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. 2023 Nov 29;103(2):103331. doi: 10.1016/j.psj.2023.103331

The signal pathway of melatonin mediates the monochromatic light-induced T-lymphocyte apoptosis in chicken thymus

Juanjuan Xiong *,†, Zixu Wang †, Yulan Dong †, Jing Cao †, Yaoxing Chen †,1
PMCID: PMC10764265  PMID: 38100948

Abstract

Our previous study revealed that under monochromatic red light (RL), the melatonin nuclear receptor reduces the proliferation activity of broiler thymic lymphocytes through the P65 signaling pathway. The main objective of this study was to investigate the signal mechanism by which RL decreases thymic lymphocyte proliferation. Initially, broilers were purchased and randomly assigned to be fed under white light (WL), red light (RL), green light (GL), and blue light (BL). Pinealectomy was performed 3 d later, and the broilers were euthanized after 14 d. The results showed that the expression of the antiapoptotic proteins Bcl-2/Bcl-xl decreased under RL, while the expression of the pro-apoptotic factor Bax/caspase-3 and the pro-inflammatory factors INF-γ/TNF-α/IL-6 increased. After pinealectomy, the expression of Bax/TNF-α/IL-6 increased in conjunction with the decrease in Bcl-2 expression. In vitro experiments demonstrated that exogenous melatonin decreased the expression of Bax/TNF-α/IL-6 in thymic lymphocytes of chicks reared under RL. This melatonin-induced effect was enhanced by SR1078 (RORα/RORγ agonist) but attenuated by SR3335 (RORα antagonist) and BAY (P65 antagonist). These findings revealed that the melatonin nuclear receptor RORα/RORγ promotes the expression of the pro-apoptotic factor Bax/caspase-3 and the pro-inflammatory factors INF-γ/TNF-α/IL-6, while inhibiting the expression of the antiapoptotic factor Bcl-2/Bcl-xl. Our research suggested the signaling pathway of monochromatic red light impacts the apoptosis of thymus lymphocytes in broiler.

Key Words: immune system, RORα/RORγ, mechanism, red light, chick

INTRODUCTION

Light in the environment can be categorized into 3 aspects: light regime, intensity, and light wavelength (Blatchford et al., 2009; Li et al., 2015a). Numerous studies have shown that light can impact the physiological functions of animals. Due to their complex visual system, broiler chickens are particularly susceptible to the effects of light on their growth and development (Rozenboim et al., 2013), especially in relation to their immune system (Kliger et al., 2000; Blatchford et al., 2009). Yang et al. (2016) stated that the immune function of poultry can be influenced by the light environment they are exposed to during their growth. Our previous research demonstrated that green light promotes T-lymphocyte proliferation in the thymus (Chen et al., 2016), as well as B-lymphocyte proliferation in the bursa of Fabricius (Li et al., 2015b).

It is well-known that immune cell apoptosis plays a crucial role in the development of immune function (Kalkavan and Green, 2018). Apoptotic processes are essential for maintaining the homeostasis of the immune system, particularly in thymus development (Hojo et al., 2019). Inappropriate apoptosis can impair immune function and negatively impact animal welfare. Various factors, including medications (Wang et al., 2017), heat (Li et al., 2014), stress (Arimoto-Matsuzaki et al., 2016), and light, can influence cell apoptosis. Studies have shown that blue light emitting diodes (LEDs) can affect apoptosis in bone marrow-derived mesenchymal stem cells (Yuan et al., 2017). Additionally, blue light can modulate cell metabolism and decrease cell viability in keloid-derived fibroblasts (Magni et al., 2020), as well as induce apoptosis in 3 epithelial cell lines (HaCaT, A431, and A549) (Laubach et al., 2019). Similarly, exposure to visible light at night has been found to increase DNA damage and cell apoptosis in mouse ovaries (Li et al., 2019). However, there is limited information available on how monochromatic light regulates T-lymphocyte apoptosis in the thymus of chicks.

