Journal of Translational Regenerative Medicine
"Translating Regeneration into Life"

Comparative Impact of Curcumin and Metformin on Reactive Oxygen Species Production and Antioxidant Gene Expression in T Cells

Document Type : Original Article

Authors

Ali Mamivand 1
Mohsen Chamanara 2
Alireza Mahboubian 3
Ali Sabberian 4
Ruhollah Mirjani 5
Zahra Hami 2
Reza Heidari 6

1 Cancer Epidemiology Research Center (AJA-CERTC), AJA University of Medical Sciences, Tehran, Iran.

2 Toxicology Research Center, AJA University of Medical Sciences, Tehran, Iran.

3 School of Business, The Ingenuity Center, University of Nottingham, Nottingham, United Kingdom.

4 Faculty of Agriculture, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.

5 Department of Genetics and Advanced Technologies, Faculty of Medicine, AJA University of Medical Sciences, Tehran, Iran.

6 Infectious Diseases Research Center, AJA University of Medical Sciences, Tehran, Iran. & Medical Biotechnology Research Center, AJA University of Medical Sciences, Tehran, Iran.

https://doi.org/10.32598/JTRM.1.1001
Abstract
Background: Excessive production of reactive oxygen species (ROS) is implicated in the pathogenesis of inflammatory and autoimmune disorders, partly through the dysregulation of T cell function. Curcumin and metformin possess well-documented antioxidant and anti-inflammatory properties, yet their combined effects on T cell oxidative stress have not been comprehensively evaluated.
Methods: Human peripheral blood T cells from healthy donors were treated with curcumin, metformin, or their combination. Intracellular ROS, superoxide, and glutathione (GSH) levels were quantified by flow cytometry. mRNA expression of key oxidative (NOX2) and antioxidant genes (CAT, SOD1, SOD2, NRF2) was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR).
Results: Both agents significantly decreased ROS levels and increased intracellular GSH compared with untreated cells. Metformin exhibited superior effects, reducing ROS by ~2.5-fold and increasing GSH nearly 3-fold compared to curcumin. Metformin also induced stronger upregulation of NRF2 and SOD2, and greater suppression of NOX2. Co-treatment produced no general synergistic effects on ROS, GSH, or most antioxidant genes, except for a significant synergistic increase in SOD1 expression.
Conclusion: Metformin outperformed curcumin in enhancing antioxidant defenses and suppressing ROS in T cells, whereas combined therapy showed limited interaction, confined to SOD1. These findings support metformin—alone or with curcumin—as a potential candidate for managing oxidative stress–driven immune disorders, warranting further in vivo and clinical evaluation.

