|Year : 2018 | Volume
| Issue : 2 | Page : 55-58
The role of serum levels of thioredoxin and thioredoxin-interacting protein in stroke
Fawaz Al-Hussain1, Muhammad Iqbal2, Mohammed Al-Quwayee2, Abdullah Bin Jurays2, Muhannad Al-Wabel2, Saqr Dayes2, Fars Al-Manie2, Tariq Al-Matrodi2, Khalid Al-Regaiey2, Shahid Bashir3
1 Department of Neurology, Faculty of Medicine, King Saud University, Riyadh, Saudi Arabia
2 Department of Physiology, Faculty of Medicine, King Saud University, Riyadh, Saudi Arabia
3 Department of Neurophysiology, Neuroscience Center, King Fahad Specialist Hospital Dammam, Dammam, Saudi Arabia
|Date of Web Publication||6-Jun-2018|
Department of Neurophysiology, Neuroscience Center, King Fahad Specialist Hospital Dammam, Dammam
Source of Support: None, Conflict of Interest: None
Aims: Serum level of thioredoxin (TRX), a redox-regulating protein with antioxidant activity, increases under oxidative stress. The present study measured serum levels of TRX and its inhibitor TRX-interacting protein (TXNIP) in patients who experienced first-ever acute ischemic stroke (AIS). Subjects and Methods: We retrospectively enrolled 45 patients who experienced AIS and 33 age- and sex-matched healthy controls. Serum TRX and TXNIP levels in stroke patients and healthy controls were analyzed by performing solid-phase sandwich enzyme-linked immunosorbent assay. Results: Our results showed that mean serum TXNIP levels were significantly higher in stroke patients than in healthy controls (P = 0.044). However, serum TRX levels were not significantly different between stroke patients and healthy controls (P = 0.405). Moreover, we observed a significant positive correlation between TRX and TXNIP levels (R2 = 0.476, P < 0.003). Conclusions: These results suggest that TRX and TXNIP are rapid, inexpensive, and convenient biomarkers of stroke. However, additional studies should be performed to validate these preliminary observations and the role of TRX and TXNIP in AIS.
Keywords: Ischemic stroke, thioredoxin, thioredoxin-interacting protein
|How to cite this article:|
Al-Hussain F, Iqbal M, Al-Quwayee M, Jurays AB, Al-Wabel M, Dayes S, Al-Manie F, Al-Matrodi T, Al-Regaiey K, Bashir S. The role of serum levels of thioredoxin and thioredoxin-interacting protein in stroke. J Nat Sci Med 2018;1:55-8
|How to cite this URL:|
Al-Hussain F, Iqbal M, Al-Quwayee M, Jurays AB, Al-Wabel M, Dayes S, Al-Manie F, Al-Matrodi T, Al-Regaiey K, Bashir S. The role of serum levels of thioredoxin and thioredoxin-interacting protein in stroke. J Nat Sci Med [serial online] 2018 [cited 2020 Jun 6];1:55-8. Available from: http://www.jnsmonline.org/text.asp?2018/1/2/55/233815
| Introduction|| |
Stroke is the third-leading cause of death worldwide and is a devastating endpoint of cerebrovascular diseases in many surviving patients. Acquired brain injuries such as stroke continuously affect patients, families, and society because of the aging of the general population and the increasing length of postinsult survival. Data on stroke prevalence in Saudi Arabia are scarce. However, a recent study reported lower prevalence of stroke in Saudi Arabia than in Western and Asian countries and increased incidence of stroke in the younger population in Saudi Arabia. Stroke is an important global health problem and a prime cause of death and disability worldwide.
Normal cellular metabolism produces low or moderate concentrations of reactive oxygen species (ROS) that participate in normal physiological processes. However, high concentrations of ROS are toxic to cellular components such DNA, proteins, and lipids, and cells cannot reverse the deleterious effects of ROS through antioxidant mechanisms. This imbalance in the oxidant status of cells is called oxidative stress and is implicated in aging and development of cancer and other neurodegenerative diseases such as cancer and atherosclerosis., Thioredoxin (TRX) system, which comprises NADPH, TRX reductase, and its substrate TRX, is an important antioxidant system. The promoter of TRX contains a cis-regulatory region that is stimulated in the presence of oxidative stress induced by ischemic reperfusion, oxidative agents, or ultraviolet irradiation.
