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. 2025 Oct 31;14:118. doi: 10.4103/abr.abr_99_24

Melatonin Reduces Radiation-induced Apoptosis Through Change of bax and bcl-2 Expression in Peripheral Blood Lymphocytes

Elham Motallebzadeh 1, Sayyed Alireza Talaei 2, Mehran Mohseni 3,✉
PMCID: PMC12646334  PMID: 41307083

Abstract

Background:

The most important harmful effect of ionizing radiation is the production of free radicals. A correlation was demonstrated between free radicals and apoptosis. Melatonin might be a direct free radical scavenger. Melatonin’s ability to modify the expression of bcl-2 and bax and apoptosis in peripheral blood lymphocytes in rats was studied.

Materials and Methods:

Rats received one total-body x-radiation dosage of 2 Gy either with or without prior treatment of melatonin at a dosage of 100 mg/kg body weight. The animals were grouped as follows: VC control (VC), 2 Gy irradiation (RT), 100 mg/kg melatonin + 2 Gy irradiation (MLT + RT), and 100 mg/kg melatonin (MLT). Animals were given intraperitoneal melatonin or an identical amount of vehicle alone 1 hour before radiation. Blood samples were collected 4, 24, and 72 hours after radiation to evaluate apoptotic lymphocytes through flow cytometric analysis using the Annexin V/PI assay, as well as to measure bcl-2 and bax expression via quantitative real-time PCR.

Results:

An elevation was noted in the RT group in the percentage of apoptotic lymphocytes compared to the VC group (P < 0.0001). In contrast, the MLT + RT group considerably reduced it compared to the RT group (P < 0.0001) in all periods. The decline in melatonin-induced apoptosis was for the bax downregulation, bcl-2 upregulation, and a lower ratio of bax to bcl-2.

Conclusion:

Melatonin can protect rat lymphocytes from x-radiation-triggered apoptosis by modulating bcl-2 and bax expression. Therefore, melatonin could serve as a potential radio-protective agent, making it a candidate for use alongside radiation in clinical trials.

Keywords: Apoptosis, ionizing radiation, lymphocytes, melatonin

INTRODUCTION

Radiation therapy is commonly used for cancer treatment utilizing high-energy particles or waves such as gamma and X-rays to target and destroy cancer cells. Its main goal is to damage the DNA within these cells, preventing them from dividing and growing.[1] Increasing use of ionizing radiation in various medical fields and occupational exposure requires radiation protection.[2] Ionizing radiation can cause the creation of reactive oxygen species (ROS) in biological systems. This process happens when radiation collides with water molecules and produces free radicals, which can then negatively impact subcellular structures, especially DNA molecules.[3,4,5] Because of the association between apoptosis and free radicals, most anti-cancer treatments such as radiotherapy cause DNA damage and apoptosis in healthy cells and tissues.[6,7]

Apoptosis is a genetically programmed mechanism of cell death. The bcl-2 family of proteins, comprising pro-apoptotic genes such as bax and apoptosis-inhibitors protein bcl-2, regulates apoptosis. The relative levels of the pro-apoptotic bax versus the anti-apoptotic bcl-2 can specify the cell’s relative sensitivity to the apoptotic stimulus. Change in the bax/bcl-2 ratio is a significant and critical factor in the cell’s decision to survive or start the apoptotic death process.[1,8,9,10] Lymphocytes are among the most sensitive cells that can undergo apoptosis easily in patients treated with radiotherapy.[11]

As cell damage triggered by ionizing radiation is mostly due to the adverse impacts of free radicals, molecules with direct scavenging features of free radicals are specifically desirable as radioprotectors.[12] Many studies have shown that melatonin (N-acetyl-5-methoxytryptamine), the pineal gland’s main secretory product, is a strong direct scavenger of free radicals. The main reason for measuring its radiation protection effect[13,14,15] is its fat-soluble nature and small size; melatonin easily passes through biological membranes and enters the cell.[16] There are research works regarding the protective function of melatonin against radiation damage in different animal and human tissues.[13,17,18,19] As shown by recent research findings, melatonin reduces apoptosis caused by X and gamma rays in the spleen lymphocytes of both Indian squirrels and mice.[13] Sharma’s results demonstrated that melatonin pre-treatment renovates apoptosis in splenocytes induced by radiation.[20,21]

This work aimed at studying the moderating impact of melatonin on 2 Gy radiation-triggered apoptosis and bcl-2 and bax expression in experimental groups 4, 24, and 72 hours after radiation in rat blood lymphocytes. According to a previous work,[14] the gene expression in the phenomenon of apoptosis caused by radiation changes the most during these times.