Keywords

Oxidative stress
Reactive oxygen species (ROS)
Metformin
Curcumin
T cells
Introduction
Reactive oxygen species (ROS) are highly reactive molecules containing oxygen that are generated naturally as byproducts of cellular metabolism. They are primarily produced by the electron transport chain in mitochondria and the cytochrome P450 system. Additionally, a significant source of ROS originates from NADPH oxidases expressed in various cell types, particularly in professional phagocytes and endothelial cells, where their dysregulation contributes to vascular pathology and oxidative stress [1, 2]. These enzymes are crucial in initiating the inflammatory response [3]. Besides their involvement in inflammation, ROS play essential roles in numerous physiological processes, including cell signaling and immune system function [4, 5]. 
While ROS are vital for maintaining normal cellular activities, an imbalance between their production and elimination can lead to oxidative stress. This imbalance can damage critical cellular components, including proteins, lipids, and DNA. Consequently, oxidative stress is implicated in the development of several diseases, including neurodegenerative disorders, cardiovascular diseases, cancer, and autoimmune conditions [6-8]. To combat ROS, cells employ intricate defense mechanisms that include antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) peroxidase, as well as non-enzymatic antioxidants such as vitamins C and E, which collectively help maintain the balance between ROS production and elimination [9, 10].
In the context of inflammation, ROS serve a dual purpose. They act as signaling molecules essential for host defense against pathogens, but can also contribute to tissue damage when their levels become excessive or dysregulated. Maintaining a precise balance of ROS is therefore critical, as excessive ROS can lead to chronic inflammation and significantly contribute to the pathogenesis of various inflammatory diseases. Current research focuses on unraveling the complex regulatory mechanisms that control ROS levels to develop targeted therapeutic strategies for inflammatory disorders [11, 12].
T lymphocytes are central to the adaptive immune system, orchestrating responses to pathogens, allergens, and tumors throughout an individual’s life [13, 14]. For their proliferation and functional responses, T cells depend on multiple metabolic pathways to generate sufficient energy and metabolites. These metabolic activities, however, often result in the production of ROS. Emerging studies have identified ROS as vital secondary messengers in T cell receptor signaling and activation, although their effects can vary across different T cell subsets. Therefore, the precise regulation of ROS production through cellular antioxidant mechanisms is essential to ensure accurate signaling and effective T cell responses [15].
Numerous studies have explored the impact of curcumin on ROS metabolism and inflammation. Curcumin, a compound renowned for its antioxidant properties, effectively scavenges ROS, including superoxide anions and HO [16, 17]. It also enhances the activity of key antioxidant enzymes, such as SOD and CAT, which are fundamental in protecting the body against oxidative stress. Due to its potent anti-inflammatory and antioxidant effects, curcumin has gained considerable attention as a potential therapeutic agent for chronic inflammatory diseases such as rheumatoid arthritis (RA) and inflammatory bowel disease, with various studies investigating its efficacy in these conditions [8, 18, 19].
Similarly, metformin is being studied for its potential effects on ROS and oxidative stress [20]. Metformin primarily functions by activating AMP-activated protein kinase (AMPK), a critical regulator of cellular energy homeostasis. Activation of AMPK leads to multiple downstream effects, including the suppression of hepatic gluconeogenesis, increased glucose uptake in peripheral tissues, and maintenance of intracellular energy balance. Notably, evidence suggests that metformin’s antioxidant effects may be linked to AMPK activation [21, 22]. This mechanism may play a significant role in managing oxidative stress at the cellular level, providing insights into metformin’s broader implications in reducing inflammation. These findings indicate that metformin may help lower oxidative stress, supported by scientific studies demonstrating its beneficial effects.
In this study, we examine the antioxidant effects of curcumin and metformin on the redox system in T cells. While previous research has investigated the impact of these two drugs on the redox systems of various tissues, their combined effects on the immune system, particularly on T cells, remain unexplored. Therefore, we focused on how curcumin and metformin influence the expression of enzymes involved in ROS production within T cells. Specifically, our investigation highlights how the combination of these drugs enhances the expression of antioxidative genes, including CAT, nuclear factor erythroid 2-related factor 2 (NRF2), and SOD (SOD1 and SOD2), which are crucial for degrading ROS and mitigating oxidative stress. Concurrently, the treatment suppresses NADPH oxidase 2 (NOX2) expression, a gene directly involved in ROS generation. Using flow cytometry, we quantitatively assess changes in ROS, superoxide anion (O₂⁻), and GSH levels in T cells following treatment, thereby corroborating the genetic expression data with empirical evidence of reduced oxidative stress. This research proposes a potential therapeutic strategy combining curcumin and metformin for conditions characterized by heightened inflammatory responses and oxidative stress, providing a molecular basis for their use by modulating ROS dynamics in immune cells.

Materials and Methods
Cell culture
Blood was obtained from healthy donors, and human peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll–Paque density gradient centrifugation. These isolated PBMCs were then placed in 24-well plates (1.5×106 cells/well) and cultured in RPMI 1640 with 10% FBS and 100 IU hIL-2 (Miltenyi Biotec). To enrich T cells, PBMCs were cultured with 3 μg/mL anti-CD3 (Miltenyi Biotec) and 10 μg/mL anti-CD28 (Miltenyi Biotec) antibodies.