TRX performs many biological functions, including ROS scavenging, thus exerting protective effects against oxidative stress; moreover, serum and plasma levels of TRX are good indicators of oxidative stress and are important biomarkers of different diseases.,,,
TRX-interacting protein (TXNIP) is an endogenous inhibitor of the TRX system. Overexpression of TRX in transgenic mice or knocking down TXNIP expression by siRNA exhibited neuroprotective effects in ischemic brain damage., Increased TXNIP expression exerts proinflammatory and proapoptotic effects in stress-related diseases such as stroke. Pharmacological inhibition of TXNIP prevented ischemic brain damage in an animal model by inhibiting oxidative stress and inflammasome activation.
Due to the association between oxidative stress and stroke, we examined whether serum TRX and TXNIP levels were associated with the pathogenesis of stroke and could be used as reliable predictive markers for the diagnosis of stroke.
| Subjects and Methods|| |
We retrospectively analyzed 45 patients diagnosed with acute ischemic stroke (AIS), complete clinical data containing laboratory and magnetic resonance imaging records on admission were accessed. In addition, this study included 33 healthy controls without any risk factors or chronic diseases. The study was approved by our institute ethics committee.
Blood collection and complete blood count analysis
Approximately 5 ml blood samples were collected from all the study participants in two tubes (2.5 ml blood sample in each tube) with and without ethylenediaminetetraacetic acid. Complete blood count was determined using Sysmex-XE 2000i automated blood cell analyzer (Sysmex, Kobe, Japan) within 1 h after collecting the blood samples. For performing biomarker analysis, serum was separated from the blood samples by performing centrifugation at 1500 ×g for 10 min, aliquoted, and stored at −80°C until further use.
Serum TRX and TXNIP levels were measured by performing sandwich enzyme-linked immunosorbent assay with human TRX and TXNIP enzyme-linked immunosorbent assay kits (Elabscience Biotechnology Co., Ltd., China), according to the manufacturer's instructions. In brief, precoated antibodies specific to TRX and TXNIP were incubated with samples, standards, and appropriate controls for 90 min at 37°C. After incubation, the samples were treated with biotinylated antibodies against TRX and TXNIP for 1 h at 37°C. After washing, antigen–antibody complexes were determined using an avidin–horseradish peroxidase conjugate. An enzyme substrate was used to hydrolyze the reaction, and the enzyme–substrate reaction was stopped by adding sulfuric solution. Intensity of the reaction was measured at 450 nm by ELISA microplate reader (BioTek Instruments, USA).
All statistical analyses were performed using the Statistical Package for the Social Sciences for Windows v20.0 (SPSS Inc., Chicago, IL, USA). Kolmogorov–Smirnov test was used to examine the normal distribution of data. The mean ± standard deviation was used for continuous variables and percentage for categorical variables as according to distribution state. Mann–Whitney U-test or Student's t-test was used to compare two independent groups according to distribution state. Spearman's correlation coefficients were used to evaluate statistical significance among nonnormally distributed variables (TRX and TXNIP).
| Results|| |
Baseline characteristics of the study subjects
The mean age of 45 patients and 33 healthy controls was 40 ± 16 and 41 ± 14 years, respectively. No significant difference was observed between the two groups with respect to age. [Table 1] summarizes the demographic and laboratory characteristics of the patients with AIS.
Our results indicated that serum TXNIP levels were significant higher in patients with AIS (3.98 ± 0.19 ng/mL [mean ± standard error mean (SEM)], n = 45) than in healthy controls (3.3 ± 0.23 ng/mL [mean ± SEM], n = 33; P = 0.044 [Figure 1]a). However, no significance difference was observed in serum TRX levels between stroke patients (11.9 ± 0.63 ng/mL [mean ± SEM], n = 45; P = 0.405) and healthy controls (11.0 ± 0.73 ng/mL [mean ± SEM], n = 33 [Figure 1]b). Moreover, a positive correlation was observed between TXNIP and TRX levels [R2 = 0.476, P < 0.003; [Figure 2].
|Figure 1: (a) Analysis of serum levels of thioredoxin-interacting protein levels in acute ischemic stroke patients and healthy controls by indirect ELISA. Serum thioredoxin-interacting protein levels were significantly higher in stroke patients than in healthy controls (P = 0.044). (b) Analysis of serum thioredoxin levels between acute ischemic stroke patients and healthy controls by indirect ELISA. There was no significant difference in thioredoxin serum levels of stroke and healthy participants (P = 0.405)|
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|Figure 2: Correlation of thioredoxin-interacting protein and thioredoxin levels in stroke patients. Comparison between thioredoxin-interacting protein and thioredoxin showed positive correlation (R2 = 0.476)|
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| Discussion|| |
The main findings of the present study are as follows: (1) TXNIP levels are significantly higher in stroke patients than in healthy controls and (2) TRX levels are not significantly different between stroke patients and healthy controls.
