Cyanidin-3-O-Glucoside alleviates atopic dermatitis and depression-like behavior by modulating immune cells in DNFB-induced mice

Abstract

Atopic dermatitis (AD) significantly impacts individuals’ daily lives. Patients with AD typically exhibit depressive-like behavior. Cyanidin-3-O-glucoside (C3G) is extensively utilized to treat inflammatory conditions, but its beneficial effect on AD has not been reported. The present study aims to evaluate the efficacy and effects of C3G on AD and depressive behavior. Mice sensitized with 2,4-dinitrofluorobenzene (DNFB) were utilized to establish the Atopic dermatitis (AD) model. The drug groups were treated with daily oral administration of 150mg/kg and 100mg/kg C3G for three weeks. We determined the number of scratching bouts and skin severity scores. Effects were analyzed by toluidine blue-staining (TB) and hematoxylin and eosin staining (HE). Changes in inflammatory factors were detected by ELISA assay. Immunohistochemistry and Flow cytometry were employed to investigate changes in immune cells. Behavioral experiments were used assess the effects on depressive-like behaviors. Compared to the control group, C3G improved AD and depressive behaviors in mice by regulating the levels of immune cells, thereby affecting the production of inflammatory factors and inflammatory cell infiltration. C3G improved AD and depressive symptoms in mice by modulating immune cell populations, particularly CD8+ T cells, and influencing the generation of inflammatory mediators.

Keywords:
Cyanidin-3-O-glucoside; Atopic dermatitis; CD8⁺ T cell; Depressive behaviors; Flow cytometry.

INTRODUCTION

Atopic Dermatitis (AD) is a widespread inflammatory cutaneous condition that is associated with erythema, mossy changes, eczematous lesions, and severe itching. It is also known as eczema(Chen et al., 2023a), AD increases the risk of depressive behavioral disorders and concentration issues, and in severe cases, can trigger suicidal ideation.(Yaghmaie, Koudelka, Simpson, 2013). The pathogenesis of AD remains to be fully unraveled, with prevailing theories underscoring a complex interplay between skin barrier dysfunctions. Immunological abnormalities are believed to have a crucial function in the development of AD (Bonamonte et al., 2019; Santamaria-Babi, 2022). The imbalance between Th1 and Th2 cells, leading to inflammation and abnormal cytokine secretion, is an important cause of the growth of AD. Both Th1 and Th2 cells play a main effect part in the growth of AD in the chronic phase, while Th2 cells are the main cell involved in the acute phase (Gittler et al., 2012; Villani et al., 2021). Eosinophils and mast cells play a key role in the chronic phase of AD by coordinating with inflammatory microenvironments, and triggering pruritus and mossy skin lesions in AD patients (Cheung et al., 2010; de la O-Escamilla, Sidbury, 2020). Aberrant IL-4 expression triggered by TH2 cell dysregulation leads to increased infiltration of eosinophils and mast cells, worsening the progression of AD (Carretero et al., 2016; Yamanishi et al., 2020).

Therefore, it is feasible to alleviate AD and depressive behaviors in patients by modulating immune cells and the cytokines that are generated by them.

It has been demonstrated that CD4⁺ T cells play a key role in generating a mixed pattern of Th1 and Th2 cytokines which influences on the development of eosinophilic inflammation in the skin (Furue et al., 2006; Leung et al., 2004). Studies have indicated a significant link between the level of CD8⁺ T cells and the extent of illness in AD, one of the allergic reactions to AD, caused by immunoglobulin E antibodies directed against antigens (Warner et al., 2006; Wollenberg et al., 2021).Circulating CLA-positive CD8⁺ T cells could lead to stimulate B cells to generate IgE (Akdis et al., 1999), thus contributing to the development of AD.

While CD4⁺ T cells dominate in AD skin lesions, the epidermis is primarily infiltrated by CD8⁺ T cells. Moreover, CD8⁺ T cells can be activated and mature into effector cells without CD4⁺ T cell assistance (Chen et al., 2023b; Hennino et al., 2007). CD8⁺ T cells can exacerbate the development of AD through cytotoxic effects, pro-inflammatory factor secretion, and barrier disruption (De Bruyn Carlier et al., 2021; Hennino et al., 2007). Therefore, we believe that AD improvement by CD8⁺ T cells is of research interest. Additionally, studies have demonstrated that a remarkable increase in CD8⁺ T cells and a decrease in CD4⁺ T cells are correlated with higher levels of depression, with CD4⁺ T cells having a more pronounced impact on depressive symptoms (Rattazzi et al., 2013). Also, CD8⁺ T cells can indirectly cause dysregulation of IL-4, which leads to dysregulation of 5-hydroxytryptamine (5-HT) levels and promotes further depressive behaviors in patients with AD (Stanciu et al., 1997).