Flow cytometry and ROS assay
ROS and superoxide detection assay kits (ab139476, USA) were utilized to assess intracellular ROS production levels. T cells (2×105 cells per well) were cultured in RPMI1640 complete media with or without curcumin, metformin, and metformin/curcumin co-treatment (7 mM). Following harvesting and washing, the cells were exposed to a permeable green probe (responsive to hydroxyl radicals [HO], hydrogen peroxide (H2O2), peroxynitrite [ONOO-], peroxy radical [ROO], and nitric oxide [NO]) and an orange probe (specifically reactive to superoxide [O2-]) at 37 °C for 30 minutes. The GSH antioxidant level was determined using a GSH assay kit (ab112132, USA). After harvesting and washing, the cells were treated at 24 °C with thiol green dye for 20 minutes. Subsequently, flow cytometry was employed for cell analysis. ROS/superoxide and GSH production were determined by measuring the difference in mean fluorescence intensity (MFI) between treated and untreated cells. Flow cytometry was performed using a BD FACS Calibur (BD Biosciences, USA) and analyzed using FlowJo software, version 7.6.1 The experiments were carried out in triplicate and replicated three times.

RNA extraction and cDNA synthesis
TriPure isolation reagent (Roche, Mannheim, Germany) was used for RNA extraction, according to the protocol. RNA concentration was quantified with the NanoDrop-2000 spectrophotometer (Thermo Fisher, U.S) and sorted at -70 °C. cDNA synthesis using 1 μg of total RNA and PrimeScript RT reagent (Takara Bio Inc., Shiga, Japan) was performed according to the protocol.

Quantitative reverse transcription polymerase chain reaction 
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was carried out using the SYBR Premix Ex TaqTM according to a previous study [23]. First, primers were designed using Oligo 7, and their specificity was checked using Primer-BLAST. Primer sequences were listed in Table 1.
 
 
All primers were designed on exon-exon junctions or had an intron larger than 2000 nucleotides between them to prevent amplification of genomic DNA contamination (if any). The melting curve was also analyzed to confirm the primers’ specificity. A serial dilution was prepared, and PCR conditions were adjusted to achieve a PCR efficiency of 2. The qRT-PCR reaction was performed according to the following conditions: incubation for 10 min at 95 °C, followed by 40 cycles of elongation, including 10s at 95 °C and 25 s at 60 °C. To exclude primer dimers or byproducts, dissociation curves were carefully analyzed to assess the specificity of the product melting peak. The PCR products were ultimately confirmed by 2% agarose gel electrophoresis.

Statistical analysis
Superoxide and GSH levels in CD4+ T cells between the treated and untreated groups were compared using a one-way analysis of variance (ANOVA) followed by a Tukey post hoc test. The Wilcoxon test was used to assess differences in expression between groups. A P<0.05 was deemed statistically significant for a confidence interval of 95%. Statistical analysis was performed using GraphPad Prism software, version 8.

Results
Curcumin treatment and significant antioxidant effects on T cells
Flow cytometry analysis revealed that curcumin treatment markedly reduced intracellular ROS levels in activated T lymphocytes compared with untreated controls (Figure 1A).
 
 
The MFI for ROS decreased substantially, indicating a significant attenuation of oxidative stress. Although a mild decrease in superoxide (O₂⁻) production was observed, this change did not reach statistical significance. Conversely, intracellular GSH levels rose significantly following curcumin administration (Figure 1B), suggesting enhanced antioxidant capacity.
Gene expression analysis by qRT-PCR showed significant upregulation of key antioxidant enzymes—CAT, SOD1, and SOD2—in curcumin-treated cells. While NRF2 expression exhibited an upward trend, it did not reach statistical significance. Importantly, curcumin significantly downregulated NOX2 expression, a critical ROS-generating enzyme, consistent with the observed reduction in oxidative stress (Figure 1C).
 
Metformin treatment demonstrates significant antioxidant effects on T Cells
Metformin treatment resulted in a pronounced reduction in ROS levels in T lymphocytes, surpassing the effect observed with curcumin (Figure 2A).
 
 
The MFI for ROS in metformin-treated cells (108) was approximately 2.5-fold lower than that of curcumin-treated cells (286). GSH levels increased significantly, nearly three-fold higher with metformin (1494 MFI) compared to curcumin (544 MFI) (Figure 2B). Superoxide production showed a slight but statistically nonsignificant decrease.
At the molecular level, metformin significantly upregulated CAT, SOD1, SOD2, and NRF2. Moreover, metformin markedly suppressed NOX2 expression to a greater extent than curcumin (Figure 2C). Collectively, these data indicate that metformin exerts more potent antioxidant effects on T cells, both by enhancing antioxidant defence genes and by more effectively inhibiting prooxidant gene expression.

