Diabetes Mellitus is a global disease with high economic and social burden.
Islet transplantation can today be offered to selected patients with type-1
diabetes who are non-responsive to drugs. Despite many advances in protocols
for pancreatic islet transplantation, yet it cannot compensate complete body
insulin requirement and thus pancreatic islets derived from multiple donors
are needed. It is believed that in the transplant setting, islets of Langerhans
are faced with various types of stress related to the isolation and transplantation
procedure which trigger a cascade of cell signaling pathways that compromise
their function and viability. For instance, apoptosis during the initial stages
of islet transplantation prior to stable engraftment results in non-functionality.
Newly transplanted islets are essentially avascular and thus islets must pass
the initial period of hypoxic ischemia until the process of revascularization
is completed. This ischemic microenvironment produces oxidative stress condition
that is detrimental to transplanted islets (Mohseni-Salehi-Monfared
et al., 2009).
Islets are essentially vulnerable to oxidative-induced damage because they
have inherently decreased antioxidant capacity (Rahimi et
al., 2005). The antigen-independent complexities of islet transplantation
increase the incidence of primary graft non-functionality and β-cell death,
thus requiring protection for islets at early stages of the transplant procedure
(Irani et al., 2009). Prevention of oxidative
stress by means of more powerful compounds may further improve the survival
of isolated islets in culture and in vivo (Mohseni-Salehi-Monfared
et al., 2009; Hasani-Ranjbar et al.,
Setarud (IMOD) is the outcome of an invention referring to a method for preparing
an herbal extract from a mixture of Rosa canina, Tanacetumvulgare
and Urtica dioica comprising selenium and urea treated by pulsed electromagnetic
field of high frequency that has been patented in USA and Europe (Novitsky
et al., 2007) for its efficacy in Human Immunodeficiency Virus (HIV)
infection by increasing CD4 and reduction of tumor necrosis factor alpha (TNF-α).
The herbs used in this complex have strong anti free-radical potentials and
useful in oxidant-related diseases (Hasani-Ranjbar et
al., 2009). The benefit of IMOD in autoimmune experimental diabetes
(Mohseni-Salehi-Monfared et al., 2010) and in
human severe sepsis (Mahmoodpoor et al., 2010)
have been recently reported. Commonly used anti-rejection drugs are excellent
at inhibiting the adaptive immune response; however, most are harmful to islets
and do not protect well from oxidative damage resulting from islet isolation
and ischemia-reperfusion injury. The aim of this investigation was to examine
probable benefit of IMOD on in vitro islet viability and function.
MATERIALS AND METHODS
Chemicals: All chemicals were purchased from Sigma-AldrichChemie (Gmbh, Munich, Germany) unless otherwise stated. IMODTM was obtained from Parsrus Research Group (Tehran, Iran). Rat specific insulin ELISA kit was purchased from (Mercodia, Sweden).
Experimental animals: Male adult Wistar rats (>12 weeks old), with a weight of approximately 250±25 g, were used as pancreatic islets donors. All of the animals were treated according to the National Health Ethical Guidelines for experimental animal studies.
Pancreatic islet isolation and culture: After accommodation of rats
to lab environment, rats were anesthetized with intraperitoneal injection of
sodium pentobarbital (60 mg kg-1) and underwent laparotomy; the common
bile duct was ligate at the duodenal end and cannulated at its the duodenal
side. Then, the rat pancreas was distended by injecting 10 mL of Krebs buffer
([in grams per liter] 8 NACL, 0.27 KCL, 0.42 NaHCO3, 0.06 NaH2PO4,
0.05 MgCL2, 2.38 HEPES, 0.22 CaCL2.2H2O, 0.5
glucose. 1H2O, pH = 7.4) into the duct. Distended pancreas was excised
carefully from duodenum and preserved in a plate within Krebs buffer solution.
The pancreas was cut into 1-2 mm pieces to increase the surface area and providing
conditions for digestive enzyme collagenase to break down the tissue surrounding
the islets. After separation of fat tissue, islets were washed by Krebs-HEPES
buffer three times. Then the extracted tissue was placed in falconin Krebs solution
and centrifuged with 1700 rpm 60 sec for two times and 0.5% BSA was added for
completion of digestion. Islets were thereafter isolated by hand picking with
sampler under a stereomicroscope and handpick islet was incubated in culture
media which contain RPMI-1640 medium, in 5% CO2 at 37°C for 15
IMOD in vitro dose optimization: Optimization of dose was done
by pretreating islets with various concentrations (0.1, 1, 10, 100, 1000 μl
L-1 or ppm) of IMOD for 24 h. IMOD solution is dispersed as vials
containing 125 mg of active ingredients in 4 mL solution. The effective in
vivo dose of IMOD is 20 mg kg-1 in rat (Baghaei
et al., 2010).