Cyanidin-3-O-glucoside (C3G), as the main anthocyanins component, has been confirmed to have significant antioxidant and anti-inflammatory effects, which can decrease neuronal damage by reducing the generation of pro-inflammatory cytokines, and molecules of oxidative stress. It also provides neuroprotection and improvement of depressive behaviors (Fang et al., 2020; Zhang et al., 2019), Literature suggests that C3G gel can improve skin hydration and decrease epidermal water loss, which is critical for reducing AD lesion severity.(Peng et al., 2020). Protection of the epidermal barrier of the skin is important for reducing the extent of skin lesions in patients (Li et al., 2023). At the same time, C3G can improve AD symptoms and depressive behavior by inhibiting IL-4 production in activated EL-4 T cells, while not affecting Th1 cytokine production (Pyo et al., 2014). Cyanidin-3-O-glucoside is an important active ingredient in black bean skin and the literature has shown that C3G plays a significant role in improving AD in A63 black beans. Can C3G be used as an effective drug to improve AD symptoms and depressive-like behavior triggered by AD? This paper aimed to investigate the ameliorative effect of C3G on AD by studying the effects of C3G on the number and function of immune cells and the modulation of inflammatory factor levels in BALB/C mice. In this paper, we intend to monitor the number of immune cells like CD4 and CD8 through flow cytometry, detect the level of inflammatory factors by ELISA, observe and count the locomotor status of mice in different experimental groups by means of behavioral experiments. The purpose of the above operation is to investigate the possible mechanism of C3G on the symptoms of AD patients and on the improvement of depression-like behavior by using the immune cells as an entry point.

MATERIAL AND METHODS

Substances and reagents

Cyanidin-3-O-glucoside (90% purity) was purchased from Xinlu Biotechnology Co. Ltd (Xi’an). Olive oil was provided by Aladdin Chemical Co. Ltd (Shanghai, China) and Dexamethasone (DEX, 98% purity) was provided by Hubei Minkang Pharmaceutical Co. Ltd (Yichang, China). 2,4-dinitrofluorobenzene (DNFB) (99% purity) was provided by Adamas Reagent Co. Ltd (Shanghai, China). RBC Lysis Buffer10X (Biolegend, USA) Cell Staining Buffer (Biolegend, USA), Flow-through antibody (Biolegend, USA) phosphate-buffered saline PBS (Biolegend, USA) 7-AAD Viability Staining Solution (Biolegend, USA).

Animals

The female BALB/c mouse, 6 weeks old and weighing between 18-22 g, was acquired from Chunyu Hong Animal Company. (Wuhan, China). We chose female mice for our study because the incidence and severity of disease have a pronounced female predilection in AD(Kanda, Hoashi, Saeki, 2019). Female mice may also show a similar tendency. The mice were raised under qualified animal laboratory environments at an ambient temperature of 22±2 °C. The dark/light environment changed continuously every half a day. These mice were provided with the right amount of food and water according to their weight. We divided the mice into four groups after a week of adaptive rearing, each group was randomly assigned 8-10 mice. The initial group was labelled as the normal group (Normal); the second group was labelled as the AD model group (Model); the third group was labelled as the positive model group (Dex group). The fourth group was the C3G high-dose group (150 mg/kg) and the fifth group was the C3G low-dose group (100 mg/ kg). DEX was chosen as the positive control drug in this experiment. The study was approved by the Academic Ethics Review Committee of Jianghan University under the ethical approval number JHDXLL2023-015 and the study complied with all regulations.

Model preparation and administration

AD lesions were induced in the mice’s dorsal skin by DNFB. One day before the DNFB modeling. We removed hair from the backs of mice (about 2 × 3 cm). A 6% sodium sulfide solution was applied to the surface to depilate 100 μL of 1.0% DNFB solution was used for the back skin on 1-3 d. In addition, 50 μL of 0.5% DNFB solution was used for the back skin on 6, 9, 12, 15, and 18d. DNFB-sensitized mice in the positive control group were given DEX 0.1 mg/kg by gastric perfusion for 6-20 d. DNFB-sensitized mice were fed with 150mg/kg (high) or 100 mg/kg (low) C3G in the drug group, respectively. The mice were given saline by gastric perfusion in the normal group and model group. (Figure 1)

FIGURE 1
A timeline for the development of the AD model.

Observation of Skin Lesions and Statistics on Organ Coefficients

There are three main indicators of clinical manifestations of AD: skin severity score, dorsal cutaneous thickness, and organ coefficient. Firstly, the skin severity score is founded on macroscopic measurements of the subsequent manifestations: erythema / bleeding, scaling / dryness, oedema / swelling, vesicles / abrasions, epidermal peeling / scratching, and scaling. The overall score is determined by summing the individual scores together: 0 (asymptomatic), 1 (mild), 2 (moderate), and 3 (severe), with a maximum total of 12 points. On the 21st day, mice were euthanized after blood was taken. We collected their dorsal skin, liver, lung, brain, heart, kidney, thymus, and spleen. Then we measured the thickness of the skin on the back and organ coefficients.