MATERIALS AND METHODS

Research units

The current experimental research was carried out on adult male Wistar rats (180–200 g), which were sourced from the animal facility of Kashan University of Medical Sciences in Kashan, Iran. Standard plexiglass cages (25 [w] × 45 [L] × 20 [h] cm) were used at 4‐rat/cage for housing animals. The experiments were conducted under a 12-hour light/12-hour dark cycle, at 22°C–24°C and humidity at 50%–55%. Animals had free access to water and standard diet food (Pars Animal Food Company, Iran).

Experimental design and irradiation

First, a 2-week adjustment period was conducted, and the rats were put in four groups randomly. The experiment involved 60 rats, with each of the four groups consisting of 15 rats: the vehicle-received control (VC), the 2 Gy total body irradiation (RT), the melatonin-treated (100 mg/kg body weight) (MLT), and the melatonin (100 mg/kg body weight) +2 Gy total body irradiation (MLT + RT).

An hour prior to the experiment, all the rats were transported to a lab near the Siemens Primus linear accelerator (Siemens AG, Erlangen, Germany).[22] In the VC group, rats only received an intraperitoneal (IP) injection of 5% absolute ethanol in 500 µL PBS, whereas the RT rats received 5% absolute ethanol in PBS before 2 Gy total body irradiation. Rats in the MLT group received an intraperitoneal injection of melatonin (freshly prepared) (Sigma–Aldrich Co., St. Louis, MO, USA) in 5% absolute ethanol solution,[14] while the MLT + RT group received melatonin before 2 Gy radiation. An hour following the injections, the animals were sedated with an intraperitoneal injection of xylazine (20 mg/kg) and ketamine (60 mg/kg). Afterward, the animals in groups 2 and 4 received total-body radiation at a single dose of 2 Gy with 200 cGy/min dose rate in the field size of 40 × 40 cm2 by the use of a source surface distance of 100 cm.[23,24] The one-hour gap between injection and exposure to radiation was chosen according to findings from earlier research.[14,25] In addition, the concentration of melatonin and the radiation dose chosen were selected according to previous research findings.[26,27] Under xylazine (10 mg/kg) and ketamine (50 mg/kg) anesthesia, five blood samples were taken on EDTA sterile tubes at each collection time of 4, 24, and 72 h from each group after radiation. Blood samples were split into two sections. One section was used to evaluate apoptotic lymphocytes through flow cytometry with Annexin V/propidium iodide (PI) double staining, while another section was employed to measure the expression levels of bax and bcl-2 by quantitative real-time reverse transcriptase polymerase chain reaction (RT2qPCR).

Flow cytometry of apoptotic lymphocytes

Consistent with our previous study, a flow cytometric analysis was performed on apoptotic lymphocytes. Briefly, lymphocytes were separated from blood samples by Ficoll-Histopaque density. Each lymphocyte sample normally involved a primary density of 1 × 106 cells/mL. The Annexin-V-FLUOS Staining Kit was used to assess necrosis and apoptosis based on the manufacturer’s guidelines. Additionally, a negative control lymphocyte sample was collected at each time point without undergoing the staining process to identify the quadrant. Lymphocyte samples’ data were collected directly after the staining process was finished. Flow cytometric analysis on a FACS Calibur flow cytometer was conducted to analyze lymphocyte samples for necrotic and apoptotic cells. A minimum of five independent lymphocyte samples were examined for each group at the indicated time points after 2 Gy radiation. In each sample, 10,000 events were calculated and evaluated.[14]

Quantitative real-time RT-PCR

In our earlier research, we employed quantitative real-time PCR (RT2qPCR) for assessing the bcl-2 and bax gene expression. Briefly, from each lymphocyte sample, RNA was taken using the High Pure RNA Isolation Kit following the manufacturer’s instructions. Expand Reverse Transcriptase (Roche Diagnostics GmbH, Mannheim, Germany) was used to synthesize cDNA through reverse transcription of 1 mg of total RNA utilizing random hexamer primers in a total volume of 20 mL.