Melatonin, an endocrine hormone, plays a role in mediating the effects of light on lymphocytes in chicks (Zhang et al., 2014; Guo et al., 2015). Moreover, melatonin possesses antioxidant properties that can alter the expression of apoptotic factors, thereby influencing cell and tissue development (Fernández et al., 2015). For instance, melatonin has been shown to significantly increase the levels of Bcl-2 family proteins, thereby inhibiting apoptotic events in vitrified bovine oocytes (Zhao et al., 2016). Tiong et al. (2019) found that melatonin reduces high glucose-induced apoptosis in Schwann cells through alterations in the Bcl-2, NF-κB, and Wnt signaling pathways. Furthermore, exogenous melatonin has been found to alleviate apoptosis induced by SiO2 in RAW264.7 cells (Zhang et al., 2019). Similarly, melatonin has been shown to decrease apoptosis induced by glucocorticoid receptor activation and inhibition of antioxidant status in peripheral blood mononuclear cells (Singh and Haldar, 2016). Despite numerous studies on the effects of melatonin on apoptosis, it remains unclear how melatonin mediates monochromatic light-induced lymphocyte apoptosis in the thymus of chicks.

Apoptosis primarily occurs through 2 pathways: the extrinsic and intrinsic pathways, which rely on apoptosis signals (Kalkavan and Green, 2018). The extrinsic pathway is activated by death receptors on the cell membrane, such as the TNF receptor superfamily, including TNFR1, TRAIL, and Fas. These receptors can be activated by ligand binding (Dickens et al., 2012). Research has shown that TNF-α binding to receptors can induce inflammation injury in venous endothelial cells, and this process can be effectively suppressed by melatonin (Lu et al., 2019). On the other hand, the intrinsic pathway is controlled by the Bcl-2 protein family and is achieved through mitochondrial outer membrane permeabilization (Kalkavan and Green, 2018). Both of these pathways lead to the activation of caspases (Li et al., 1997), and the subunits of caspase play important roles in apoptosis through different signaling pathways (Fan et al., 2005). The main purpose of this study is to figure it out whether melatonin through these apoptosis pathways participate in the effect of monochromatic light on lymphocytic apoptosis in chicks.

MATERIALS AND METHODS

Animal Treatments

A total of 120 commercial posthatching day (P) 0 Arbor Acre male broilers were purchased from Beijing Huadu Breeding Company (Beijing, China), newly hatched chickens were randomly divided into 4 groups, including WL (400–700 nm), RL (660 nm), GL (560 nm), and BL (480 nm). Three days later, each light color group was divided into 3 groups (pinealectomy group, sham operation group, and intact group). The pinealectomies were performed as follows: the anesthetized birds were placed on a stereotaxic instrument (SR-60, Narishige, Tokyo, Japan), a small mid-sagittal incision was made in the skin above the cranium and a small portion of the skull was then removed. The pineal was removed with forceps, and the wound was then closed with surgical sutures and treated with a topical antibiotic ointment. Sham surgeries were performed exactly the same way as the pinealectomies, but the pineal was left intact.

The light cycle consisted of 23 h light followed by a 1 h darkness period, which was controlled using a mechanical timer. the light density was 15 ± 0.3 lx at the level of the bird's head. Room temperature was maintained at 32 ± 2°C in the first wk and then reduced by 1°C every 2 d until it reached 30°C in the second wk. Feed and water were provided ad libitum during the entire experimental period. The diet was formulated to meet or exceed the nutrient recommendations of the National Research Council for poultry (1994).

Then, 14-day-old broilers were euthanized by cervical dislocation, removed the left thymus aseptically, and save it in 2 parts. One part of the thymus preserved in a low temperature (−80°C) assessed for mRNA and protein levels by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting.

The animal trial was raised in accordance with the Animal Welfare Committee of China Agricultural University (Approval No. CAU20171114-2).

RT-qPCR

The total RNA was purified from the thymus of chicks using a reverse transcription kit (Thermo Fisher Scientific, Boston, MA). For each sample, 2 µg of the total RNA was taken for reverse transcription according to the manufacturer's instructions. The transcribed cDNA samples were stored in a −20°C in a refrigerator.

PCR amplification test in briefly. PCR amplification system contains 2 μL cDNA sample, 0.4 μL target primer, 10 μL RT-qPCR SYBR Green Mix (Vazyme, Nanjing, China), and 7.2 μL ddH2O; mixed system and centrifugation in 4°C. Put the mixture into the instrument for amplification (Roche, LightCycler 480 System, Switzerland). The program was set as 95°C for 10 min, 95°C for 30 s by 40 cycles, annealing at 57°C for 30 s, and extension at 72°C for 30 s. cGAPDH was used as internal reference for standardization. The PCR primers are listed in Table 1.

Table 1.

Sequences of apoptosis primers used for RT-qPCR.

