Embryonic antitumor modulator of mkrtchyan (EATM) As a new diabetes perventive compound

Introduction

Diabetes mellitus is one of the most serous problems of medicine. The total number of people with diabetes is estimated as 171 million in 2000 projected and is going to rise to 366 million in 2030 [1]. The prevalence of diabetes is higher in men than women, but there are more women with diabetes than men. The most important demographic change to diabetes prevalence across the world appears to be the increase in the proportion of people >65 years of age. Medications used to treat diabetes include insulin [2]. Everyone with type 1 diabetes and some people with type 2 diabetes must take insulin every day to replace what their pancreas is unable to produce [3,4]. Unfortunately, insulin can’t be taken in pill form because enzymes in the stomach break it down. In recent years, researchers have focused increasing attention on transplantation of pancreas or Islet cell transplantation for people with type 1 diabetes [5]. But all these procedures are difficult, dangerous and expensive. That’s why search for new antidiabetic compounds, which are free of above mentioned shortcomings, is extremely actual.

It is gratifying to mention that outstanding scholars in the area of medicine and health protection set their face to the idea of preventive medicine. As a compound, which possess antidiabeticpreventive properties was chosen EATM - embryonal antitumor modulator, created by prof. L.Mkrtchyan [6], an immunomodulator of embryonic genesis to be applied in extremely low doses once a year by adults and elderly of a high oncological risk group (pre-cancerous pathology, heavy smokers, family inclination /susceptibility to cancer, etc.). In many of its parameters EATM becomes similar to anticancer preventive vaccine.The author supports the idea that biologically active substances, which are applied for prophylaxis of human health, are more preferable than the chemically synthesized compounds . The EATM possess wide spectrum of action, in nonspecific way activates the immune system of the organism. Exept antitumor, it reveals also neuroprotective action [7]. Ultimate safety of EATM was proved in strictly controlled and manifold repeated experiments performed in compliance with the requirements of the former USSR Pharmacological Committee, Russian Federation Agency on Medicines Safety and GLP. EATM is composed of a wide pool of protein bodies isolated exceptionally from normal embryonal substances in a state maximum close to the native one [8]. Increased oxidative stress is a widely accepted participant in the development and progression of diabetes and its complications . Diabetes is usually accompanied by increased production of free radicals [9-12] or impaired antioxidant defenses [13-16]. As a result of the streptozotocin action, B cells undergo the destruction by necrosis. Antioxidants reveal some protective effect in diabetes patients [17,18].

MATERIALS AND METHODS

To induce experimental diabetes in animals is widely used streptozotocin[Scudelski]. 102 male male albino rats 170-200 g were used in this study. The rats were housed at 25 ± 2°C under 12 hour cycles of dark and light and were allowed standard food and water ad libitum. The rats were fasted for 18 hours before induction of diabetes. Diabetes was induced by a singleintraperitoneal injection of freshly prepared streptozotocin (55 mg/kg) dissolved in 0.1M citrate buffer (pH 4.5). Streptozotocin (Sigma) (STZ)-injected rats exhibited symptoms of diabetes mellitus such as polyuria, polydipsia, polyphagia, and weight loss after 48 hours post STZ administration. Two days after the injection of STZ, fasting blood glucose concentration was measured using blood samples from the heart. Animals with blood glucose concentrations 200 mg/dl were considered diabetic and used in this study. Subsequently, fasting blood glucose was measured weekly in each rat. The rats were divided into 3 groups. The 1-st group - control animals, the 2-nd group - STZ injected group, the 3-rd group - was i/p injected EPOM in 1 mg/rat dose,a week after they were injected STZ , 2 weeks after last injection the animals were sacrificed, blood and liver tissue excised and processed. The EPOM was kindly granted by prof.L.Mkrtchyan. The Yerevan State Medical University Ethics Committee approved this study.

Blood glucose concentration (in mg/dl)was determined with Glucose liquichrom Delta G-COL (Cat.N DSG106) diagnostic tests. The amount of daily consumpted water (in ml per kg of rats weight) and food (in gr per kg of rats weight) was measured. The concentration of malondialdehyde was measured according Yoshika et al. [19], a-tocopherol -by Duggan [20] and superoxide dismutase activity -by Nishikimi et.al.[21], middle molecular peptides in blood plasma- by Gabrielyan et al.[22], protein content - by Lowry et al [23]. Determination of the level, NADPH depended on02- -productive and ferrihemoglobin (ferriHb)-restoring activity of prooxidantmetalloproteins (isoforms of NADPH oxidases from erythrocyte membranes, serum, component of cell of the body’s immune system - spleen, as well as from suprol, cytochrome b5 and cytochrome c) and the activity of key antioxidant metalloproteins (total fraction of Cu, Zn-SOD and Mn-SOD, catalase ) was carried out by Simonyan et al.[24-26].Liver tissues were stained with Hematoxylin and Eosin (H&E), and with PAS (Periodic Acid Shiff) reaction. Pancreatic tissues were stained with H&E and with Aldehyde fuchsin. 

