Significance of propolis administration for homeostasis of CD4+CD25+ immunoregulatory T cells controlling hyperglycemia
© Rifa’i and Widodo; licensee Springer. 2014
Received: 24 June 2014
Accepted: 5 September 2014
Published: 15 September 2014
In the present study, we examined the effect of ethanolic soluble derivative of propolis (EEP) extract on immunological function in diabetic mouse models with the aim of highlighting the role of regulatory T cell, the change of cell surface molecule, and in vivo productions of IFN-γ. Murine models of diabetes mellitus (DM) were created by injecting normal mice with S961 peptide. Normal BALB/c mice were injected intraperitoneally with peptide S961 300 mg/kg body weight (BW) twice a day for eight days. On day 15, the spleen was isolated; then, cell surface molecules and regulatory T cells were analyzed using flow cytometry. The histopathological changes were monitored in the liver of treated and control mice. Afterward, we tested the ability of propolis as an immunomodulator that initiate normality metabolism and homeostasis. The propolis decreased blood sugar levels and increased the number of naïve T cells expressing CD62L molecule by suppressing the development of effector cells in diabetic mice. However, the propolis stimulated development of effector cells, which was indicated by increasing the number of CD4+CD25+ T cells in normal mice. Moreover, the propolis increased the production of IFN-γ in normal mice, whereas in mouse models of diabetes mellitus propolis tends to suppress the production of IFN-γ. The histological analysis of the liver shows that at a dose of 50–200 mg/kg BW propolis does not show a toxic effect so that the dose is categorized safe. Therefore, the ethanolic soluble derivative of propolis (EEP) extract warrant for further exploited as an antidiabetic agent that safe for human.
KeywordsDiabetes Mice Propolis S961 Peptide Regulatory T cells
Diabetes mellitus is the worst and fastest growing metabolic disorder in the world. Now it is known that the heterogeneity of this disorder also increases so that an appropriate therapy becomes a significant challenge. Severe metabolic imbalances and non-physiologic changes in many tissues can occur in diabetes, where oxidative stress plays a critical role in the etiology. Reactive oxygen species (ROS) is involved in diabetes, and it contributes oxidative damage, particularly to nerve, liver, heart, kidney, eyes, large and small blood vessels and immunological system (Oršolić and Bašić2008; Yue et al.2003; Obrosova et al.2003; Loprinzi et al.2014).
Diabetes mellitus is currently stretching across the entire world. It penetrates population not only in poor and developing countries, but also in developed ones. The prevalence of this disease is known to be increased. In every ten seconds, one person dies from diabetes mellitus. Indonesia is one country having the greatest biological biodiversity in the world. It has natural resources that potentially increase the population welfare as well as provide a wide range of materials to cure various diseases (Vikram and Jena2010; Syamsudin et al.2008; Kusumawardani2011; Castaldo and Capasso2002). So far, the treatment of various diseases including type-2 diabetes mellitus has a dependency on the synthetic drugs whose safety is still being debated. On the other hand, a lot of herbal medicines claimed to relieve and even to cure type 2 diabetes mellitus. Unfortunately, most of traditional medicine society claimed the various herb treatments have an efficacy without scientific evidence. By considering the case of diabetes mellitus, particularly type-2 diabetes that continues to grow, it is necessary to do adequate research to find a useful drug to ameliorate or even to cure the disease (Murata et al.2004; Khalil2006; McLennan et al.2008; Sforcin2007; Lotfy et al.2006; El-Sayed et al.2009; Sartori et al.2009).
There has been a growing public opinion arguing that propolis can cure diabetes mellitus. Propolis is complex resinous material collected by honey bees from buds and exudates of certain plant sources neighbouring their hives. The main types of flavonoids contained in propolis are pinocebrin, galangin, chrisin, and caffeic acid phenethyl ester. The use of propolis as an alternative healing therapy for type-2 diabetes mellitus has been claimed to alleviate the disease. Previous study states that propolis improves normal homeostasis by balancing the body’s condition through the enhancement of the immune system (Oršolić and Bašić2008; Yue et al.2003; Obrosova et al.2003; Loprinzi et al.2014; Ganong2005; Mahler dan Adler1999; Cetin et al.2010; Arora et al.2009; Bailey1999; Kang et al.2010; Sforcin2007). However, the mechanism action of propolis to modulate the immune system to face type-2 diabetes mellitus cannot be explained. Water extracts of propolis have been studied to prevent the destruction of beta cells by inhibiting the activation of IL-1β and NO synthase activity. Administration with water or ethanolic extract of propolis for seven weeks in mice can decrease glucose, triglyceride, and total cholesterol levels in the blood, thus it is alleged that propolis can control glucose levels and modulate glucose and lipid metabolism. Propolis is also alleged to decline the lipid peroxidation output, and to function as a scavenger for free radicals in rat models of diabetes mellitus. Propolis possesses many functions of biological activities and also has used in folk medicine. Administration of propolis to mouse models of DM suggests homeostasis maintenance, so that further activated cell can be controlled by involving regulatory T cells from both CD4 and CD8 T cells (Oršolić and Bašić2008; Yue et al.2003; Obrosova et al.2003; Sforcin and Bankova2011; Sawicka et al.2012; Rifa’i et al.2004; Lee et al.2008).