Genes Primer sequences (5ʹ–3ʹ) Accession No. Product size (bp)
Bcl-2 F: GAT GAC CGA GTA CCT GAA CC
R: CAG GAG AAA TCG AAC AAA GGC		 NM_205339.2 114
Bax F: TCC TCA TCG CCA TGC TCA T
R: CCT TGG TCT GGA AGC AGA AGA		 XM_015274606.1 107
Bcl-xl F: GAT GCG CGA AAG GTC GGA T
R: CCC GGT TAC TGC TGG ACA TT		 NM_001025304.1 141
Caspase-3 F: TGG CCC TCT TGA ACT GAA AG
R: TCC ACT GTC TGC TTC AAT ACC		 NM_204725.1 139
GAPDH F: ATC ACA GCC ACA CAG AAG ACG
R: TGA CTT TCC CCA CAG CCT TA		 NM_204305 124
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Abbreviations: F, forward primer; R, reverse primer.

Western Blot

The tissue or cells were lysed with RIPA buffer, and performed according to the protein assay kit (CW0014, CWBIO, Beijing, China). The total protein lysate was separated by SDS-polyacrylaminde gel electrophoresis (SDS-PAGE) and transferred to a polyvin vlidene difluoride membrane (Millipore, Billerica, MA). The membrane was blocked with 5% skim milk and then incubated with primary antibody overnight at 4°C. Primary antibodies against Bcl-2 (Orb10173, 1:1000, Biorbyt, Cambridge, UK), Bcl-xl (#2764, 1:2000, CST, MA), Bax (Orb334986, 1:1000, Biorbyt, Cambridge, UK), caspase-3 (#9661, 1:2000, CST, MA) were chosen in the experiment. β-actin was used as a control (CW0096, 1:4000, CWBIO, Beijing, China). The bands were incubated with HRP-conjugated antirabbit or antimouse IgG secondary antibodies (goat antimouse IgG-HRP diluted 1: 6000; goat antirabbit IgG-HRP diluted 1: 6000; CoWin Biotech Co., Inc, Jiangsu, China) at room temperature for 2 h. Target proteins were visualized using an automatic chemiluminescence image analysis system (5200, Tanon, Shanghai, China) and quantified by Image Analysis software (Gel-Pro Analyzer 4.5; Media Cybernetics, Rockville, MD).

ELISA

The commercial chicken ELISA kit (IL-6, CSB-E08549Ch, CUSABIO, Wuhan, China; INF-γ, SEA049Ga, Cloud-Clone, Wuhan, China; SEA133Ga, TNF-α, Cloud-Clone, Wuhan, China) were used to detect the cytokines concentration in tissues or cell secretions. All experiments followed the ELISA kit manufacturer's protocol. The standard or sample (50 μL) was added to each well and incubated with 50 μL of detection reagent A for 1 h at 37°C. Then, the medium in each well was discarded, and a biotin antibody (100 μL) was added to each well, followed by incubation for 30 min at 37°C. Washed the wells 5 times. And 90 μL of substrate solution was added for 20 min at 37°C, followed by the addition of 50 μL of stop solution. The OD values were then read at 450 nm. The OD value of the standard was calculated as a standard curve by the log function. The sample's concentration was calculated according to the standard curve.

Lymphocyte Culture

The lymphocytes of the thymus from 40 chicks were isolated in a sterile environment. Cell suspensions were distributed in 6-well (6 × 106 cells/well). The control group was cell suspension only, while the experimental groups were added melatonin (10−9 M, Sigma-Aldrich, St. Louis, MO) under the stimulation of ConA (20 µg/mL, Sigma-Aldrich), the experiment groups were added chemical blocker for 30 min prior to the addition of ConA and melatonin. Cells were cultured at 37°C for 48 h, then cell proteins and secretions were collected for the next step.

The chemical blocker contains: 5 μM SR3335 (a selective RORα inverse agonist; MCE, NJ), 10 μM SR1078 (a nonselective agonist of the nuclear receptor RORα and RORγ; MCE, NJ), 1 μM BAY (an antagonist of P65; MCE, NJ).

Statistical Analyses

All experiments were performed at least 3 times. In vivo data were analyzed using 2-way ANOVA, and in vitro data were analyzed using 1-way ANOVA, followed by a multiple comparison posthoc least significant difference test, and error bars represent the Standard Error of Mean (S.E.M), unless otherwise specified. All of the data were analyzed by SPSS version 17.0 software (SPSS, Inc., Chicago, IL) followed by Duncan's multiple rang tests. For all tests, the P-value showed ≤0.05 was considered significant statistically.

