BIOTROPIKA Journal of Tropical Biology
https://biotropika.ub.ac.id/
Vol. 11 | No. 3 | 2023 | DOI: 10.21776/ub.biotropika.2023.011.03.04
A COMPARATIVE PROFILE OF FREE RADICALS, ENDOGENOUS ANTIOXIDANTS, AND CYTOKINES IN MOUSE MODEL OF TYPE 1 DIABETES MELLITUS
Fikriya Novita Sari1), Rizky Senna Samoedra1), Setyaki Kevin Pratama1), Sri Rahayu1), Aris Soewondo1), Muhaimin Rifa’i1)*
ABSTRACT
Diabetes mellitus is a metabolic disorder characterized by high blood glucose (hyperglycemia). Hyperglycemia will cause the body to undergo physiological changes such as free radical, antioxidant and inflammation alteration. This research aims to compare the profile of free radicals, endogenous antioxidants, and cytokines in mouse models of type 1 diabetes mellitus (T1DM). Mice were separated into two different groups, normal and diabetic mice groups. The normal group was a group of mice that were not induced to have diabetic conditions, while the diabetic mice group was induced to be diabetic using streptozotocin injection. Blood glucose levels were checked every three days for 14 days, while the immune response was evaluated after 14 days using flow cytometry.
Data analysis was done using SPSS software with T-test analysis. This research showed that the increasing ROS represented by MDA would trigger inflammation in T1DM represented by the increasing TNF-α along with IFN-γ and reducing anti-inflammatory cytokines represented by IL-10. Interestingly, SOD expression, which is an endogenous antioxidant, is also increased in the diabetic mice group, and we conclude that it is some sort of adaptive response of the diabetic mice group against the increasing ROS.
Keywords: antioxidant, free radical, inflammation, type 1 diabetes mellitus
INTRODUCTION
A metabolic disorder called diabetes mellitus (DM) is characterised by abnormally high amounts of blood sugar (hyperglycemia). Diabetes mellitus has been a significant problem in the world because of its increasing prevalence year by year. In 2021, there were about 8.4 million individuals worldwide with type 1 diabetes (T1DM), with a death rate of 6.7 million [1]. In T1DM, damaged pancreatic cells result in insulin insufficiency, which leads to hyperglycemia [2].
High glucose circulating in the blood of diabetic patients is toxic because it triggers the formation of reactive oxygen species (ROS) that leads to oxidative stress, which causes cellular damage [3].
ROS (such as -HO, O2- and H2O2) have reactive unpaired electrons in their outer orbitals that can modify RNA, proteins, or other biomolecules like oxidised polyunsaturated fatty acids (PUFA), and the reaction will produce malondialdehyde (MDA) [4]. MDA is able to be used as a biomarker for increased ROS in the body. ROS that increases continuously without being accompanied by balanced levels of internal antioxidants such as glutathione peroxidases (GPx), catalase (CAT), superoxide dismutase (SOD) and will trigger inflammation by transcription factor activation that is crucial for proinflammatory cytokine release [5].
A high level of proinflammatory cytokine will impact tissue damage that worsens β cell eradication by aggravating immune cell invasion to target and forcing the apoptosis of β cells, which is
associated with increasing risk of diabetes complications. This study aims to compare the profile of free radicals, endogenous antioxidants, and cytokines in the mouse model of type 1 diabetes mellitus.
METHODS
Experimental design. The Animal Care and Use Committee of Universitas Brawijaya examined and approved all procedures used in this study with ethical approval number 016-KEP-UB- 2021. A total of 20 male mice (Mus musculus) BALB/c strain aged eight weeks old with an average body weight of 38.03 g from Gadjah Mada University, Indonesia, were used. Each mouse was maintained in the Animal Physiology Laboratory at Universitas Brawijaya and was given a week of acclimatization before the procedure. Then, the mice are separated into two groups (n = 10 for each group), which are normal and diabetic (DM) groups. Then, the immune response was evaluated after 14 days of DM and blood glucose was measured every three days for 14 days using an Easy Touch glucometer (Bioptik Technology Inc., Taiwan).