Co-treatment with metformin and curcumin does not demonstrate synergistic effects
When both drugs were applied simultaneously, ROS levels decreased significantly and GSH levels rose compared with untreated cells (Figure 3A), mirroring the trends seen with single-agent treatments.
 
 
However, the magnitude of change was comparable to metformin alone, and no statistically significant difference was observed in ROS or GSH levels relative to either monotherapy. Superoxide production showed a slight, non‑significant rise compared with monotherapy groups (Figure 3B).
Gene expression profiling revealed that combined treatment induced increases in CAT, SOD1, SOD2, and NRF2, and suppressed NOX2, resembling the patterns observed with the individual treatments (Figure 3C). Notably, SOD1 was the only antioxidant enzyme to show a significant synergistic increase under co-treatment, suggesting a specific but limited additive effect.

Comparative analysis across treatment groups
Direct comparison of the three experimental groups—curcumin, metformin, and their combination—confirmed metformin’s superior efficacy in lowering ROS and boosting GSH levels (Figure 4A).
 
 
ROS production in curcumin-treated cells (286 MFI) was approximately 2.5 times higher than in metformin-treated cells (108 MFI). GSH levels were almost three times higher in the metformin group (1494 MFI) than in the curcumin group (544 MFI). NRF2, SOD2, and NOX2 expression differed significantly between curcumin and metformin groups, favoring metformin. In contrast, CAT and SOD1 levels were slightly higher with curcumin, but the difference was not statistically significant. As expected from the gene-level data, the only clear collaboration in the co-treatment group was observed for SOD1 expression (Figure 4B).

Discussion
In this study, we examined the antioxidant effects of curcumin and metformin on T cells, with a particular focus on their ability to modulate ROS production and alter the expression of key genes involved in ROS metabolism. Our results clearly demonstrate that both agents significantly reduced intracellular ROS levels—as evidenced by decreased fluorescence intensity in flow cytometry assays—with metformin-treated T cells exhibiting approximately 2.5-fold lower ROS fluorescence (108 MFI) than curcumin-treated cells (286 MFI). Both treatments enhanced intracellular antioxidant capacity by increasing GSH levels, though metformin induced a nearly three-fold greater rise than curcumin (1494 MFI vs 544 MFI). This stronger effect may be linked to metformin’s potent activation of AMPK and its consequent impact on upregulation of NRF2 and SOD2, along with more substantial suppression of NOX2 expression.
These findings fit within the broader context of ROS biology in immune cells. ROS play a dual role in immune function—facilitating pathogen eradication by damaging microbial membranes and DNA. At the same time, excessive accumulation can impair T cell function, intensify inflammation, and contribute to the development of autoimmune disorders [24]. Elevated ROS levels have been reported in diseases such as RA, multiple sclerosis (MS), and inflammatory bowel disease (IBD) [25-27]. For instance, IBD is characterized by abnormally high ROS levels in the colon, and increased ROS in T cells during relapse phases of MS has been documented, along with elevated expression of ROS-producing enzymes [25-27]. Therefore, agents that reduce ROS in T cells are likely to improve disease prognosis and attenuate pathological inflammation.
In our study, curcumin significantly decreased ROS levels in T cells and upregulated antioxidant enzymes CAT, SOD1, and SOD2, while NRF2 expression increased but did not reach statistical significance. These observations align with previous research in other cell types, such as endothelial cells in diabetic patients, where curcumin treatment reduced NOX2 expression—a key enzyme in ROS production [28]. Besides its antioxidant role, curcumin is well known for its anti-inflammatory effects, including inhibition of pro-inflammatory cytokines and chemokines and downregulation of inflammatory gene expression [29].
Consistent with its reported immunomodulatory properties, metformin in our study exhibited more potent antioxidant effects than curcumin. Compared with curcumin, metformin led to markedly greater suppression of NOX2, significant upregulation of NRF2 and SOD2, and a nearly three-fold larger increase in GSH levels. Metformin has been shown to inhibit pro-inflammatory cytokines (interleukin-1 beta [IL-1β], IL-1β, IL-6, Tumor necrosis factor [TNFα]) and promote IL-10 expression in activated macrophages [30], as well as shift T cell differentiation towards regulatory T cells (Tregs) while suppressing Th1 and Th17 cell subsets via AMPK activation [30]. These mechanisms, in combination with its antioxidant activity, may explain the superior performance of this compound in reducing ROS in T cells observed here.
When comparing the two agents directly, a broadly similar pattern of antioxidant gene expression emerged. Still, the magnitude of change differed substantially, favoring metformin for ROS suppression, GSH enhancement, NRF2 and SOD2 upregulation, and NOX2 suppression. Co-treatment with both compounds did not produce synergistic effects for most measured outcomes. The sole exception was SOD1 expression, which showed a statistically significant synergistic increase under combined treatment. This lack of broader synergy could indicate overlapping modes of action or saturation of shared signaling pathways, such as those mediated by AMPK or NRF2-dependent antioxidant responses.
It should be noted that these findings are based on in vitro T cell cultures, which may not fully replicate the complexity of immune responses in vivo. The lack of synergy across most parameters suggests that alternative dosing strategies, sequential administration, or formulation changes may be needed to maximize potential combinational benefits. Future studies involving in vivo models and clinical trials are necessary to validate these results and further investigate the molecular interplay between curcumin and metformin in regulating oxidative stress in immune-mediated disorders.