RESULTS

Monochromatic Light Affected the mRNA Expression of Apoptosis Factor in Thymus

As shown in Figure 1, the results demonstrated that GL significantly increased the mRNA levels of Bcl-2 by 17.48 to 22.70% (P = 0.000–0.001) compared to the other 3 light conditions in the intact group. Similar results were observed in the sham-operated group (5.10–25.54%, P = 0.001–0.766) (Figure 1A). Furthermore, GL exhibited higher levels of the antiapoptotic factor Bcl-xl mRNA by 60.35% (P = 0.001), 83.92% (P ≤ 0.001), and 46.23% (P = 0.003) compared to WL, RL, and BL, respectively, in the intact group (Figure 1B). Similar findings were observed in the sham-operated group. However, pinealectomy reduced the levels of Bcl-2 and Bcl-xl by 1.01 to 18.83% (P = 0.002–0.816) and 15.60 to 53.89% (P = 0.000–0.417), respectively, in the thymus under the 4 light treatments compared to the corresponding sham operation group. No significant differences were observed among the different lights after pinealectomy (P > 0.05).

Figure 1.

Figure 1

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Effect of monochromatic light on the mRNA expression levels of apoptosis factor in the thymus of chicks. (A) Bcl-2 mRNA expression; (B) Bcl-xl mRNA expression; (C) Bax mRNA expression; (D) caspase-3 mRNA expression. Data are presented as the means ± SEM. Different letters on the column indicate significant differences (P < 0.05) between treatment groups. *represents a significant difference in the pinealectomy group (P < 0.05) compared with the corresponding intact and sham operations. Abbreviations: BL, blue light; GL, green light; RL, red light; WL, white light.

The results for the 2 pro-apoptotic factors, Bax and caspase-3 mRNA levels, showed contrasting outcomes (Figures 1C and 1D). The GL group exhibited the lowest levels, while RL significantly increased Bax mRNA levels by 4.80 to 35.09% (P = 0.006–0.645) compared to the other lights in the intact or sham operation groups. Similarly, RL increased caspase-3 levels by 7.83 to 29.61% (P = 0.025–0.445) compared to the other lights in the intact or sham operation groups. Pinealectomy increased the levels of Bax and caspase-3 by 11.11 to 38.31% (P = 0.004–0.602) and 29.98 to 61.72% (P = 0.000–0.202), respectively, in the thymus under the 4 light treatments compared to the corresponding sham operation group. No significant differences were observed among the different lights after pinealectomy (P > 0.05).

Monochromatic Light Affected the Protein Expression of Apoptosis Factor in Thymus

The results indicate that GL increased the protein levels of Bcl-2 by 25.60% (P = 0.000) compared to WL, RL, and BL, which increased the levels by 41.04% (P = 0.000), and 3.28% (P = 0.544), respectively, in the intact group (Figure 2A). GL also increased Bcl-xl levels by 26.37 to 54.31% (P = 0.009–0.384) compared to the other lights (Figure 2B). The protein levels of both Bcl-2 and Bcl-xl in the sham operation groups were similar to those in the intact group. However, pinealectomy decreased the Bcl-2 protein levels by 3.37 to 24.20% (P = 0.000–0.762) compared to the corresponding monochromatic light in the sham operation, and Bcl-xl levels were decreased by 8.71 to 31.88% (P = 0.006–0.478), with no significant difference among the light treatments (P > 0.05, Figures 2A and 2B).

Figure 2.

Figure 2

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Effect of monochromatic light on the protein levels of Bcl-2, Bcl-xl, Bax, and caspase-3 in the thymus of chicks. (A) Bcl-2 protein levels; (B) Bcl-xl protein levels; (C) Bax protein levels; (D) caspase-3 protein levels. Data are presented as the means ± SEM. Different letters on the column indicate significant differences (P < 0.05) between treatment groups. *represents a significant difference in the pinealectomy group (P < 0.05) compared with the corresponding intact and sham operations. Abbreviations: BL, blue light; GL, green light; RL, red light; WL, white light.

Green light reduced the protein levels of Bax by 23.17 to 31.31% (P = 0.048–0.228) compared to other lights in the thymus, while RL increased the protein levels of Bax (Figure 2C). Similarly, RL increased caspase-3 protein levels by 7.26 to 20.32% (P = 0.041–0.394) in the intact or sham operation group (Figure 2D). After pinealectomy, the protein levels of Bax increased by 1.99 to 91.58% (P = 0.000–0.904) compared to the corresponding sham operation, and caspase-3 levels decreased by 16.27 to 33.23% (P = 0.000–0.702), with no significant differences among the light treatments (P > 0.05).