Induction of diabetic mice. Diabetic mice induction uses streptozotocin (STZ) in a single high dose of 145 mg/kg body weight (Bioworld, USA). A single high dose of STZ as a diabetogenic agent was injected to induce type 1 diabetic mice [6]. STZ was given once with intraperitoneal
Submitted : December, 26 2022 Accepted : January, 20 2024
Authors affiliation:
1) Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya, Indonesia
Correspondence email:
How to cite:
Sari, FN, Samoedra RS, Pratama SK, Rahayu S, Soewondo A, Rifa’i M. 2024. A comparative profile of free radicals, endogenous antioxidants, and cytokines in mouse model of type 1 diabetes mellitus.
Biotropika: Journal of Tropical Biology 11 (3): 156-162.
injection. Mice fasted for four hours before receiving STZ administration. STZ was dissolved in a citrate buffer with a pH of 4.5 and then injected into mice at no more than 10 minutes [7]. The blood glucose level was measured on the seventh day post-STZ administration. Diabetes condition was determined in mice with blood sugar above 200 mg/dL [8].
Spleen and liver isolation. The cervical dislocation method was used to kill the mice after induction of DM. Following the dissection of mice, the whole liver and spleen were separated and homogenised using 3 mL phosphate buffer saline (PBS). A microtube was then filled with homogenate and centrifuged at 2500 rpm for five minutes at 10°C. After removing the supernatant, 1 mL PBS was used to resuspend the pellet. Then, about 50 μL sample was taken and placed in a new microtube for antibody staining. Spleen cells were used to evaluate cytokine-secreting lymphocytes, whereas hepatocytes were isolated to analyze MDA and SOD expression.
Antibody staining and flow cytometry. A microtube with 50 µL of isolated spleen cells was added with 50 µL of anti-mouse CD4 and CD25 antibodies for extracellular staining. They were incubated for 20 minutes at 4°C afterwards. For intracellular staining, 50 µL of fixation buffer was added to the microtube samples and incubated at 4°C for 20 minutes. The wash buffer solution (BioLegend, USA) of 200 μl was added for permeabilization and centrifuged for 5 minutes at 10°C at 2500 rpm. TNF-α, IFN-γ and IL-10 antibodies were employed as intracellular antibodies to stain the cell after the supernatant was discarded. The substances were prepared for flow cytometry analysis (BD FACS-Calibur, USA) by adding 400 μl of PBS to the cuvet. BD Cellquest ProTM was used to examine the data obtained [9].
The liver samples were stained directly using intracellular (MDA and SOD) antibodies with the same intracellular staining protocol as previously described. The flow cytometry data from spleen
samples were gated in specific lymphocyte populations and analyzed with a quadrant dot plot.
Flow cytometry data from liver samples were gated in whole living cells and analyzed with a histogram plot.
Data analysis. SPSS 21.0 program was used to examine the data statistically using a t-test with a p-value ≤ 0.05. The data examined using this test was blood glucose levels and the percentage of cells expressing MDA, SOD, CD4+TNF-α+, CD4+IFN-γ+, and CD4+CD25+IL-10+, respectively.
RESULTS AND DISCUSSION
Blood glucose levels. After testing blood glucose levels every three days for 14 days, this study's DM group and normal group were shown to have significantly different blood glucose levels. A single high-dose injection of STZ was successful in inducing DM as indicated by the measurement 0- day, showed a blood glucose level of 400 mg/dL and was stable until the 14-day measurement. The final measurement for the blood glucose levels was 485 mg/dL for the DM group and 149 mg/dL for the normal group (Figure 1). This result showed that blood glucose levels were indeed elevated beyond normal levels (>200 mg/dL) in diabetic conditions.