Oxidative stress is associated with the pathogenesis of many diseases, including cancer, neurodegenerative disease, aging, and stroke. Production of free radicals (ROS) contributes to brain damage in stroke and reperfusion stroke. The brain is more prone to oxidative stress than other organs because of the presence of polyunsaturated fatty acids; high consumption of oxygen by brain cells; and decreased activities of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione reductase, thus making the brain an easy target for attack by ROS. Accumulating evidence suggests that oxidative stress plays an important role in stroke and reperfusion stroke.,, Toxic effects of the oxidative system are neutralized by the action of the enzymatic and nonenzymatic antioxidant system. The TRX/TXNIP system in the central nervous system regulates many biological processes and signaling pathways in the brain. TRX is a redox protein that exerts antioxidant effects through TRX reductase and NADPH, a reducing agent. Effects of TRX are regulated by TXNIP, which in turn regulates the cellular redox state.
In the present study, no significant difference was observed in TRX levels between stroke patients and healthy controls. A recent study reported that TRX levels were elevated in Chinese patients with AIS and were associated with stroke severity and lesion volume. This study suggested that TRX can be considered as a novel independent diagnostic marker for AIS. Our results are not in agreement with the above study. Possible reasons for the discrepancy in study results is the small sample size included in our study compared with that included in the study by Wu et al. (312 patients) and difference in the population cohort between these two studies. Therefore, further studies involving a large sample size should be performed to replicate the findings of the present study.
In the present study, TXNIP levels were statistically higher in stroke patients than in healthy controls. TXNIP is an endogenous inhibitor and regulator of the TRX system. It is possible that stroke patients in the severe phase of the disease develop high oxidative stress. Significantly elevated TXNIP expression is associated with proinflammatory and proapoptotic responses in stress-related disease models of neurotoxicity, metabolic disorder, and stroke., An earlier study had shown elevated levels of tumor necrosis factor-alpha mRNA and protein in ischemic brain. It is believed that during ischemic stroke, NOD-like receptor protein 3 inflammasome in neurons and glial cells has a role in detecting tissue damage and induces inflammatory response. A recent animal study showed that TXNIP contributed to the development of acute ischemic brain injury by inducing redox imbalance and inflammasome activation and that therapeutic inhibition of TXNIP prevented ischemic brain injury by reducing inflammatory processes. Thus, TXNIP induces neurotoxicity by releasing proinflammatory cytokines and altering antioxidant status.,
The present study is the first to report significantly increased TXNIP levels in AIS patients and to suggest that TXNIP levels can be used as a useful biomarker in this region. However, it is unclear whether significantly high TXNIP levels in AIS patients have any pathological significance, i.e., whether they are a risk factor of AIS onset or simply an indicator of oxidative stress and inflammation. Therefore, further large-scale studies involving increased number of patients are required to investigate the role of TXNIP in AIS.
- TRX and TXNIP levels were measured in a small population. Therefore, further studies involving a large sample size should be conducted to validate the results of the present study
- TRX and TXNIP levels were determined at a single time point. These findings did not reflect when and how the levels of these markers change and it is possible that our data might underestimate the fluctuations in the levels of oxidative stress. Therefore, serial measurements of circulating TRX and TXNIP levels should be performed under pre- and post-AIS conditions
- TRX and TXNIP levels were measured using the serum and not the cerebrospinal fluid. Therefore, it is unclear whether these levels reflect similar changes in the central nervous system.
| Conclusions|| |
Novel biomarkers are needed to determine the level of disease in stroke patients. To the best of our knowledge, this is the first clinical study to report increased TXNIP levels in acute ischemic stroke patients in local population.
Financial support and sponsorship
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group No (RG- 1438-008).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Feigin VL, Norrving B, George MG, Foltz JL, Roth GA, Mensah GA, et al.
Prevention of stroke: A strategic global imperative. Nat Rev Neurol 2016;12:501-12.
Alahmari K, Paul SS. Prevalence of stroke in Kingdom of Saudi Arabia – Through a physiotherapist diary. Mediterr J Soc Sci 2016;7:228-33.
Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O. Oxidative stress and antioxidant defense. World Allergy Organ J 2012;5:9-19.