Conclusion
In conclusion, both curcumin and metformin reduced oxidative stress in human T cells by lowering ROS levels and enhancing antioxidant defenses. Metformin consistently outperformed curcumin, showing approximately 2.5-fold greater ROS suppression, nearly 3-fold higher GSH elevation, stronger upregulation of NRF2 and SOD2, and more potent suppression of NOX2 expression. While co-treatment with both compounds did not produce broad synergistic effects across ROS, GSH, or most antioxidant genes, SOD1 expression emerged as a noteworthy exception, displaying a clear synergistic enhancement. These findings suggest that metformin, alone or in combination with curcumin, may be a promising therapeutic candidate for immune-related disorders characterized by elevated oxidative stress. Nevertheless, the lack of general synergy and the in vitro nature of this study underscore the need for further in vivo work and clinical evaluation to determine optimal therapeutic strategies and elucidate the molecular interactions underlying these effects.

Ethical Considerations
Compliance with ethical guidelines
There were no ethical considerations to be considered in this research.

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.

Authors' contributions
Conceptualization and study design: Ali Mamivand and Reza Heidari; Data collection, data analysis and interpretation, writing, and final approval: All authors.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors would like to express their gratitude to the Central Research Laboratory at Aja University of Medical Sciences, Tehran, Iran, for providing the necessary resources and support for this research.


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    2. Kato K, Hecker L. NADPH oxidases: Pathophysiology and therapeutic potential in age-associated pulmonary fibrosis. Redox Biology. 2020; 33:101541. [DOI:10.1016/j.redox.2020.101541] [PMID]
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    5. Chakrabarti S, Visweswariah SS. Intramacrophage ROS Primes the Innate Immune System via JAK/STAT and Toll Activation. Cell Reports. 2020; 33(6):108368. [DOI:10.1016/j.celrep.2020.108368] [PMID]
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    7. Das A, Chakrabarty S, Nag D, Paul S, Ganguli A, Chakrabarti G. Heavy water (D(2)O) induces autophagy-dependent apoptotic cell death in non-small cell lung cancer A549 cells by generating reactive oxygen species (ROS) upon microtubule disruption. Toxicology in Vitro. 2023; 93:105703. [DOI:10.1016/j.tiv.2023.105703] [PMID]
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    9. Menezes LB, Segat BB, Tolentino H, Pires DC, Mattos LMM, Hottum HM, et al. ROS scavenging of SOD/CAT mimics probed by EPR and reduction of lipid peroxidation in S. cerevisiae and mouse liver, under severe hydroxyl radical stress condition. Journal of Inorganic Biochemistry. 2023; 239:112062. [DOI:10.1016/j.jinorgbio.2022.112062] [PMID]
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