The physiological changes in mice to hyperglycemia after injection of STZ are due to the fact that STZ can enter pancreatic β cells through glucose transporter 2 (GLUT 2), which works by forming highly reactive free radicals which cause damage to cell membranes, proteins and DNA genetic material, resulting in distract insulin production by pancreatic β cells. In addition, STZ causes dephosphorylation of ATP, which leads to apoptosis and necrosis of pancreatic β cells, then leads to an increase in blood sugar levels [10]. High blood glucose over time can affect tiny and large blood vessels, increasing the risk for microvascular and macrovascular complications [11].
Figure 1. Blood glucose level on normal and DM mice from day 0 to day 15
High blood glucose or hyperglycemia can lead to higher ROS production through various mechanisms and also lead to chronic inflammation.
In addition, hyperglycemia may impair the micro- RNA (miRNA) production that causes vascular damage brought on by advanced glycation end products (AGE) and decreased angiogenesis that causes diabetic vascular disease. Hyperglycemia can also be caused by impaired insulin secretion due to ROS [12]. Chronic hyperglycemia accelerates the production of AGE resulting from non-enzymatic protein glycation. The production of ROS and inflammatory cytokines is subsequently encouraged by the increased build-up of AGE and free fatty acids (FFA) [13].
ROS effects and endogenous antioxidants. In this research, our findings found that the relative number of cells expressing MDA in normal and DM groups were significantly different (p≤0.05).
The relative numbers of cells expressing MDA in normal groups were 14.75% and 24.87% in the DM group (Figure 2a). This demonstrated that the number of cells expressing MDA was statistically higher in the DM group than in the normal group.
MDA itself can be used as a diagnostic marker for oxidative stress. MDA can also be a biomarker for increased ROS within the body. MDA is known to
be increased in diabetic conditions [14]. This is because, in diabetic conditions, there is an increase in ROS that usually comes from hyperglycemic conditions [12]. An imbalance of ROS and antioxidant activity within the body will lead to ROS accumulation that can directly damage cells and tissues [15].
The relative number of cells expressing SOD in this study between the normal and DM group were also significantly different. The normal group have 2.58% of its cells expressing SOD, while the DM group have 4.76% of its cells expressing SOD (Figure 2b). Interestingly, this means that the relative number of cells expressing SOD which represents one of the endogenous antioxidants in the body was significantly higher in the DM group than in the normal group. This result, supported by Ezekwesili et al., showed SOD increasing in diabetic conditions [16]. However, this could also mean that the increasing SOD levels were some adaptive response towards the increasing ROS to protect β cells from destruction [17]. This finding is consistent with our MDA data, which demonstrates that the DM group had a larger relative number of cells expressing MDA compared to the normal group in our research (Figure 2a).
Figure 2. Percentage (a) MDA and (b) SOD on normal and DM mice showed increasing expression on DM mice; *, p≤0.05 by independent sample t-test
One of the risk factors for diabetes is oxidative stress, which can result from patients' lifestyles and excessive food consumption. A 5 to 10-fold rise in ROS can be caused by consuming too many calories. Additionally, ROS are known to damage proteins, lipids, nucleic acids, and are strongly linked to diabetes, particularly T1DM. SOD is one of the methods of controlling ROS. Antioxidants are the obvious defense against ROS. Free radicals can be neutralised by antioxidants, which lowers the formation of reactive oxygen species (ROS) and helps the body achieve equilibrium once more [18].
ROS effects and inflammatory cytokines expression. In this research, the expression of inflammatory cytokines, TNF-α and IFN-γ secreted by CD4 T cells are significantly different between normal and DM groups (Figure 4a and 4b). In the normal group, the relative number of CD4+T cells secreting TNF-α and IFN-γ are 0.49%
and 0.27%, respectively. Meanwhile, for the DM group, CD4+T cells secreting TNF-α and IFN-γ levels are 1.22% and 0.65%, respectively. These findings demonstrated that diabetes increases the production of inflammatory cytokines.