CD4+CD25+ regulatory T cells (Treg) are of central importance for the immune tolerance network and, malfunction of this T cell population can either lead to impaired or increased suppression apparently resulting in an array of distinct diseases. The role of CD4+CD25+ regulatory T cells in diabetes mellitus is still being debated. These cells are reported to produce IL-10, TGF-β, and IL-4 to stop T cells activation. Some investigators reported that activation and proliferation of Treg correlate with their ability to suppress diabetes, suggesting that existence of Treg is important to maintain homeostasis (Bluestone and Tang2005; Mahmoud and Al-Ozairi2013; Afzal et al.2014). However, another investigator state that Treg was not involved in the development of diabetes mellitus (Afzal et al.2014). In peripheral blood of DM patient showed a significant increase of IFN-γ, TNF-α, and IL-8. These cytokines contribute to aggravate DM patient and in early stage also cause in the development of insulin resistance (Mahmoud and Al-Ozairi2013; Aoi et al.2013). In the other hand propolis is known to contain high-level nutrient factor including vitamins, polyphenols, and amino acids that would be expected to improve insulin sensitivity. Thus, intake of propolis to decline the expression of inflammatory molecules is one of strategies to ameliorate patient with hyperglycemia. The present study was designed to investigate the effect of ethanolic soluble derivative of propolis extract on T cell activation, blood glucose uptake, apoptosis of splenic cell, regulatory T cell development, and IFN-γ production.
Results and discussion
It is very interesting that the CD4+CD25+ T cells were increased in the mouse model of diabetes mellitus. There are some possibilities why CD4+CD25+ T cells increase in the mouse model of diabetes mellitus. First, an increase of CD4+CD25+ T cells is a manifestation of regulatory T cell enhancement to prevent further cell activation. This fact is consistent with the data shown by Figure 1 in which T cells in mice model of diabetes mellitus were dominated by lymphocytes that lose CD62L molecule, so that the suppressor cells were required to overcome the activated cells. Second, the increase in T cells with CD4+CD25+ marker may be due to the cells being activated and proliferating so that they require CD25 molecule to bind IL-2. Between the two possibilities above, the second possibility may be closer to the truth. This idea is supported by evidence that administering propolis to mice model of diabetes mellitus decreases the expression of CD25 molecules. This fact is consistent with data in Figure 2 showing that the administration of propolis led to naïve cells (CD62L) were more dominant. The domination of naïve cells reflects that the homeostasis in an individual is running well so that mature T cells migrating from thymus to peripheral lymphoid tissue are not activated. The reduction of activated T cells in DM mice after receiving propolis is corresponding with rules of physiological framework. The activated cells will undergo apoptosis when the cells are no longer needed (Rifai’i2010; Lee and Rifa’i2011).
Administration of Ethanolic soluble derivative of propolis (EEP) extract to diabetic mice able to decrease of blood sugar levels, suppress the development of effector cells and production of IFN-γ. The propolis also does not show a toxic at a dose of 50–200 mg/kg BW that warrant for an antidiabetic agent that safe for human.
Materials and methods
In this study we used 8-weeks-old BALB /c, which were maintained in the pathogen free facility, Biology Department, Faculty of Sciences, Brawijaya University, Malang, Indonesia.
Preparation of ethanol extract of propolis
Propolis was obtained from Lawang, East Java, Indonesia. GC-MS analysis has shown that Ethanolic propolis extract (EEP) contained (percentage of total ion current): Benzoic acid 0.41, Phenylic acid 95.62, D-glucofuranuronic acid 0.56, 4-oxo-2-thioxo-3-thiozolidinepropionic acid 0.79, 1-Naphtalenemethanol 95.62, Patchoulene 0.27, D-mannitol 0.51, Threitol 0.86, Glycerol 0.86 (Syamsudin et al.2009). Preparations of propolis extract consisted of three phases including drying, extracting, and evaporating. The drying process began by washing the sample, cutting it into small pieces, and putting them in the oven with a temperature of 40–60°C. Before the extraction process, samples were dried and then crushed by a blender. 100 grams of dry samples were weighed and put in 1 L Erlenmeyer glass, soaked with ethanol to the volume of 1 L. Sample in ethanol was stirred for ± 30 minutes and allowed to stand overnight to settle. Then, solution containing the active substance was filtered with filter paper. Soaking process was repeated three times and the last stage was evaporation. Extraction solvent was inserted into 1 L evaporation flask. Then, water bath was filled with water up to a full circuit and then installed according to an equipment protocol and set to a temperature of 90°C. Ethanol was allowed to drip in the flask (±1.5–2 hours/flask containing ± 900 mL). Extraction results obtained roughly one tenth of dried natural materials (ten grams extract/100 gram’s sample).