Titan HotTaq EvaGreen® qPCR Mix (BioAtlas, Riia, Estonia) was utilized on the Bio-Rad real-time PCR detection system (Hercules, CA, USA) to perform RT2qPCR amplifications. For each group, five separate blood samples were evaluated at 4, 24, and 72 hours following irradiation. Each sample underwent testing in triplicate. The comparative 2-∆∆T[28] method was employed to assess relative fold alterations in the expression of bcl-2 and bax as target genes, normalized against an endogenous reference (GAPDH gene), and a pertinent untreated and unirradiated control. ∆∆CT was the difference between average ∆CT (vehicle control) and average ∆CT (treatment group), with ∆CT representing the difference between the mean CT of the related gene and the mean CT internal control gene.[14]

Statistical analysis

Each data point indicates the mean ± SD from a minimum of five independent experiments for each group. A two-way repeated measures ANOVA and Tukey’s post-hoc test were performed, and the significance threshold was considered as P < 0.05.

RESULTS

Figures 1–7 present the relative expression of bcl-2 and bax genes, the ratio of bax to bcl-2, and the analysis of apoptotic lymphocytes across four groups at all time points.

Figure 1.

Figure 1

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The experimental groups displayed RT² qPCR analysis of the fold change in bax at different periods following irradiation (in relation to VC). Values are presented as mean ± SD of five independent samples. A significant difference in comparison to the VC (**** P < 0.0001) and RT (### P < 0.001) groups

Relative expression of bcl-2 and bax/bcl-2 ratio

Figure 1 illustrates that in the RT group, bax expression significantly rose during the first four hours in comparison with the VC group (10.56 ± 0.86-fold; P < 0.0001) and then stabilized at comparable levels at both 24- and 72-hours post-irradiation. In the MLT + RT group, bax expression was notably reduced in comparison with the RT group at 4, 24, and 72 hours after radiation (6.93 ± 0.67-fold, 6.83 ± 0.76-fold, and 4.93 ± 0.49-fold, respectively; P < 0.001) [Figure 1].

Conversely, in the RT group, bcl-2 showed a significantly lower expression during the first four hours than the VC group (0.67 ± 0.05-fold; P < 0.001) and remained at that reduced level at both 24 and 72 hours after irradiation. In the MLT + RT group, expression of bcl-2 showed a considerably higher compared to the RT group at 4, 24, and 72 hours after irradiation (0.87 ± 0.01-fold, 0.89 ± 0.03-fold, and 0.92 ± 0.01-fold, respectively; P < 0.01, P < 0.0001, and P < 0.001, respectively) [Figure 2]. The reduction in bax activation and the elevation in expression of bcl-2 led to a notable decline in the ratio of bax to bcl-2 in the MLT + RT group when in comparison with the RT group across all time points (8.57 ± 0.77 vs. 15.62 ± 2.18 at 4 h, 7.72 ± 1.34 vs. 15.53 ± 1.91 at 24 h, and 5.33 ± 0.66 vs. 14.18 ± 1.59 at 72 h; P < 0.0001) [Figure 3].

Figure 2.

Figure 2

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The experimental groups displayed RT² qPCR analysis of the fold change in bcl-2 at different periods following irradiation (in relation to VC). Values are presented as mean ± SD of five independent samples. A significant difference in comparison to the VC (*** P < 0.001) and RT (## P < 0.01) groups in four time periods, (#### P < 0.0001) in 24 hours, and (### P < 0.001) in 72 hours, respectively

Figure 3.

Figure 3

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The experimental groups displayed RT² qPCR analysis of the fold change in bax/bcl-2 ratio at different periods following irradiation (in relation to VC). Values are presented as mean ± SD of five independent samples. A significant difference in comparison to the VC (**** P < 0.0001) and RT (#### P < 0.0001) groups

Apoptotic lymphocytes analysis

Figures 4 and 5 illustrate that four hours after irradiation, the RT groups exhibited a significant rise in the proportion of apoptotic lymphocytes (Annexin V + and PI-) when compared to the VC group (55.08 ± 0.95% vs. 4.52 ± 0.77%; P < 0.0001). In contrast, the MLT + RT group demonstrated a reduction in the proportion of apoptotic lymphocytes relative to the RT group (29.4 ± 1.22%; P < 0.0001).

Figure 4.

Figure 4

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Melatonin’s effect on apoptosis triggered by radiation in the peripheral blood lymphocytes of rats was investigated. The rats took a single total body radiation dose of 2 Gy (RT), either with or without prior treatment with melatonin (MLT). Flow cytometric assay was used to analyze necrotic and apoptotic lymphocytes 4 hours after irradiation. Sample dot plots from one of five independent tests involving Annexin V and PI staining. Necrotic lymphocytes (PI+ and Annexin V+) are located in the upper right quadrant and Apoptotic lymphocytes (PI− and Annexin V+) are represented in the lower right quadrant

Figure 5.