Administration of anti-TNF-α was shown to reduce insulin resistance in T1DM non-obese diabetic (NOD) mice. Moreover, short-term treatment of anti-TNF-α monoclonal antibodies (mAb) can also restore immune self-tolerance [19]. TNF-α could enhance cell adhesion by activating endothelial cells, upregulating the MHC I and II in the islet, and activating antigen presenting cells (APCs) and T cells [20]. TNF-α could also activate nuclear factor kappa-beta (NF-κβ) activation, which will raise the formation of ROS and inflammation [12].
Furthermore, more inflammation means more autoreactive T cells (CD4 and CD8 T cells), and they have crucial involvement in the pathogeneses that cause β cell destruction in T1DM [11].
Meanwhile, IFN-γ and TNF-α are inflammatory cytokines secreted by autoreactive CD4+T cells for the destruction of β cells. It is also the reason that T1DM is called an autoimmune disease. It has been shown that blocking IFN-γ in IFN-γ specific antibodies or soluble receptors has been reported to prevent spontaneous T1DM in non-obese diabetic (NOD) mice, whereas transgenic production of IFN-γ may worsen autoimmune disease [20].
Figure 4. Percentage (a) TNF-α and (b) IFN- γ of CD4+ on normal and DM mice showed increasing expression in DM mice; *, p≤0.05 by independent sample t-test
ROS has the potential to affect adaptive immune maturation. This can be attained via controlling the activator protein-1 (AP-1), mitogen-activated protein kinase (MAPK), and nuclear factor-B (NF-κβ) pathways, which are crucial for innate immune system cells like macrophages and dendritic cells to produce inflammatory cytokines such as TNF-α and IFN-γ.
Inflammatory cytokines will promote adaptive immunity contribution to the cellular death of pancreatic tissue. Therefore, ROS and inflammatory cytokines secreted by the innate immune system will work together to create an immune response by linking innate and adaptive immune mechanisms [20]. In this study, it has been shown that in the DM group, the relative numbers of TNF-α and IFN-γ secreting CD4 T cells are substantially higher than in the normal group. This means the result of the study is in line with the previously shown MDA data (Figure 2) that represents ROS, which also means more ROS in the DM group can lead to more inflammatory cytokines. Inflammation is, without a doubt, one of the major physiological alterations in T1DM because of the recruitment of other effector T cells by the increasing amounts of inflammatory cytokines. Recruited effector T cells will then be responsible for killing β-cells through granzyme and perforin and the toxicity of cytokines [21].
ROS effects and anti-inflammatory cytokines expression. The proportion of regulatory T cells (Tregs) that secrete IL-10 was considerably higher in the normal group compared to the DM group. The values were 9.53% and 4.61% for the normal and the DM group, respectively (Figure 5). Among the cytokines that Tregs release to inhibit immunological response is IL-10. The expression of Tregs that secrete IL-10 is declining or is below average [22]. IL-10 is one of the main cytokines expressed by regulatory T cells to suppress immune response. A systemic
increase of IL-10 is also known to alleviate the symptoms of T1DM in NOD mice, including decreasing pancreatitis and blood glucose [23].
The levels of IL-10 expressed by Tregs in the DM group in this study correlate with the MDA data (Figure 2a), which represents ROS in this study. IL-10 protects against endothelial dysfunction in diabetes, which is caused by an increase in the free radical O2-. Moreover, IL-10 also have the ability to inhibit ROS in monocytes and neutrophils. Meanwhile, inflammatory cytokines can activate xanthine oxidase in tissue culture. Xanthine oxidase can produce O2- while IL-10 can inhibit the production of inflammatory cytokines [24]. Our findings indicated that in the DM group, the disruption of homeostasis in T1DM leads to ROS overproduction, represented by MDA. It, in turn, lowers the level of IL-10 produced by Tregs and increases the level of inflammatory cytokines such as TNF-α and IFN-γ secreted by CD4 T cells.