Measurement of the number of scratching bouts

Groups of mice were placed in cardboard boxes with 3*3 compartments. A video camera was located above the box to record the behavioral activities of the mice. After the animals were acclimatized in the box for more than 2 h. The mice were filmed for 15 min in a closed and unoccupied environment. The video was played back after filming to record the number of scratching behaviors exhibited by the mice before and after treatment, starting from lifting the forelimbs and hindlimbs and ending with putting the forelimbs and hindlimbs down or putting them into the mouth, with continuous itching regarded as one itch.

Experiments on animal behavior

Mice were placed in a soundproofed and temperature-controlled laboratory at approximately 22° for 2 h. Then all behavioral tests were performed at midday, avoiding sleep breaks.

Elevated Zero Maze

The experimental site consists of a circular platform measuring 45 cm (diameter) by 6 cm (width) by 40 cm (height) The circular maze is divided into four sectors of the same area, two of which have closed arms and the other two have open arms. After 5 min of adaptation, mouse movements were recorded using an overhead camera. The distance moved and the time invested in the closed and open arms and the central zone were measured and analyzed. It was used to detect the degree of depression in mice.

Hanging tail test

Each group of mice was hung in a glass cylinder and secured with a tape wound 1cm from the end of the mouse’s tail. They were considered to be in a static state when the mice were suspended on the cord and no longer moving back and forth in a continuous motion. During the experiment, each mouse was recorded for 6 minutes. The suspension time of mice was analyzed by SuperTst tail suspension assay test equipment and analysis system-XR-XX203 (Shanghai Xin rong Information Technology Co. Ltd. Shanghai, China).

Forced Swimming Test

Each mouse in the group was put in a glass cylinder with a stable water temperature of 20°. A camera device connected to a computer was used to record the movement of the mice in the water for six minutes, and the floating time of the mice in the water was analyzed by SuperFst forced swimming experimental testing equipment and analysis system XR -XQ202 (Shanghai Xinrun Information Technology Co. Ltd. Shanghai, China).

Histological analysis

We fixed the collected dorsal skin of mice with a 4% formaldehyde solution, followed by embedding in paraffin wax after 24 h. The sections were then stained with hematoxylin and eosin (H&E) and toluidine blue (TB). Select special fields of view under the microscope for analysis, and the epidermal thickness and mast cell count were measured using Image J software.

Immunohistochemical staining

Mice skin paraffin sections were put under 60° for melting wax, followed by xylene dewaxing and gradient ethanol dehydration. The tissue sections underwent antigen repair with antigen repair buffer (pH 6.0) for 30 minutes, followed by incubation with the primary CD8 antibody and corresponding secondary antibody, respectively. DAB was used to develop the color and hematoxylin was used for re-staining. The stained sections were examined using a microscope at 200x magnification. ImageJ software was used to analyze the proportion of protein immunostained areas to the total epidermal immunostained area by randomly selecting 5 different areas in 3 sections.

Enzyme-linked immunosorbent assay

On the day of dissection, the experimental mice were euthanized. and the blood from the mice was taken and refrigerated at 4 °C for 1 h, after which it was centrifuged for 10 minutes to obtain serum for subsequent analysis. Taking the lesioned dorsal skin of mice, subcutaneous fat was removed, washed with saline, and filter paper was used to swab dry, weigh, shear, and add 9 times the amount of saline of the weighed mass. The sample was mixed with 100 μL of PBS at a ratio of 10 mg to get the skin homogenate. serum levels of immunoglobulin E (IgE), Interleukin-4 (IL-4), Interleukin-10 (IL-10), Interleukin-15 (IL-15), and skin levels of Interleukin-1(IL-1) were measured by enzyme-linked immunosorbent assay (ELISA) kits (Bolf Bio-technology Co. Ltd. Wuhan, China).

Flow cytometry

After dissecting the mice, their spleens were removed and made into a single-cell suspension with PBS containing 1% BSA, centrifuged for five minutes, and removed the supernatant. Additionally, the ACK lysis buffer was added to the cell suspension to lysate the erythrocytes, after which it was left at room temperature for 5 minutes. After that, 10 mL of PBS with 1% BSA was added to stop the lysis process. Then we centrifuged it for five minutes and removed the supernatant. The cells were resuspended by adding 15 mL of PBS containing 1% BSA. Repeat the above centrifugation, and discard the supernatant. In addition, we added fluorescently labelled antibodies (anti-CD4, anti-CD8, etc.), kept the mixture away from light, and incubated for half 1 h. PBS solution containing BSA was added to the cell suspension, repeated the above centrifugation, and discarded the supernatant. PBS solution was added again to re-suspend the cells. Finally, the number of immune cells such as CD4⁺ T cells and CD8⁺ T cells was measured by flow cytometry and analyzed by Prism and SPSS software.

Pearson correlation analysis

The Pearson correlation analysis was employed to measure the association between immune cells, inflammatory cytokines, and depressive-like behavior.