Figure 5

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Flow cytometry was used to assess apoptotic lymphocytes at 4, 24, and 72 hours after irradiation. The proportions of apoptotic lymphocytes in the case groups are presented. Values are presented as mean ± SD of five independent samples. A significant difference compared to the VC (**** P < 0.0001) and RT (#### P < 0.0001) groups

Furthermore, 24 hours after radiation, flow cytometric analysis revealed results that were nearly identical to the levels of apoptotic lymphocytes observed at 4 hours after irradiation in case groups [Figures 5 and 6]. As shown in Figures 5 and 7, at 72 hours post-radiation, RT groups revealed a significant rise in the proportion of apoptotic lymphocytes (Annexin V + and PI-) than VC groups (50.97 ± 0.5% vs. 4.66 ± 0.87%; P < 0.0001). However, in MLT + RT groups, the proportion of apoptotic lymphocytes decreased compared to the RT group (21.07 ± 0.92%; P < 0.0001). The VC and MLT groups showed comparable levels of apoptotic lymphocytes at 4, 24, and 72 hours post-irradiation. Additionally, x, the proportion of necrotic lymphocytes (Annexin V+ and PI+), showed an insignificant (<2%) and minimal level in all groups at the specified time points after exposure to radiation [Figures 4, 6, and 7].

Figure 6.

Figure 6

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Melatonin’s effect on apoptosis triggered by radiation in the peripheral blood lymphocytes of rats was studied. The rats took a single total body radiation dose of 2 Gy (RT) either with or without prior treatment with melatonin (MLT). Flow cytometric assay was used to assess necrotic and apoptotic lymphocytes 24 hours after irradiation. Sample dot plots from one of five independent tests involving Annexin V and PI staining. Necrotic lymphocytes (PI+ and Annexin V+) are located in the upper right quadrant, and apoptotic lymphocytes (PI− and Annexin V+) are represented in the lower right quadrant

Figure 7.

Figure 7

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Melatonin’s effect on apoptosis triggered by radiation in the peripheral blood lymphocytes of rats was studied. The rats took a single total body radiation dose of 2 Gy (RT) either with or without prior treatment with melatonin (MLT). Flow cytometric assay was used to assess necrotic and apoptotic lymphocytes 72 hours after irradiation. Sample dot plots from one of five independent tests involving Annexin V and PI staining. Necrotic lymphocytes (PI+ and Annexin V+) are located in the upper right quadrant, and apoptotic lymphocytes (PI− and Annexin V+) are represented in the lower right quadrant

DISCUSSION

Lymphocytes are the most susceptible cells that can be significantly impacted by apoptosis in patients undergoing radiotherapy.[29,30] The bcl-2 family contains pro-apoptotic proteins such as bax and apoptosis-inhibiting proteins such as bcl-2, which play a role in regulating apoptosis. Equilibrium between these proteins is essential for controlling the activation and deactivation of the cellular apoptotic machinery. Any alteration in the ratio of pro-apoptotic and anti-apoptotic factors influences cell death.[1]

This study examines the molecular mechanisms behind melatonin’s protective effect against apoptosis induced by radiation by analyzing the gene expression of key proteins related to apoptosis, including bax and bcl-2. Our findings are similar to our previous study with a dose of 8 Gy gamma radiation, and we showed that a dose of 2 Gy, which is a clinically prevalent fractional dosage, could alter the bcl-2 and bax expressions.[14,21]

In our study, after 2 Gy x-irradiation in rats, there was a rapid increase in apoptotic lymphocytes, with significant changes lasting up to 72 hours. Furthermore, radiation produced the up-regulation of bax and downregulation of bcl-2 and consequently an elevation in the ratio of bax to bcl-2 in lymphocytes and showed consistency with the rise in apoptosis in all periods in post-irradiation. Our findings align with previous studies showing similar effects on spleen lymphocytes after radiation exposure. Jang et al.,[21] found that an increase in apoptosis of spleen lymphocytes after 2 Gy X-ray irradiation was followed by a decrease in bcl-2 expression level and thus an increase in the ratio of bax to bcl-2.