Monochromatic Light Affected the Expression of Inflammatory Factor in Thymus

The concentrations of INF-γ in the RL group were higher by 19.24% (P = 0.045), 25.28% (P = 0.013), and 33.80% (P = 0.002) compared to the WL, GL, and BL groups, respectively. Additionally, RL significantly increased the expression of INF-γ by 21.44 to 36.38% (P = 0.001–0.030) in the sham operation group. However, pinealectomy increased INF-γ concentrations of by 6.59 to 36.16% (P = 0.000–0.746). No significant differences were observed in the pinealectomy group (P > 0.05) (Figure 3A).

Figure 3.

Figure 3

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Effect of monochromatic light on the concentrations of inflammatory factor in the thymus of chicks. (A) INF-γ concentrations; (B) IL-6 concentrations; (C) TNF-α concentrations. Data are presented as the means ± SEM. Different letters on the column indicate significant differences (P < 0.05) between treatment groups. * represents a significant difference in the pinealectomy group (P < 0.05) compared with the corresponding intact and sham operations. Abbreviations: BL, blue light; GL, green light; RL, red light; WL, white light.

The concentrations of IL-6 and TNF-α in the RL group showed an increase of 16.76 to 43.78% (P = 0.005–0.057) and 17.34 to 25.87% (P = 0.004–0.015), respectively, compared to other light treatments in the intact or sham operation groups. However, GL significantly reduced the expression of both IL-6 and TNF-α. Meanwhile, the concentrations of IL-6 and TNF-α increased by 58.47 to 89.91% (P = 0.000–0.466) and 12.73 to 33.58% (P = 0.003–0.962), respectively, after pinealectomy compared to the corresponding sham operation groups. No significant differences were detected among the various light treatments in the pinealectomy group (P > 0.05) (Figures 3C and 3D).

Effect of RORα and RORγ on the Expression of Apoptosis Factor

In cultured thymic lymphocytes, SR1078 (10 μM), SR3335 (5 μM), or BAY (1 μM) were added 30 min before the addition of ConA (20 μg/mL) + melatonin (10−9 M) group. The ConA + melatonin group exhibited a significant increase of 38.72% (P = 0.006) in Bcl-2 protein levels in thymic lymphocytes compared to the control group, while Bax protein levels decreased by 32.91% (P = 0.004). Furthermore, compared to the melatonin + ConA group, the protein levels of Bcl-2 decreased by 41.73% (P ≤ 0.001) in the melatonin + ConA + SR1078 group (a nonselective agonist of the nuclear receptors RORα and RORγ), while Bax protein levels increased by 32.33% (P = 0.004). On the other hand, a selective RORα inverse agonist (SR3335, 5 μM) increased Bcl-2 protein levels by 26.98% (P = 0.006) and decreased Bax levels by 19.14% (P = 0.029) (Figures 4A and 4B). It is important to note that SR1078 or SR3335 alone had no effect on the expression of Bcl-2 and Bax (P > 0.05).

Figure 4.

Figure 4

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Effect of the RORα/RORγ/P65 on the Bcl-2/Bax and TNF-α/IL-6 expression. (A) Protein levels of Bcl-2; (B) protein levels of Bax; (C) TNF-α concentrations; (D) IL-6 concentrations. SR3335: RORα antagonist (5 μM); SR1078: RORα and RORγ agonist (10 μM); BAY: P65 antagonist (1 μM); ConA: 20 µg/mL, Melatonin: 10−9M. Data are presented as the means ± SEM. Different letters on the column indicate significant differences compared with the control group (P < 0.05).

The expression of TNF-α and IL-6 were also detected (Figures 4C and 4D). The addition of SR1078 or SR3335 alone to the cultured thymic lymphocytes had no effects on the concentrations of TNF-α or IL-6 (P > 0.05). The addition of exogenous melatonin reduced the TNF-α concentrations by 30.56% (P ≤ 0.001), and IL-6 were decreased by 20.17% (P ≤ 0.001). Meanwhile, those effects were enhanced by SR3335 (TNF-α, 23.40%, P = 0.041; IL-6, 14.42%, P = 0.035). In contrast, administration of SR1078 increased the concentrations of TNF-α by 44.30% (P = 0.003), and IL-6 by 18.92% (P = 0.007) in T-lymphocytes.