Statistical analyses

Each experiment was conducted a minimum of three times so that we could ensure the accuracy and authenticity of experimental data. All statistical analyses were conducted utilizing the GraphPad Prism software. (version 9.5; USA) The experimental results were presented by the mean ± standard deviation (SD). We accessed one-way ANOVA and Dunnett test to measure the significance of each result value. In addition, p < 0.05 was used as the criterion for statistical significance of differences.

RESULTS

Effects of C3G on clinical characteristics of AD mice

On the 20th day of our study, the experimental mice in each group were weighed, and the weight of the mice in each group did not show any significant variance. On the 21st day, the mice were euthanized and dissected for analysis. As shown in Figure 2, the difference in weights of the liver, kidney, heart, lung, and thymus was not statistically significant within each group of mice. This indicates that C3G is safe and well tolerated (Figure 2 A-F). On the 9th and 18th days of the experiment, the degree of skin lesions on the back of the mice was observed and scored. By comparison with the blank group, the skin severity scores of the model group were significantly higher, suggesting that the modeling had been successful (Figure 2 H). On 18d, the skin severity scores of the C3G high-dose group (P < 0.001) and the DEX group (P < 0.05) were significantly different from the model group, indicating that both drugs had good therapeutic effects and that the therapeutic effects of the C3G high-dose group were even more favorable.

FIGURE 2
Effects of Cyanidin 3-O glucoside (C3G) on organ coefficient, weight of body, skin thickness, and skin severity score. (A) Weight of the body on the 20th day (B) Liver weight (C) Lung weight (D) Heart weight (E) Kidney weight (F) Spleen weight (G) Thymus weight (H) Skin severity scores on the 9th and 18th days of the experiment. (I) The number of scratching bouts on the 8th and 17th days. Data are presented as mean ± SD (n = 5), *P < 0.05, **P < 0.01, *** P < 0.001, ****P < 0.0001.

In scratching experiment, on the 8th day of the experiment, both model and drug groups had very high significantly increased scratching compared to control group,, indicating that modelling was successful and effective(Figure 2 I) (P < 0.0001). Compared to the model group, C3G group significantly reduced the number of scratching bouts of the mice (P < 0.001). On the 17th day of our study, both high and low-dose C3G groups showed the same trends, though not statistically significant (Figure 2 I). In addition, except for the model group, other groups did not differ from the blank group. These findings demonstrate that oral administration of DEX and C3G significantly improved the AD symptoms in mice, alleviating itching and pain in the dorsal skin lesions. However, the symptoms still did not disappear completely, and the mice still had the phenomenon of scratching the back skin.

Effects of C3G on depressive-like behaviors and anxiety of AD mice

On 20d, the mice underwent a Forced Swim Test (FST), Elevated Zero Maze (EZM), and Tail Suspension Test (TST). The number of itching was recorded on the 10th and 18th days to measure the healing benefits of C3G on depressive behaviors and atopic dermatitis in AD mice. In the FST test, our model group floated considerably longer than the C3G high-dose group (Figure 3 A) (P < 0.01). In the EZM test, by comparison with the blank group, the ratio of the distance travelled in the open area to the total distance travelled was considerably increased for mice in the model group (Figure 3 C) (P < 0.01). Additionally, by comparison with drug groups, the difference was smaller but still lower than the drug group. By comparison with the blank group and the C3G drug groups, there was a significant difference in the ratio of time spent moving in the open area to the total time spent in the model group (Figure 3 D) (P < 0.05). By comparison with our AD model group, C3G drug groups served to alleviate the depression-like behavior of mice. The C3G high and low dose groups and the blank group maintain the same trend of change.

FIGURE 3
Effects of Cyanidin 3-O glucoside (C3G) on depression-like behavior of mice(A) Floating time of FST. (B) Resting time of mice in TST (C) The ratio of the distance travelled to the total distance travelled by mice that were in the open area in the EZM test. (D) The ratio of the time the mouse was in the open area to the total time in the EZM test. Data are presented as mean ± SD (n = 5), *P < 0.05, ****P < 0.01, P < 0.001, P < 0.0001.

Effects of C3G on skin histopathology of AD mice

The mitigating effect of C3G on AD was further explored through histopathological observation. The Figure 4 A shows the extent of skin lesions on the back of mice on 11 and 19d. On 11d, all groups of DNFB-induced model mice except the normal group showed erythema and extensive scarring on the back skin, with epidermal hyperplasia, hyperkeratosis, and inflammatory cell infiltration. On 19d, the drug groups showed better lesion improvement than the model group. In addition, H&E staining of the dorsal lesional skin was conducted to examine the morphological variations in AD mice. The experimental results indicated that both the thickness of the epidermis and dermis in the model group had a notable increase and C3G could dose-dependently decrease the epidermal thickening and attenuate the histopathological damages mentioned above. (Figure 4 C, F) (P < 0.0001) Mast cell infiltration was demonstrated by Toluidine blue staining. Mast cell infiltration was substantially increased in the model mice. By comparison with the model group, C3G drug groups showed suppressed mast cell infiltration. (Figure 4 B, E). In conclusion, we confirmed by immunohistochemistry staining (IHC) that compared to the normal group, DEX- and C3G-administered mice increased in the number of CD8⁺T cells. But it is not significantly different. (Figure 4, G)