CONCLUSION
This study showed significantly different values from parameters of blood glucose, antioxidant, free radical, pro-inflammation and anti-inflammation between normal and diabetic mice. In diabetic mice, glucose levels and MDA, which is a marker for ROS accumulation, were elevated. This increase has a correlation with antioxidants and inflammatory cytokines, such as the upregulation of TNF-α and IFN-γ as proinflammatory cytokines and the downregulation of IL-10 in diabetic mice.
Intriguingly, SOD increases in this study along with MDA in the DM group, and this may be viewed as the DM group's adaptive response to the rising ROS. Based on this research, the related parameters can be further observed as one of the targets for developing anti-diabetic drugs.
Figure 5. Percentage IL-10 of CD4+CD25+ on normal and DM mice showed decreasing expression in DM mice; *, p≤0.05 by independent sample t-test
REFERENCES
[1] Gregory GA, Robinson TIG, Linklater SE, Wang F, Colagiuri S, de Beaufort C, Donaghue KC; International Diabetes Federation Diabetes Atlas Type 1 Diabetes in Adults Special Interest Group; Magliano DJ, Maniam J, Orchard TJ, Rai P, Ogle GD (2022) Global incidence, prevalence, and mortality of type 1 diabetes in 2021 with projection to 2040: a modelling study. The Lancet Diabetes & Endocrinology 10(10):
741–760. doi: 10.1016/S2213- 8587(22)00218-2.
[2] Burrack AL, Martinov T, Fife BT (2017) T cell-mediated beta cell destruction:
autoimmunity and alloimmunity in the context of type 1 diabetes. Frontiers in
Endocrinology 8(343). doi:
10.3389/fendo.2017.00343.
[3] Samarghandian S, Azimi-Nezhad M, Farkhondeh T (2017) Catechin treatment ameliorates diabetes and its complications in streptozotocin-induced diabetic rats. dose- response 15(1): 155932581769115. doi:
10.1177/1559325817691158.
[4] Juan CA, Lastra JMP de la, Plou FJ, Pérez- Lebeña E (2021) The chemistry of reactive oxygen species (ROS) revisited: outlining their role in biological macromolecules (DNA, lipids and proteins) and induced pathologies. International Journal of
Molecular Sciences. doi:
10.3390/ijms22094642
[5] Hardiany NS, Sucitra S, Paramita R (2020) Profile of malondialdehyde (MDA) and catalase specific activity in plasma of elderly woman. Health Science Journal of Indonesia
10(2): 132–136. doi:
10.22435/hsji.v12i2.2239.
[6] Yan L-J, Wu J (2015) Streptozotocin-induced type 1 diabetes in rodents as a model for studying mitochondrial mechanisms of diabetic & beta; cell glucotoxicity. Diabetes, Metabolic Syndrome and Obesity: Targets
and Therapy 181. doi:
10.2147/DMSO.S82272.
[7] Lestari ND, Anita CR, Wahyu IA, Nilamsari RV, Jatmiko YD, Widodo N, Rahayu S, Rifa’I M (2022) Bioactivity of Moringa oleifera and albumin formulation in controlling TNF-α and lFN-γ production by NK cells ⅰn mice model type 1 diabetes.
Jordan Journal of Biological Sciences 15(02):
205–208. doi: 10.54319/jjbs/150207.
[8] Oka R, Shibata K, Sakurai M, Kometani M, Yamagishi M, Yoshimura K, Yoneda T (2017) Trajectories of postload plasma glucose in the development of type 2 diabetes
in Japanese adults. Journal of Diabetes Research 2017: e5307523. doi:
10.1155/2017/5307523.
[9] Adharini WI, Nilamsari RV, Lestari ND, Widodo N, Rifa’I M (2020) Immunomodulatory effects of formulation of Channa micropeltes and Moringa oleifera through anti-inflammatory cytokines regulation in type 1 diabetic mice.
Pharmaceutical Sciences 26(3): 270–278.
doi: 10.34172/PS.2020.43.
[10] Firani NK (2017) Metabolisme karbohidrat:
tinjauan biokimia dan patologis. Universitas Brawijaya Press.