Cui et al.[31] discovered that following 4 hours of total body exposure to 2–8 Gy of gamma radiation, there was a rapid increase in apoptotic lymphocytes, with the expression of the bax gene peaking 24 hours post-radiation. The expression of bcl-2 decreased at 3 hours following radiation, and the decline reached its lowest of 24 hours afterward. Another study conducted by Ghorbani et al. examined various doses ranging from 20 mGy to 1000 mGy by γ-rays in rat peripheral blood lymphocytes. Their results demonstrated that 1000 mGy irradiation groups significantly increased the bax/bcl-2 ratio at 24 hours post-irradiation.[29,32]

Some studies have indicated a time-dependent expression,[33] particularly within the first 5 hours, with a return to baseline levels occurring within 20 hours.[34] In Azimian’s research, however, these changes persisted much longer, gradually diminishing over the course of a week. They reported that low doses of gamma radiation (ranging from 20 to 100 mGy) resulted in a reduction in the ratio of bax to bcl-2 in peripheral blood lymphocytes observed from 4 to 168 hours post-radiation. Although the impact of high doses in altering the bax/bcl-2 ratio compared to low doses of gamma radiation is inverse, the period of bax and bcl-2 expression changes is the same.[35] Furthermore, Wei et al.[36] showed that an 8 Gy dose of irradiation caused apoptotic damage in mouse spleen tissue by downregulating the expression of the anti-apoptosis protein bcl-2 and upregulating pro-apoptotic proteins such as bax, effects that lasted until day 28. Additionally, Yu-Rong Li examined the hematopoietic system in mice subjected to a 6 Gy dose 7 days after the exposure.

Following radiation, expression of bcl-2 was repressed while bax expression increased, which aligns with previous studies, indicating the ability of radiation to induce apoptosis in bone marrow cells.[37]

Alvarez et al.[38] observed similar patterns in thymus and spleen tissues, noting that changes in bax and bcl-2 expression occurred as early as 1 hour post-exposure, peaking at 3 hours after total body irradiation at doses as low as 0.05 Gy. This underscores the importance of both the specific gene involved and the organ being examined in understanding the cellular response to radiation.

Our results showed that pretreatment with 100 mg/kg of melatonin decreased the expression of bax at the specified time points after radiation and enhanced the expression of bcl-2 at all periods following irradiation.

Furthermore, a notable reduction in the ratio of bax to bcl-2 was noticed at all periods following irradiation. This is significant because the ratio of bax to bcl-2 is often a key factor in regulating apoptosis.

The notable reduction in the ratio of bax to bcl-2 for melatonin treatment suggests a protective mechanism against radiation-induced apoptosis. By suppressing the opening of mitochondrial permeability transition pores, melatonin can help maintain mitochondrial integrity, resulting in the prevention of the cytochrome C release into the cytosol. This action effectively inactivates the caspase cascade, which is crucial for the apoptotic process, ultimately leading to reduced apoptosis in irradiated cells. In our study, a comparison of apoptosis across all periods between the case groups showed that pretreating with a 100 mg/kg dose of melatonin considerably decreased 2 Gy X-irradiation-induced apoptosis in lymphocytes of rats.

Our results are consistent with those presented by Kumar et al.,[13] which demonstrated that melatonin modulates the expression of apoptosis-related proteins (bcl-2 and bax) in the spleen and bone marrow of mice. Their research highlighted the anti-apoptotic capacity of melatonin, which appears to be linked to proteins related to the G-CSF pathway.

The findings from Jang et al.[21] highlight the protective function of melatonin against radiation-induced apoptosis, particularly at a dose of 4 Gy. The study demonstrated that melatonin administration led to a rise in the expression of bcl-2 and a decline in the ratio of bax to bcl-2 that was correlated with a decline in p53 mRNA and protein levels. This suggests that melatonin can mitigate the radiation-triggered apoptotic response.

Moreover, the current study indicates that even lower doses of radiation, such as 2 Gy- commonly used in clinical settings, can also influence the expression of apoptotic-related genes such as bcl-2 and bax. The results emphasize the potential of melatonin as a radioprotective agent, particularly for preserving the hematopoietic system during radiotherapy. This could have significant implications for improving patient outcome by reducing the adverse effect of radiation on blood cell production. Overall, these findings support further exploration of melatonin as a therapeutic adjunct in radiation treatment protocols.

Ethics approval and consent to participate

All the experimental procedures were carried out according to the principles and guidelines of the Ethics Committee, Deputy of Research and Technology, Kashan University of Medical Sciences. (IR.KAUMS.NUHEPM.REC.929).

Conflicts of interest

There are no conflicts of interest.

Acknowledgments

This study was supported by the Deputy of Research and Technology, Kashan University of Medical Sciences, Kashan, Iran.

Funding Statement

This manuscript is supported by Kashan University of Medical Sciences, Grant/Award Number: 92025.

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