FIGURE 4
Effects of Cyanidin 3-O glucoside (C3G) on the skin thickness of the extent of skin lesions and the extent of mast cell infiltration on the back of DNFB-induced AD mice. (A) Photographs of dorsal skin representative samples from the mice. (B) Images obtained by toluidine blue staining on the back skin of mice (C) Hematoxylin and eosin (H&E) staining dorsal skin images (D) Immunohistochemical analysis of CD8⁺ T cells. (E) Microscopic counting of mast cells regarding the back skin of mice. (F) Dorsal skin thickness. (G) The quantitative analysis of the relative percentage of CD8⁺ T cells in the surface area of the back skin. Data are presented as mean ± SD (n = 5). *P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.

Effects of C3G on IL-1, IgE, IL-15, IL-4, IL-10 levels of AD mice

In this study, Dex was used as a comparison to analyze the pharmacological effects of C3G. ELISA assay was used to study the effects of C3G on inflammatory cytokines and chemokines in AD mice. IL-1 leads to increased inflammatory cell infiltration (Abramovits, Bejarano, Valdecantos, 2013), IL-4 promotes T-cell activation (Chiricozzi et al., 2020), IL-15 enhances the killing activity of CD8⁺ T cells (Weng et al., 2002), and IL-10 inhibits eosinophil survival and the production of inflammatory substances. These inflammatory factors play an important role in the development of AD (Kim, 2022; Yamanaka, Mizutani, 2011). By the change of these inflammatory factors, the improvement of the C3G group and for the inflammation can be observed. By measuring the levels of IgE, IL-15, IL-4, and IL-10 within the serum and the level of IL-1 within the skin to explore whether C3G alleviated AD by regulating the quantity of IgE and the cytokines regarding Th1 and Th2. The results showed that the quantity of pro-inflammatory factors was up-regulated in AD mice. The levels of IgE and IL-15 were notably elevated compared to those of the drug group. IL-10, which plays an important role in inhibiting AD lesions, was up-regulated in the C3G and DEX groups of mice. The C3G drug groups significantly inhibited the levels of IgE (P < 0.05) (Figure 5, D), IL-4 (P < 0.001), and IL-15 (P < 0.001), and substantially raised the amount of the inflammation-suppressing factor IL-10 (P < 0.05) in the AD mice (Figure 5, B, C, E), while the C3G high-dose group substantially decreased the number of IL-1 (P < 0.05), but did not restore the levels of the factors to those in normal mice. On the contrary, IL-1 levels were substantially increased in the C3G low-dose group. (P < 0.05) (Figure 5, A) In comparison with the AD model group, both C3G high and low-dose groups showed improvement in AD, but the ELISA results did not indicate any notable difference between the C3G groups except for IL-1.

FIGURE 5
Effects of Cyanidin 3-O glucoside (C3G) on the levels of IgE and the cytokines regarding Th1 and Th2 levels in AD mice. (A) IL-1 levels in the skin (B) Intra-serum IL-15 levels (C) Intra-serum IL-4 levels (D) Intra-serum IgE levels (E) Intra-serum IL-10 levels. Data are presented as mean ± SD (n = 5). *P < 0.05, * * P < 0.01, *** P < 0.001 and **** P < 0.0001.

Effects of C3G on immune cell levels in the spleen of AD mice

CD4⁺ T-helper cells and CD8⁺ cytotoxic T-cells, which are common T-lymphocytes in the study of the pathogenesis of AD. We monitor the number of each immune cell by Flow cytometry to research the regulatory effects of C3G on immune cells. In addition, we selected the C3G high-dose group, which had more favorable results in the previous experiments, to participate in the Flow Cytometry experiments. The analysis of Figure 6 B demonstrated that CD8⁺ T cells was significantly lowered in the drug group of mice. Moreover, CD4:CD8 was too high in the DEX-administered mice probably related to DEX being an immunosuppressant (Figure 6 C). CD4:CD8 ratio in the C3G group had the same down-regulation trend as the normal group. By comparison with the model group, CD8 / CD28 was much higher in the C3G group, suggesting that the CD28 molecule was down-regulated in C3G-administered mice (Figure 6 D). CD8 / PD1 (Programmed death 1) reflected much higher activated of CD8⁺ T cells in drug group mice than in the model and blank groups (Figure 6 E) The infiltration of CD8⁺ T cells in the lesional skin was an ephemeral occurrence. The level of CD8⁺ T cells increased in the 24-hour period, but it decreased after 48 h (Hennino et al., 2007). According to the time of mice dissection, the results for the present experimental drug group showing a reduction in CD8⁺ T cells but an increase in their activation are consistent with the literature. NK killer cells were elevated in the C3G drug groups and the normal group, Additionally, by comparison with the model group, the NK cells decreased in number in the DEX group. The reason for this result may be linked with the over-immunosuppression of DEX (Figure 6 F). Then macrophages decreased in number in both normal and drug-group mice. Slowing down the further development of AD (Figure 6 G). C3G high dose group inhibited the developmental process of AD by greatly decreasing the number of CD8⁺ T cells (P < 0.05), macrophages, CD4: CD8 ratio (P < 0.05), elevating the CD8 / CD28 ratio (P < 0.001), the number of NK killer cells attenuated AD lesions in mice and maintained a consistent change trend with the normal group.

