyloidogenic substance - egg albumin: glycerol: gelatin: Mycobacterium tuberculosis is 1:1:1:0,1 respectively.
Drug:
The composition of curcuminoids obtained dry powder extraction from the rhizomes of Curcuma longa with 70% ethanol during the day at a ratio of 1:5, and then the supernatant was separated by filter evaporated on a steam bath for 3 hours. Next, the dry residue was evaporated distribute individually depending on the weight of the rats at a dose of 1 mg / g was mixed with corn oil in a ratio of 1:3 to obtain a gel mass. The resulting pharmacological composition is administered per os. Table 1. Animals' groups
The design of the experiment:
Animals were divided into three groups (Table 1). Group 1 and 2 was modeled systemic amyloidosis within
On day 31 of the group 1 starts to get a pharmacological composition at a dose of 1 mg / g in terms of dry matter per os. Group 2 received 1 ml normal saline NaCl per os, a group 3 is intact.
On day 51 of the experiment, all animals are displayed according to the requirements of humanity. [16]
Biochemical studies:
Biochemical study used a serum with the anticoagulant heparin. Determination of IL-6, IL-8 was performed by ELISA on the machine «Anthos 2020» with the reagents byfirm"Cytokine". Protein fractions were determined by concentrated solutions of potassium degidrofosfat on photoelectric colorimeter KFK-3 at a wavelength of 670 nm.
Histological studies:
On histological examination were taken lungs, kidneys, liver, spleen, excision of a full thickness tissue fixing pieces of the size of 1 cm in 10% neutral formalin. For histological examination, prepare medicines according to [12]. Subsequently, paraffin blocks are manufactured with serial sections 5 microns thick. For light-optical microscopy is used microscope "Micromed-1" with fotocamera Levenhuk C130 and circular polarizing filters and the BlueLine and Flama. The slides were stained with hematoxylin and eosin [10] with which to evaluate the overall plan of the structure body, the presence or absence of inflammation, damage and repair cells. To assess the presence of amyloid and its localization is used coloring histologic specimens kongo red [6], in which the density is analyzed and the number of lesions in the target kongo red the slides using polarizing filters.
Statistical studies:
Statistical processing of the results produced by the software package Microsoft Office Excel
2007.
Results:
Biochemical studies:
The results of the protein fractions shown in Table 2, a statistically significant difference in terms of albumin, alpha-2 globulin and gamma-globulins of the groups 1, 2, 3
Group Introduce substances Number of animals
Group 1 Amyloidogenicsubstance + curcuminoidsindose 1 mg/gperos 16
Group 2 Amyloidogenicsubstance 16
Group 3 Intact 16
30 days, a group of 3 intact.
In the investigation inflammatory cytokines in Table 3, IL-6 in the group 1 - 1,95 ± 0,72 ng / ml group 2 - 5,27 ± 0,92 ng / ml group 3 - 3,8 ± 1,2 ng / ml. In the study of IL-8, group 1 - 12,27 ± 6,5 pg / mL, group 2 - 71,85 ± 32,8 pg / ml, group 3 - 31,85 ± 17,75 pg / ml. Statistically significant differences between groups 1 and 3, p <0.05.
Table 1.Proteins fractions in serum, in%
Group Albumin a - 1 a - 2 P Y
Group 1 (n=16) 34,86±3,2* 9,4±1,7 16,5±3,7* 30±3 6±2,2*
Group 2(n=16) 19,25±6,5* 8,75±4,6 27,5±2,1* 32±4,2 12,25±1*
Group 3(n=16) 50±3,7* 15,5±4,8 7,75±1,8* 24,8±5,4 4,75±1,4*
The symbol * indicates significant differences between the groups, p <0.05
Table 2.Changes in the level of IL-6 and IL-8
Group IL-6, Hr/M^ IL-8 nr/M^
Group 1 (n=16) 1,95±0,72* 12,27±6,5*
Group 2 (n=16) 5,27±0,92 71,85±32,8
Group 3 (n=16) 3,8±1,2* 31,85±17,75*
The symbol * indicates significant differences between the groups, p <0.05
Histological studies:
It were investigated 240 histological specimens. Group 2 - in the specimens, staining with hematoxylin and eosin in the liver: in the course of the sinusoids in the lobules expressed deposition of amorphous eosinophilic masses. Hepatic beams dramatically thinned, atrophic hepatocytes. In the kidney, the deposition of amorphous masses of pink along the capillary loops glomerular basement membrane tubules. In mild diffuse lymphocytic infiltration, deposition of eosinophilic peribronchial masses. In the deposition of amyloid in the spleen, lymph follicles as an amorphous pink solid
In specimens staining liver kongo red: amyloid deposition brick red during sinusoidal capillaries in the walls of blood vessels. Hepatocytes were thinned, hypo - and atrophic. In the lung lesions kongofilii peribronchial, stroma, in the walls of blood vessels. Polymorphocellular focal and diffuse infiltration of the stroma. Amyloid deposition in kidney brick red during capillary loops glomerular basement membrane of tubules stroma in the vessel walls. In the spleen, expressed diffuse deposition of amyloid brick red pulp in the spleen along the vascular wall in the interior of the spleen capsule (see Fig. 1,2,3,4)
Group 1 in the specimens stained with hematoxylin-eosin - deposits of eosinophilic mass is observed.When painting kongo red 22% specimens were showed positive amyloid lesions in the liver along the sinusoids in the peribronchial lung. All Microscopic lung marked decrease in lymphocyte infiltration foci kongofilii the number and area of the stroma and in the course of the vascular walls. In the slides kidneys preserved glomerular integrity, amyloid deposition occurs along the vessels in 18% of cases. In 12% of the spleen was found Microscopic lesions kongofilii.
Group 3 - without the structural and functional features that correspond to the normal structure of the organ.
Discussion:
According to the results of biochemical analysis of an increase in the number of pro-inflammatory cytokines in the group 2 speaks of actively flowing inflammation in simulated disease. Increasing the amount of alpha-2 globulin and gamma globulins and reduction of serum albumin in the animals of group 2 indicates chronic inflammation and nephrotic syndrome, which indirectly reflects the pathogenetic mechanisms amyloidosis [8.11]
According to the results of biochemical tests we set the positive effect of curcuminoids: to reduce the levels of IL-6 and IL-8 in group 1, as well as stable high rates of protein fractions (Tab.1 and 2). Probably, these effects are realized through the reduction of the inflammatory response phase, possibly through suppression of signal transduction factor STAT-3 [2,6,7,9].
According to histological analysis amyloidosis foci were predominantly found in the group 2, which did not receive curcuminoids. The lungs, liver and kidneys of two focal density amyloidosis 4-5 times higher specimens of lungs, livers, kidneys than in the group 1, treated curcuminoids. We suggest that curcuminoids have the ability to inhibit the aggregation of amyloid, and promote lysis or other process aimed at removing already formed lesions.
Conclusions:
As a result, we have installed an anti-inflammatory and anti-amyloidogenic effect of curcuminoids on the model of systemic amyloidosis. Expected to further study the mechanism of action and histophysiologies amyloid deposits in the tissues in systemic amyloidosis.
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