[11] Erener S (2020) Diabetes, infection risk and COVID-19. Molecular Metabolism 39
101044. doi:
10.1016/j.molmet.2020.101044.
[12] Luc K, Schramm-Luc A, Guzik TJ, Mikolajczyk TP (2019) Oxidative stress and inflammatory markers in prediabetes and diabetes. J Physiol Pharmacol 70(6). doi:
10.26402/jpp.2019.6.01
[13] Hodgson K, Morris J, Bridson T, Govan B, Rush C, Ketheesan N (2015) Immunological mechanisms contributing to the double burden of diabetes and intracellular bacterial infections. Immunology 144(2): 171–185.
doi: 10.1111/imm.12394.
[14] Pan HZ, Zhang L, Guo MY, Sui H, Li H, Wu WH, Qu NQ, Liang MH, Chang D (2010) The oxidative stress status in diabetes mellitus and diabetic nephropathy. Acta Diabetologica 47(S1): 71–76. doi:
10.1007/s00592-009-0128-1.
[15] Charlton A, Garzarella J, Jandeleit-Dahm KAM, Jha JC (2020) Oxidative stress and inflammation in renal and cardiovascular complications of diabetes. Biology 10 (1): 18.
doi: 10.3390/biology10010018.
[16] Ezekwesili C, Ogbunugafor H, Ezekwesili- Ofili J (2012) Anti-diabetic activity of aqueous extracts of Vitex doniana leaves and Cinchona calisaya bark in alloxan–induced diabetic rats. International Journal of Tropical Disease and Health 2: 290–300. doi:
10.9734/IJTDH/2012/1693.
[17] Thomas B (2014) Serum levels of antioxidants and superoxide dismutase in periodontitis patients with diabetes type 2.
Journal of Indian Society of Periodontology 18(4): 451–455. doi: 10.4103/0972- 124X.138686.
[18] Black HS (2022) A synopsis of the associations of oxidative stress, ROS, and antioxidants with diabetes mellitus.
Antioxidants 11(10): 2003. doi:
10.3390/antiox11102003.
[19] Koulmanda M, Bhasin M, Awdeh Z, Qipo A, Fan Z, Hanidziar D, Putheti P, Shi H, Csizuadia E, Libermann TA, Strom TB (2012) The role of TNF-α in mice with type 1- and 2- diabetes. PLOS ONE 7 (5): e33254.
doi: 10.1371/journal.pone.0033254.
[20] Padgett LE, Broniowska KA, Hansen PA, Corbett JA, Tse HM (2013) The role of reactive oxygen species and proinflammatory cytokines in type 1 diabetes pathogenesis.
Annals of the New York Academy of Sciences 1281 (1): 16–35. doi:
10.1111/j.1749-6632.2012.06826.x.
[21] Weir GC (2023) Islet inflammation can be linked to the disruption of proinsulin processing in type 1 diabetes but not in type 2 diabetes. The Journal of Clinical Endocrinology & Metabolism 108(2): e21–
e22. doi: 10.1210/clinem/dgac638.
[22] Qiao YC, Shen J, Hong XZ, Liang L, Bo CS, Sui Y, Zhao HL (2016) Changes of regulatory T cells, transforming growth factor-beta and interleukin-10 in patients with type 1 diabetes mellitus: A systematic review and meta-analysis. Clinical Immunology 170: 61–69. doi:
10.1016/j.clim.2016.08.004.
[23] Bijjiga E, Martino AT (2011) Interleukin 10 (IL-10) regulatory cytokine and its clinical consequences. J Clin Cell Immunol. doi:
10.4172/2155-9899.S1-007
[24] Steen EH, Wang X, Balaji S, Butte MJ, Bollyky PL, Keswani SG (2020) The role of the anti-inflammatory cytokine interleukin- 10 in tissue fibrosis. Advances in Wound
Care 9(4): 184–198. doi:
10.1089/wound.2019.1032.