Allen CL, Bayraktutan U. Oxidative stress and its role in the pathogenesis of ischaemic stroke. Int J Stroke 2009;4:461-70.
Chan PH. Role of oxidants in ischemic brain damage. Stroke 1996;27:1124-9.
Nakamura H. Thioredoxin and its related molecules: Update 2005. Antioxid Redox Signal 2005;7:823-8.
Taniguchi Y, Taniguchi-Ueda Y, Mori K, Yodoi J. A novel promoter sequence is involved in the oxidative stress-induced expression of the adult T-cell leukemia-derived factor (ADF)/human thioredoxin (Trx) gene. Nucleic Acids Res 1996;24:2746-52.
Xie Z, Sun J, Li H, Shao T, Wang D, Zheng Q, et al.
Plasma and synovial fluid trxR levels are correlated with disease risk and severity in patients with rheumatoid arthritis. Medicine (Baltimore) 2016;95:e2543.
Burke-Gaffney A, Callister ME, Nakamura H. Thioredoxin: Friend or foe in human disease? Trends Pharmacol Sci 2005;26:398-404.
Al-Gayyar MM, Abdelsaid MA, Matragoon S, Pillai BA, El-Remessy AB. Thioredoxin interacting protein is a novel mediator of retinal inflammation and neurotoxicity. Br J Pharmacol 2011;164:170-80.
Griffiths HR, Bennett SJ, Olofsson P, Dunston CR. Thioredoxin as a putative biomarker and candidate target in age-related immune decline. Biochem Soc Trans 2014;42:922-7.
Nakamura H, De Rosa S, Roederer M, Anderson MT, Dubs JG, Yodoi J, et al.
Elevation of plasma thioredoxin levels in HIV-infected individuals. Int Immunol 1996;8:603-11.
Drake C, Boutin H, Jones MS, Denes A, McColl BW, Selvarajah JR, et al.
Brain inflammation is induced by co-morbidities and risk factors for stroke. Brain Behav Immun 2011;25:1113-22.
Ishrat T, Mohamed IN, Pillai B, Soliman S, Fouda AY, Ergul A, et al.
Thioredoxin-interacting protein: A novel target for neuroprotection in experimental thromboembolic stroke in mice. Mol Neurobiol 2015;51:766-78.
Aon-Bertolino ML, Romero JI, Galeano P, Holubiec M, Badorrey MS, Saraceno GE, et al.
Thioredoxin and glutaredoxin system proteins-immunolocalization in the rat central nervous system. Biochim Biophys Acta 2011;1810:93-110.
Saeed SA, Shad KF, Saleem T, Javed F, Khan MU. Some new prospects in the understanding of the molecular basis of the pathogenesis of stroke. Exp Brain Res 2007;182:1-0.
Tsai NW, Chang YT, Huang CR, Lin YJ, Lin WC, Cheng BC, et al.
Association between oxidative stress and outcome in different subtypes of acute ischemic stroke. Biomed Res Int 2014;2014:256879.
Patenaude A, Murthy MR, Mirault ME. Emerging roles of thioredoxin cycle enzymes in the central nervous system. Cell Mol Life Sci 2005;62:1063-80.
Mohamed IN, Hafez SS, Fairaq A, Ergul A, Imig JD, El-Remessy AB, et al.
Thioredoxin-interacting protein is required for endothelial NLRP3 inflammasome activation and cell death in a rat model of high-fat diet. Diabetologia 2014;57:413-23.
Wu MH, Song FY, Wei LP, Meng ZY, Zhang ZQ, Qi QD, et al.
Serum levels of thioredoxin are associated with stroke risk, severity, and lesion volumes. Mol Neurobiol 2016;53:677-85.
Kim GS, Jung JE, Narasimhan P, Sakata H, Chan PH. Induction of thioredoxin-interacting protein is mediated by oxidative stress, calcium, and glucose after brain injury in mice. Neurobiol Dis 2012;46:440-9.
Liu T, Clark RK, McDonnell PC, Young PR, White RF, Barone FC, et al.
Tumor necrosis factor-alpha expression in ischemic neurons. Stroke 1994;25:1481-8.
Devi TS, Lee I, Hüttemann M, Kumar A, Nantwi KD, Singh LP, et al.
TXNIP links innate host defense mechanisms to oxidative stress and inflammation in retinal muller glia under chronic hyperglycemia: Implications for diabetic retinopathy. Exp Diabetes Res 2012;2012:438238.
[Figure 1], [Figure 2]