Insulin secretion: Isolated islet divided in several groups. Each group contained ten islets in 1 mL Krebs medium alone or in combination with different concentrations of IMOD. After 24 h incubation, medium which contain different concentrations of IMOD was removed and islets were washed twice by Krebs-HEPES buffer and preincubated for 30 min at 37°C at a level of 2.8 mM glucose. Then, all groups were treated in two glucose concentrations of 2.8 and 16.7 mM as basal and stimulant doses, respectively and subsequently incubated for 30 min at 37°C. The supernatants were collected and stored in separate microtubes. Insulin assays were performed with rat insulin ELISA kit (Mercodia, Sweden) according to the manufacturer's protocol.
Metabolic activity of islets by MTT: 3-4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT), a yellow tetrazole is reduced to purple formazan in living cells. After 24 h incubation with different concentrations of IMOD, medium was removed and islets were washed twice by Krebs-HEPES buffer. Then 20 μL from MTT solution was added to islets and incubated for 4 h. At the end, 100 μL DMSO solution was added to culture medium and after 30 min, the absorbance was determined at 570 nm by ELISA reader.
Reactive Oxygen Species (ROS) assay: The intracellular formation of
reactive oxygen species was measured using 2',7'-dichlorodihydrofluorescin diacetate
(DCFH-DA). The non-fluorescent compound DCFH-DA penetrates into the cell and
is cleaved by intracellular esterase, resulting in the formation of 2',7'-dichlorodihydrofluorescin
(DCFH), the oxidation of which (due to oxidative stress) generates the fluorescent
compound dichlorofluorescein. Ninty six wells plate were used to measure ROS.
After homogenization of each ten islet isolated from pancreatic tissue by extraction
buffer and centrifuging the homogenized solution (with 5000 rpm/5 min), the
extracted solution was separated and collected in separate micro-tube. To measure
ROS, 162 μL of the buffer assay with 10 μL DCFH-acetate with in each
of the plates sink down into each well of plates. Then, 50 μL of homogenized
samples was added to it. After incubation the samples for 15 min at 37°C
for 60 min to absorb the changes (excitation: 488 nm/emission: 528 nm), changes
in absorbance were measured by ELISA fluorimeter. All values were standardized
by the amount of total protein in each well (Momtaz et
Protein assay: To measure total protein concentration of islets, diluted samples were mixed with Bradford reagent dye and after 5 min, the absorbance was measured at 595 nm by the spectrophotometer. Albumin was used as standard.
Statistical analysis: Data were analyzed by StatsDirect statistical software version 2.7.8. Results are expressed as the Mean±SEM of from three separate tests examined duplicates. One-way analysis of variance followed by Tukey'sposthoc multiple comparisons were used as the statistical tests. The p<0.05 was considered statistically significant.
Effect of IMOD on islets insulin release: As seen in Fig. 1, administration of IMOD in 0.1 ppm increased insulin secretion in basal glucose (2.8 Mm) and stimulated (16.7 mM) concentrations. However, administration of IMOD (1 ppm) had no significant effect on insulin secretion from islet in basal state but significantly increased insulin secretion from islets at high glucose concentration (16.7 mM) when compared with the matched control. IMOD at doses of 10, 100 and 1000 ppm significantly decreased secretion of insulin in both basal and stimulated glucose states.
Effect of IMOD on islets metabolic activity (MTT assay): Results are shown in Fig. 2, as percent change in relation to control. IMOD significantly increased viability in 0.1, 1, 10, 100 and 1000 ppm, among them the maximum change was observed in 1 ppm. Despite increase in viability of islets in lower doses of IMOD, viability was progressively decreased in concentrations of 10,100 and 1000 ppm significantly different from that of 1 ppm.
Effect of on islets ROS: Results are shown as percent change in relation to control (Fig. 3). IMOD significantly increased ROS in 100 and 1000 ppm. However, there was no significant change in ROS at doses of 1 and 10 ppm but a significant reduction at dose of 0.1 was observed. Despite the lack of significant differences in ROS levels at doses of 1 and 10 ppm, the observed changes in the concentrations studied were dose-dependent.
||Effects of IMOD on release of insulin from isolated rat islets.
After 24 h incubation in the exposure of various concentrations of IMOD,
islets were incubated for 30 min in the presence of various concentrations
of glucose as basal (2.8 mM) or stimulant (16.7 mM). Data are expressed
as Mean±SEM of 3 different experiments (each experiment was performed
in duplicate). *Significantly different from control group (p<0.05);
**Significantly different from control group (p<0.01)
||Effects of IMOD on viability of isolated rat islets. Data
are expressed as Mean±SEM of 3 different experiments (each experiment
was performed in duplicate). **Significantly different from control group
(p<0.01); ***Significantly different from control group (p<0.001)
||Effects of IMOD on production of ROS in isolated rat islets.