FIGURE 6
Effects of Cyanidin 3-O glucoside (C3G) on immune cell levels in the spleen of AD mice. (A) The number of CD4 cells (B) The number of CD8 cells (C) The ratio of the number of CD4 to CD8 cells (D) The ratio of the number of CD8 to CD28 (E) The ratio of the number of CD8 to PD-1. (F) The ratio of CD3 cells to CD27 cells (G) CD11B+ F4 / 80+ macrophage assay marker numbers. Data are presented as mean ± SD (n = 4). *P < 0.05, * * P < 0.01, *** P < 0.001, and **** P < 0.0001.

Correlation analysis between clinical features of AD, immune cells, inflammatory factors, and depressive behavior

We performed Pearson correlation analyses from flow cytometry data, ELISA results, skin severity scores, the number of scratching bouts, and depressive-like behaviors to investigate potential links between immunity, depression, and AD (Figure 7).The analyses reveled a linear positive correlation between skin severity scores, the number of scratching bouts and the number of CD8⁺ T cells (Figure 7), (r1 = 0.37, r2 = 0.21). In contrast, skin severity scores and the number of scratching bouts were negatively correlated with the CD8/CD28 (Figure 7) (r1 = -0.42, r2 = -0.47) and with the CD8/PD1 ratios (r1 = -0.59, r2 = -0.33, p1 < 0.01). Among the inflammatory factors, both the skin severity scores and the number of scratching bouts were positively correlated with IL-4 (Figure 7) (r1 = 0.46, r2 = 0.63, p1 < 0.05, p2 < 0.001) and IL-15 levels (r2 = 0.69, p2 < 0.001). Furthermore, IL-15 levels and the number of scratching bouts were negatively correlated with the ratio of open travelled area to total travelled area of the EZM (Figure 7) (r1 = -0.74, p1< 0.01, r2= -0.78, p2 < 0.01). FST immobility time showed a strong positive correlation with the number of scratching bouts (Figure 7) (r = 0.54, p < 0.05). Significant positive correlations were also observed between inflammatory factors IL-4, IL-15 and IgE (r1 = 0.71, r2 = 0.47, p1 < 0.001, p2 < 0.05), IL-10 was negatively correlated with IgE (r1 = -0.45, p1 < 0.05), and positive correlation of IL-4 and IL-15 and negative correlation of IL-10 (r1 = 0.59, r2= -0.45, p1 < 0.01, p2 < 0.05).

FIGURE 7
Heatmap representation of the association among clinical characteristics of AD, depressive-like behaviors, Th1 / Th2-related cytokines, and immune cells. DS represents Dermatitis Scores,N 1st represents the number of scratching bouts recorded for the first time, and N 2nd represents number of scratching bouts recorded for the second time. DS, N2nd, CD4, CD8, CD4: CD8, CD8/CD28, CD8/PD1, IgE, IL-4, IL-15, IL-10, EZM, TST, FST are consistent with the Pearson correlation applied. Data are presented as mean± SD for each group (n = 5). *P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.

DISCUSSION

Atopic dermatitis (AD) is a common chronic long-term skin disorder with the main clinical signs being inflammation, intense and persistent itching, and scratching. As the disease progresses, it can also trigger increased depression-like behaviors in patients. The breakdown of the skin barrier further increases permeability to external antigens and allergens, with Langerhans cells and dendritic cells presenting antigens and prompting the occurrence of Th2-type immune response, which in turn elevates of IgE levels. IgE induces the activation of mast cells to promote mast cells’ release of inflammatory factors and granule mediators by binding to their surface FcɛRI. At the same time, CD8⁺ T cells in AD patients are recruited to allergen-exposed sites, promoting the infiltration of eosinophils and Th1 / Th2 cells into the skin, which contributes to inflammation production (Hennino et al., 2007; Wollenberg et al., 2021). C3G has strong oxidative properties. Excess reactive oxygen species(ROS) exacerbates skin barrier disruption and inflammatory conditions in AD. Furthermore, the strong oxidative properties of C3G are effective in alleviating AD symptoms. However, no article has been published to show the therapeutic effect of C3G on AD. In this article, we demonstrate for the first time the impacts of C3G in the improvement of DNFB-induced AD-like skin symptoms and related depressive behaviors, revealing the interactions between C3G, immune cells and cytokines, and AD.