RESULTS AND DISCUSSION

In the final stages of DM (21 day of experiment) in the diabetic group death of the animals was 60-70%, indicating of an essential breach of endocrine pancreatic function and intoxication of an organism. In the case of preventive injection of EATM death of the animals was reduced to 35-40%, while there was a significant normalization of the appearance and behavior of animals, reduction of polyuria and polyphagia. As is shown in table 1, the level of glucose in normal rats (control) was 130mg/dl. In diabetic rats it increased 4 times and reached 494mg/dl. In the case of EATM profilactic injection it remained on the 173mg/dl level, which is approximately 3 times lower than in diabetes rats, and only by 33% more than in control group (table 1).

Table 1. The effect of EATM on the level of glucose, as well as water and food consumption in blood in streptozotozin induced diabetes rats

What about water and food consumption, it was also different in different experimental animal groups. According to data, presented in table 1, the water consumption in diabetic rats was increased 3 times, whreas in preventive group it was by 24% lower than in diabetic rats and 2,3 time higher than in control. The food consumption in diabetic rats was 67% higher than in control, whereas in EATM pretreated rats it was only by 31% higher than in norm and by 22% lower than in diabetes. The changes in the content of the pro- and antioxidant indices are presented in table 2. According to our data, the content of MDA in the blood of diabetes rat increased by 33%, whereas inprofilactic group it was lower by 26% compared with norm and 40% lower than in diabetic group. What about MDA in liver, it’s content in diabetes rats was 2,5 times higher than in control. Administration of EATM definitely prevented the increase of MDA level, it was only 40% higher than in control and 1,8 times lower than in diabetic rats.

The activity of the main antioxidant enzyme SOD in diabetes rats in blood increased by 50%, in the case of EATM administration it was higher than in control rats only by 19%, and lower than in diabetes rats by 13%. In liver the SOD activity in comparison to control was increased by 24%, whereas in protected group it was saved at control level. The content of the main tissue antioxidant a-tocopherol in diabetes rats blood lowered by 26%. In the case of EATM injection it’s level remained like in intact rats. The middle molecular peptides (middle molecules), which are indices of inflammation or intoxication, in STZ diabetes rats blood are increased by 35%, while in protected group they are lower even than in control group. The subsequent changes took place against a background of typical shifts of the level of metalloproteins of prooxidant activity - MPA (isoforms of acidic nature of NADPH oxidases or cytb558, localized in the blood serum, EM, cell membranes of spleen-CMS, and mitochondria of spleen cells -MSC, the nuclei of spleen cells-NSC, as well as cyt c,cyt b5 and superoxide- producing lipoprotein serum - suprol) (table 3-5) and metalloproteins of antioxidant activity of – MAA, (Cu, Zn-SOD and catalase from cytosol of erythrocytes., the total fraction of Cu, Zn-SOD andMn-SOD and fraction of catalases from cytosol of spleen cells)(table 6).

Table.2. The effect of EATM on the level of MDA in blood, MDA in liver, activity of SOD in blood, SOD in liver , concentration of a-tocopherol in blood, content of middle molecules in streptozotocin induced diabetes mellitus in rats

As is shown in table 3, the level of SOD NADPH oxidases (or cytb₅₅₈) significantly increases (from 81.5 to 225%,) in the EMI, CMS , MSC, especially in NSC. The level of cytc is significantly increased from spleen cells, indicating the decrease of the stability of mitochondria at SOD. From MPA only the level of cytb₅ from the cytosol of red blood cells is being decreased.

Table 3.The EATM effect (%) on the levels of MPA in streptozotocin induced diabetes rats (p ‹0,05, n = 6)

As shown in table 4, the 02-- producing activity of NADPH oxidase fraction, isolated from cell membranes of spleen-CMS, increases sharply. The increase in this activity is more expressed in NADPH oxidases from erythrocyte membranes-EM, nuclei of spleen cells- NSC, MSC, as well as from blood serum. In DM, theO₂^--producing activity of suprol is being significantly reduced . The latter may be associated with increased lipid peroxidation of phospholipid residues of suprol, produced by O₂^-of suprol [27]. The EATM has a positive effect, bringing the O₂^- producing activity of isoforms cytb₅₅₈(or NADPH oxidase) of acidic nature with different cell localization to the norm.