Data are expressed as Mean±SEM of 3 different experiments (each experiment
was performed in duplicate). *Significantly different from control group
(p<0.05); ***Significantly different from control group (p<0.001)
The present results show that IMOD in dose of 0.1 and 1 ppm enhances the function of islets by increasing insulin secretion in the stimulated state; however only in dose of 0.1 ppm it increased insulin secretion in basal state. In this study, we showed that IMOD increases viability of cells in all doses used in comparison to controls where the maximum effect was observed by dose of 1 ppm. Although, the current study confirms that IMOD in doses of 100 and 1000 ppm increases oxidative cell damage in pancreatic beta cells but, in the dose of 0.1 ppm its antioxidative effects was evident.
Enhancement of insulin secretion at high glucose level (16.7 mM) supports that
IMOD does not disturb islet cells membrane but provokes glucose-induced insulin
secretion in beta cells. Insulin secretion in stimulated condition may indicate
cell membrane stability and indicate functional viability. The unexpected effects
of IMOD at higher doses were seen in MTT assay which gives the impression on
sensitivity of islets to IMOD and the fact that proper doses of IMOD must be
used in vitro. IMOD increases viability in all concentration despite
a decrease in insulin release in doses above 1 ppm (10 to 1000 ppm). These findings
suggest that effect of IMOD on secretion of insulin is independent of its effect
on islet viability and may be due to its secretory stimulation or other unmeasured
factors. Another possible reason for the discrepancy is that insulin content
may have been overestimated due to non-specific intracellular insulin release
into the culture medium from damaged islet cells during incubation especially
in basal state.
In comparison of effects of IMOD on insulin secretion and viability, IMOD increased insulin secretion only at 0.1 and 1 ppm but increased viability in all doses. This means that viability is a complex factor independent of insulin secretion.
When insulin secretion and ROS production are compared, it is seen that by increasing of ROS in doses higher than 1 ppm results in lower release of insulin. This suggests that ROS causes islet cells damage and reduces their functional viability.
The current study confirmed that IMOD in doses of 100 and 1000 ppm induces oxidative cell damage in islets most probably through pro-oxidant activities of islets. We have demonstrated that IMOD in doses of 100 and 1000 ppm functions as a pro-oxidant to activate pancreatic beta cell damage but despite an increase in ROS, viability of cell increases.
Also, based on observed antioxidative effects in dose of 0.1 ppm, low dose
of IMOD may increase islets antioxidative enzymes such as catalase, superoxide
dismutase and glutathione peroxidase in favor of defense against oxidative stress.
Previous studies have demonstrated antioxidative activity of IMOD (Mohseni-Salehi-Monfared
et al., 2009; Baghaei et al., 2010;
Agha-Hosseini et al., 2011), or its main component
Urtica dioica (Mehri et al., 2011). In experimental
colitis, IMOD remarkably reduced histological scores of colitis through controlling
of TNF-α, interleukin-1β, myeloperoxidase and lipid peroxidation (Baghaei
et al., 2010). In addition, in experimental immune model of diabetes,
IMOD decreased pancreatic lipid peroxidation, myeloperoxidase, TNF-α and
interleukin-1β (Mohseni-Salehi-Monfared et al.,
2009). Therefore, in vivo data do not support dual function of IMOD
as an anti-oxidant or pro-oxidant while as shown in the present in vitro
study, nanomolar concentrations of IMOD may exert beneficial antioxidant activity
whereas pro-oxidant activity may be generated at micromolar concentrations.
However, we found evidence of antioxidative activity at nontoxic concentrations.
Therefore, oxidative or antioxidative effects of IMOD in vitro depend on doses that are used. Of course, this effect of IMOD should be tested in other cell lines in vitro to examine if it is cell-dependent or not. Taken together, the present study indicates that IMOD in doses of 100 and 1000 ppm mediates formation of highly toxic radicals which in turn induces oxidative cell damage in the rat islets. A clear understanding of the pro-oxidant mechanisms of IMOD or antioxidant at physiological levels needs to be established by measuring apoptotic and necrotic pathways (NF-κB-DNA binding) and cytokines. Finally, measurements of islets viability, insulin secretion as well as ROS conclusively demonstrate that pretreatment with IMOD at dose of 0.1 ppm rescues islets from death or dysfunction via scavenging ROS. Pretreatment with IMOD may improve transplant outcome and graft function but must be tested in a good model. Its effect in insulin release maybe promising in treatment of diabetes.
This study was partially supported by Endocrinology and Metabolism Research Institute and Pharmaceutical Sciences Research Center of TUMS. Authors thank ParsRus research group for providing IMOD.