DNFB successfully induced AD lesions on the back of the mice. It caused local epidermal thickening, scaling/dryness, and erythematous ulceration in the skin of the diseased mice. According to the results of the organ index, C3G administration demonstrated a favorable safety profile. In this article, the results of skin severity scores, the number of scratching bouts, and histological analyses revealed that the AD mice whose oral administration of 150 mg/kg or 100 mg/kg of C3G significantly alleviated the skin lesions, reduced itch counts, decreased epidermal thickness, inhibited the aggregation and infiltration of inflammatory cells and mast cells.

Cytokines secreted by helper T cells 2 (Th2) are of great importance in the development of allergic diseases. Among them, IL-4 is a crucial cytokine that promotes Th2 cell differentiation and leads to Th1 / Th2 imbalance. In addition, IL-4, as an important cytokine triggering the inflammation of AD, could increase the liberation of histamine and thymic stromal lymphopoietin (TSLP) by binding to the IL-4R on the sensory nerve, leading to severe itching (Chiricozzi et al., 2020; Oetjen et al., 2017). Furthermore, Th2 cells produce IL-10, which effectively inhibits the survival of eosinophilic granulocytes and the production of pro-inflammatory cytokines such as IFN-γ(Kim, 2022; Yamanaka, Mizutani, 2011). Conversely,, IFN-γ promotes the infiltration of inflammatory cells into inflamed tissues and stimulates the proliferation of keratin-forming cells, resulting in epidermal hyperplasia (Kim, 2022; Yamanaka, Mizutani, 2011). In conclusion, the results of the ELISA of our experiment further confirmed that the C3G drug groups played an important role in improving AD symptoms in mice by down-regulating IL-4 level and up-regulating IL-10 level compared to the model group. Additionally, IL-15 is associated with IL-4 expression. At the same time, it stimulates B cell growth and differentiation by chemotaxis of T lymphocytes and differentiated NK cells. B cells cause overproduction of IgE and sustained activation of mast cells, leading to chronic inflammation (Piccini et al., 2023), The results of the ELISA of our experiment showed that C3G down-regulates IL-15 levels, thereby lowering IL-4 expression and attenuating the clinical manifestations of AD. What is more, IL-1 is one of the major regulators of immune and inflammatory responses. In the AD development process, allergen activation triggers the liberation of IL-1β from the epidermis, resulting in increased infiltration of pro-inflammatory factors and inflammatory cells (Abramovits, Bejarano, Valdecantos, 2013; Iznardo, Puig, 2022), exacerbating the development of inflammation. The results of the ELISA of our experiment showed the C3G drug group downregulated IL-1 in the experiment, hindering this phenomenon. In addition, the significant increase in IL-1 levels in the C3G low-dose group may be an early manifestation of immune activation. Low doses of C3G may first stimulate the body’s immune defense mechanism to initiate a local immune response resulting in an increase in IL-1, whereas high doses of C3G may directly lead to a decrease in IL-1 levels by exerting its anti-inflammatory effects and inhibiting immune cell activation and function. As a result, we found that the level of IL-1 in the skin and the levels of IgE, Interleukin-15, Interleukin-4, within the serum of mice were significantly reduced and IL-10 increased after administration of C3G, which inhibited the vicious cycle of inflammatory factors and alleviated AD symptoms.

In sensitized mice, inflammatory cell infiltration was exacerbated by recruiting CD8⁺ T cells to the site of allergen exposure, while the increased concentration of CD4⁺ T cells released pro-inflammatory factors (Bak et al., 2012). Thus, the CD4 / CD8 ratio rises because of exacerbation of dermatitis in AD patients (Kägi et al., 1994). The results of the Flow cytometry of our experiment have shown that the experimental drug C3G effectively reduces CD8⁺ T cell content and decreases the CD4 / CD8 ratio. In addition, the CD28 molecule binds antigen-presenting cells to spread co-stimulatory signals, thereby promoting the activation of T lymphocytes and proliferation. The CD28 molecules were required for the deepening of stimulus-induced atopic dermatitis (Kondo et al., 1996). Moreover, PD-1 and its ligands have a significant impact on maintaining equilibrium within the immune system and inhibit T-lymphocyte activation and function (Murakami, Riella, 2014). This article CD8 / PD1 reflects cytotoxic T cell depletion in the atopic dermatitis environment. High expression of CD8 / PD1 in the drug group showed increased depletion of CD8⁺ T-lymphocytes, where PD-1 is located, thus inhibiting it from causing an inflammatory response. Furthermore, AD patients’ blood generally lacks natural killer (NK) cells. NK cells can release different types of cytokines, which are involved in the developmental process of atopic dermatitis. The increase in NK cells helped to re-establish an immune balance which can improve AD. In the later stages of inflammation, altered macrophage function accelerated the development of AD (Nakai, 2021). Our experimental results showed that the C3G drug groups significantly down-regulated the level of CD28 factor, up-regulated the level of NK cells, up-regulated the ratio of CD8 / PD1, decreased the number of macrophages, and reduced the pro-inflammatory activity, which has a significant impact on the process of AD development. This result is in keeping with the findings of the literature mentioned above, confirming the feasibility of C3G in the improvement of AD by immunomodulation. In addition, C3G can improve the excessive immune reaction caused by DNFB. The results of flow cytometry showed a very low CD4/ CD8 ratio in the DEX mice, suggesting DEX treatment is linked to immunodeficiency, which aligns with the known side effects of DEX. However, C3G does not appear to cause excessive immunosuppression. Thus oral C3G may become an effective and novel therapeutic approach to improve AD without excessive suppression of immune function.