Table 4. The EATM effect (%) on the levels of NADPH- depended Оշ^--producing activity of MPA in streptozotocin induced diabetes rats (p ‹0,05, n = 6)

FerriHb-restoring activity of these NADPH oxidase considerably is reduced in DM, especially for the NADPH oxidases of EM and for extra-cellular NADPH oxidases (table 5), indicating a significant breach of oxygen homeostasis. In this case, EATM virtually does not change the oxygen homeostasis (only ferroHb or oxyHb, but neither ferriHb nor metHb, is able to transport molecular oxygen to the cells).

Table 5.The EATM effect (%) on the ferriHb-restoring activity of MPA in streptozotocin induced diabetes rats (p ‹0,05, n = 6)

As for MAA, we can see that in DM activity of Cu,Zn-SOD fraction from the cytosol of red blood cells and spleen cells (the total fraction of Cu, Zn-SOD and Mn-SOD) is decreased in almost the same range (table 6). Analogically, catalase activity in the cytosol of red blood cells and spleen is reduced.

Table 6. The EATM effect (%) on the superoxide dismutase and catalase activity of MAA in streptozotocin induced diabetes rats (p ‹0,05, n = 6)

So, administration of STZ arise the classic picture of diabetes mellitus. Preliminary administration of EATM prevents increase of glucose level, development of characteristic for diabetes features. The balance of pro- and antioxidant systems compounds is saved about the physiological level. What about metalloproteins, the results indicate, that in DM, there is a decrease of stability of EM, CMS, MSC, and especially of NSC and enhancement of cleavage (releasing) of isoformscyt b₅₅₈ from heterogeneous phase (from membranes) to a homogeneous phase (in solution). This is facilitated by increasing peroxidation of lipids ofbiomembranes in DM [28-30].

This is confirmed by a significant decrease in the process of releasing of cyt b558 from EM and increased stability of the latter under the influence of drugs with antioxidant activity (Cu, Zn-SOD, catalase, ceruloplasmin, as well as a synthetic analogue of proline- rich polypeptide - galarmin of neurosecretory granules of hypothalamus) [31].

The increased level of serum (extracellular) cyt b₅₅₈ is possibly associated with decreased stability of red blood cells that occurs in various pathological conditions, especially in malignant tumors [31]. Mechanism of increasing the level of cyt c in mitochondria , the spleen cells in DM may be as a result of enhancement of peroxidation of lipids.

The morphological study of the pancreas showed that in intact rats islets are revealed often. The cellular elements of the islets with abundant, weak-eosinophilic cytoplasm, are compact, in case of staining with aldehyde-fuxin the colors are intyensive. STZ induced rats revealed definite destruction of it’s structure. Focal discomplexation ofacini. In some sinuses “amputation” of the apical part of cytoplasm ofthe endocrine pancreocytes is noticed. The insular apparatus is represented by rare mostly small islets. The cellular elements of the islets are located loosely, their cytoplasm is poor, appears optically empty. The cellular elements of the islets by staining aldehyde-fuchsin negative. In protective group the discomplexation of acini as compared with diabetic group was less spread. Vacuolization of the cytoplasm of individual endocrine pancreocytes was observed. Islet apparatus differs a little from that of intact rats (quantity, sizes, characteristics of cellular elements). When stained with aldehyde-fuxsin in the islets P-cells are revealed, the quantity of which does not differ much from that in intact rats.

The liver architecture is presented in pic.1. In intact rats it was revealed that the structure of liver is preserved and easily identifiable (a). Within the cytoplasm of almost all hepatocytes the glycogen accumulation is reveled, which is seen as magenda-colored intracytoplasmic material. In diabetic group (b) - the liver architecture is preserved and easily identifiable. The intracytoplasmic glycogen accumulation is absent in all hepatocytes, without any zonal predilection. In preventive group (c) - the liver architecture is preserved and easily identifiable. The quantity of hepatocytes with intracytoplasmic glycogen as well as the concentration of the glycogen is less than in control group. The glycogen-containing hepatocytes have no any zonal predilection, and the overall distribution of glycogen has a mosaic pattern.

Pic.1. The effect of EATM on the glycogen content 

Pic.2. The effect of EATM on the glycogen content in liver by H&E method

In the case ofEATM injectionall the studied parameters are notably normalized. According to our data, the mechanism of such effect of EATM is not related to its antioxidant action (EATM does not possess SOD -mimetic or catalase-mimetic activity in vitro). It is more likely that such an effect of EATM is associated with stimulation of antioxidant systems in vivo. 

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