Besides, P65 antagonist BAY (1 μM) increased the protein levels of Bcl-2 by 24.76% (P = 0.009) compared with melatonin + ConA group (Figure 4A), while BAY decreased Bax, TNF-α and IL-6 expression about 23.20% (P = 0.020), 30.92% (P = 0.011) and 15.30% (P = 0.025) (Figures 4B and 4D).

DISCUSSION

We conducted in vitro and in vivo experiments to investigate the impact of monochromatic light on thymus lymphocyte apoptosis and elucidated the signaling mechanism by which melatonin mediates this process. First, our findings demonstrated that monochromatic green light reduced lymphocyte apoptosis, while red light promoted lymphocyte apoptosis in the thymus of chicks. These results align with our previous data indicating that red light reduces lymphocyte activity in chicks. Similar results have been found in studies (Rozenboim et al., 2004; Zhang et al., 2016). Second, we further focus on the mechanism of red light on the immune function of chickens. We have observed an increase in the expression of melatonin nuclear receptors under monochromatic red light (Xiong et al., 2019), a hypothesis that was confirmed by our in vitro experiments in this study. To further explore the signaling mechanism underlying the effects of monochromatic light on lymphocyte apoptosis, we observed that red light significantly decreased the mRNA and protein levels of the antiapoptosis factor Bcl-2/Bcl-xl, while increasing the levels of the pro-apoptosis factor Bax/caspase-3. This observation indicates that monochromatic red light induces thymus lymphocyte apoptosis through the Bcl-2 family and caspase pathway.

Although there are limited studies focusing on the effect of light on apoptotic proteins, some research has shown that Bcl-2 expression is higher in the dark phase than in the light phase, while Bax expression increases during light exposure in the rat brain (Montes-Rodríguez et al., 2009). Additionally, blue light has been shown to induce damage to retinal pigment epithelial cells through the regulation of caspase-3 and Bcl-2 (Sparrow and Cai, 2001). Despite these findings, we have identified a link between red light and lymphocyte apoptosis factors. Furthermore, we observed that monochromatic red light also promotes the expression of inflammatory factors INF-γ/TNF-α/IL-6 in the chicken thymus. Similarly, red light increased the gene expression of IL-6 in 2-wk-old chickens compared to those incubated under blue light (Kankova et al., 2022).

Melatonin treatment has been found to increase Bcl-2 protein expression and create conditions that inhibit apoptosis in mouse liver cells (Michurina et al., 2021). This indicates that the melatonin nuclear receptor RORα plays a crucial role in mediating the regulatory factors of monochromatic red light and inflammatory factors. Based on the aforementioned experimental results, we examined the relationship between melatonin nuclear receptors and apoptosis factors. In vitro experiments, melatonin increased the protein expression of Bcl-2, while decreasing the Bax protein level and concentrations of the proinflammatory factors INF-γ/TNF-α/IL-6 in thymus lymphocytes of broilers reared under red light. However, this effect was attenuated by the addition of the nuclear receptor RORα and RORγ agonist. In summary, the melatonin nuclear receptors RORα/RORγ mediate the effects of monochromatic red light on thymic apoptosis through the Bcl-2 family and INF-γ/TNF-α/IL-6 pathway. In mouse Leydig cells, melatonin inhibits apoptosis via the RORα/p53 pathway (Li et al., 2020).

Our previous study demonstrated that the nuclear receptor RORα promotes NF-κB nuclear translocation by upregulating IκB phosphorylation, which reduces lymphocyte proliferation (Xiong et al., 2019). Building upon this research, further investigations showed that under monochromatic red light, after blocking the P65 pathway, the expression of both apoptotic and proinflammatory factors decreased, while the expression of the antiapoptotic factor Bcl-2 increased. This suggests that the melatonin nuclear receptor regulates cell apoptosis through apoptotic and proinflammatory factors via the P65 signaling pathway.

CONCLUSIONS

The melatonin nuclear receptor RORα/RORγ mediates the effect of monochromatic red light on thymocyte apoptosis through the P65 signaling pathway, by promoting the expression of proapoptotic factor Bax/caspase-3, proinflammatory factor INF-γ/TNF-α/IL-6, and inhibiting the expression of antiapoptotic factor Bcl-2/ Bcl-xl. Taken together, our data provide new mechanistic insights into possible signaling pathways about monochromatic red light to promote lymphocytes apoptosis in chicks.

ACKNOWLEDGMENTS

This work was supported by the Beijing Natural Science Foundation (6222019) and the Chinese National Natural Science Foundation (32172801, 32372954).

DISCLOSURES

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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