In addition, it has been shown that AD is associated with increased depressed mood. It further exacerbates depressive behaviors in mice by increasing CD8⁺ T lymphocytes and decreasing CD4⁺ T lymphocytes (Rattazzi et al., 2013). Oral administration of C3G has been shown to provide some degree of improvement in depressive behaviors. In the results of the FST test in our experiment, a notable difference was found between the model mice and oral high-dose C3G mice, by comparison with the AD group, the other groups showed a decrease in the suspension time. The results of the TST experiment were not significant. In addition, we found that sensitized mice had scratching on their backs during TST experiments, this behavior would result in shorter suspension times. In the FST experiment, cold water at 23° was used to create the experimental environment. Lowering the temperature of the water may help reduce itching by controlling the activity of thermoreceptors in the skin, which makes the experimental data reflect the depressed behavior of mice more realistically. Additionally, the results of EZM test showed the mice of C3G drug groups moved significantly more time and distance in the open area than the model group which maintained the same trend as the blank group. It suggests that C3G plays an important role in anti-depressant-like behavior in mice. In conclusion, combined with the findings from correlation analysis which showed that C3G played an ameliorative effect on mice’s depressive-like behaviors while improving AD through decreasing the level of CD8⁺ T lymphatic.

In conclusion, we determined correlations between inflammatory factors, depressive-like behavior, immune cells, and clinical characteristics of AD by Pearman correlation analysis. The results found that skin severity score and number of second scratching bouts were positively correlated with CD8⁺ T cells levels. However, they were negatively correlated with CD8 / PD1, CD4 : CD8, and CD8 / CD28. This suggests that a reduction in the number of CD8⁺ T cells and an exacerbation of the depletion state can improve AD symptoms, which is consistent with reports in the literature that CD8⁺ T cells exacerbates AD development through cytotoxic effects, proinflammatory factor secretion, and barrier disruption. Correlation results showed that IL-4 significantly exacerbated skin severity scores and number of scratching bouts, which was associated with IL-4 promoting histamine release (Chiricozzi et al., 2020). IL-15 positively correlating with CD8⁺ T cells inducing increased CD8⁺ T cells activity, further contributing to exacerbation of AD (Weng et al., 2002). Among them, IL-15 and IL-4 were significantly and positively correlated with IgE, while CD8+ T cells were significantly and positively correlated with IgE. This implies that C3G can influence the development of inflammatory factors by significantly affecting the number and activity of CD8⁺ T cells, using IgE as a mediator. Depressive-like behaviors were significantly negatively correlated with skin severity scores and number of scratching bouts, confirming the association between AD and depression. Moreover, IL-15 exacerbated the development of inflammation making it significantly associated with depressive-like behaviors. In conclusion, there is a strong association between AD clinical characteristics and depressive behaviors and inflammatory factors and immune cells. C3G improve AD and depressive behaviors in mice by decreasing the number and activation of CD8⁺ T cells, as well as decreasing the levels of pro-inflammatory factors.

SUMMARY

In the article, combined results of clinical characteristics, organ index, behavioral experiments, flow cytometry, ELISA, and correlation analyses showed that C3G can improve AD and depressive-like behaviors while also relieving itching mainly by altering the number and functionality of immune cells (predominantly CD8⁺ T cells), thereby down-regulating the creation of the inflammatory factors and the infiltration of macrophages and eosinophils. The results of these experiments also provide a theoretical and scientific basis for C3G to improve AD while improving the depression-like behavior of experimental mice. This paper provides new ideas for C3G to improve AD through immune pathways. However, the specific mechanisms by which C3G regulates immune cells and inflammatory factors and thus alleviates AD still need to be further investigated in depth.

FUNDING INFORMATION

This research was supported by the Research Fund of Jianghan University (Grant No. 2023KJZX25).

DATA AVAILABILITY STATEMENT

Use of data not disclosed.

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Correspondence: X. Cao Department of pharmacy School of Medicine Jianghan University No.8 Triangle Lake Road, Economic Development Zone, Wuhan, China, E-mail: [e-mail]

DECLARATION OF COMPETING INTEREST

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.

Associate Editor:

Roberta Stilhano

Publication Dates

• Publication in this collection
  05 Oct 2026
• Date of issue
  2026

History

• Received
  03 Dec 2024
• Accepted
  02 Aug 2025