Induction of type 2 diabetes mellitus with peptide S961 and propolis treatment
Normal BALB/c mice were injected intraperitoneally with peptide S961 300 mg/kg BW twice a day for eight days. Mice were divided into five groups including positive and negative control groups. The dose of propolis each 50, 100, and 200 mg/kg respectively was administered by oral gavage to BALB/c mice once a day from the first day of peptide injection until the day 14. Glucose level was measured every other day. On day 15, the spleen was isolated, then cell surface molecules, intracellular cytokine, and regulatory T cells were analyzed by flow cytometry.
Oral treatment with dextrose and sucrose
Mouse models of diabetes mellitus received dextrose (2 g/kg BW) by force-fed to maximize diabetes condition. 150 grams of pure sugar/sucrose were put into the Erlenmeyer flask added with 1350 mL of distilled water and shaken to homogenize the solution. Sucrose solution was given to groups of mouse models of diabetes mellitus as they had ad libitum drinking. Sucrose solution was given simultaneously with the injection of peptide and replacement of sucrose solution was performed daily for two weeks.
Measurement of blood sugar levels
Measurement of blood sugar level was done every other day with One Touch Glucometer. Mice were put into a trap, and the blood was taken from their tails. Blood was dropped into a glucostick screen installed on glucometer and noted within 5 seconds.
Isolation of lymphoid cells and flow cytometry analysis
Spleen was washed with sterile PBS twice and put on petri dish containing sterile PBS. Spleen organ was pressed by using syringe holder. Single cell solution was filtered with a sterile wire and put into a 15 mL polypropylene. Suspension in polypropylene was added with PBS up to 10 mL and then put in a centrifuge (2500 rpm, 4°C for five minutes). Then the supernatant was discarded, and the obtained pellet was resuspended with 1 mL of sterile PBS. Single cell suspension containing 2 ~ 3 × 106 cells was washed with PBS and stained with FITC-conjugated anti-mouse CD4, PE-conjugated anti-mouse CD8, PE-conjugated anti-mouse CD25, PE-conjugated anti-mouse CD62L and anti- mouse CD62L (Rifa’i et al.2004; Bluestone and Tang2005).
Intracellular cytokine staining was performed with a Cytofix/Cytoperm kit (BD-Biosciences Pharmingen) according to the protocol provided by the manufacturer. Cells were incubated with FITC-conjugated anti-mouse CD4, PE-conjugated anti-mouse CD8, PE-Cy5-conjugated anti-mouse anti-interferon (IFN)-γ antibodies. Pellets with approximately 2 ~ 3 × 106 cells were stained with FITC-conjugated anti-mouse CD4, PE-conjugated anti-mouse CD8 for 30 minutes. After incubation, the suspension was washed, and pellet was resuspended in cytofix buffer (200 μL) for 20 minutes in dark conditions, 4°C, then resuspended in 1 mL wash-perm and centrifuged again at 2500 rpm at 4°C for 5 minutes. Supernatant was discarded, and the obtained pellet was subjected to intracellular staining with anti-mouse anti-interferon (IFN)-γ for 30 minutes.
Examination of apoptotic cells
Double staining for cellular DNA using propidium iodide (PI) and FITC- conjugated annexin V binding was performed as follows. After washing twice with PBS, 2 ~ 3 × 106 cells were resuspended in annexin-V binding buffer and FITC-conjugated annexin-V was added to a final concentration of 1 μg/mL cell suspension. PI (10 μg/mL in annexin-V binding buffer) was added 15 minutes before running in Flow Cytometry. PI concentration was adjusted in a final concentration of 1 μg PI/mL cell suspension. The mixture was incubated for 15 minutes in the dark at room temperature and then analyzed with Flow Cytometry (Caliber using CellQuest program).
The obtained data were analyzed by CellQuest software and tested by statistical analysis ANOVA (Analysis of Variance) with P < 0.05. All results were presented as mean of ± SD values of five mice in each group.
We would like to thank Directorate General of Higher Education, Ministry of National Education and Culture of Republic Indonesia for the provided grant for this research.
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