Rosa roxburghii Tratt juice inhibits NF-κB and increases IL-2 to alleviates the Foxp3-mediated Tregs imbalance in the peripheral blood of arseniasis patients

Abstract

Arsenic can cause immune inflammation, which is the basis of arsenic-induced damage to multiple organs and systems. Forkhead box P3 (Foxp3)-labelled CD4⁺CD25⁺ regulatory T cells (Tregs) play an essential role in maintaining immune homeostasis. Nuclear factor-κb (NF-κB) and Interleukin-2 (IL-2) are critical regulators of Foxp3. Rosa roxburghii Tratt (RRT) is an edible medicinal plant with anti-inflammation effects. In this study, a control group (n = 41) and an arseniasis group (n = 209) were recruited, and screened subjects from the arseniasis patients for RRTJ (n = 46) or placebo (n = 43) to explore the possible mechanism by which RRT alleviates immune inflammation. The results indicated that RRTJ can inhibits NF-κB and increases IL-2, and alleviates the Foxp3-mediated Tregs imbalance in the peripheral blood of arseniasis patients. In summary, these findings suggest a novel intervention or therapeutic target for immune inflammation in arseniasis patients and provide new evidence that RRTJ inhibits immune inflammation.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-023-01384-0.

Introduction

Arsenic (As) is a naturally occurring toxic element that is widely distributed in nature. More than 200 million people around the world are exposed to arsenic. Environmental arsenic pollution is one of the world's most serious environmental and health problems (Bundschuh et al., 2022; Podgorski and Berg, 2020). Arsenic exposure can cause systemic chronic diseases in multiple organs and systems, posing a severe threat to human health. The immune system, the body's essential defence system, is one of the systems targeted by arsenic toxicity (Huang et al., 2019). Mounting evidence suggests that damage to immune function due to arsenic exposure, such as changes in the contents of immunoglobulins and the complement system, induces abnormal secretion of immune factors and persistently inhibits T lymphocyte proliferation and activation, resulting in an imbalanced subset of lymphocytes; this increases the risk of damage to multiple organs and systems in the body (Li et al., 2017; Martin-Chouly et al., 2011). Therefore, it is of vital significance to explore the mechanism and molecular targets of immune inflammation caused by arsenic. Moreover, it is crucial to identify potential edible or medicinal substances that may act on these targets, in order to perform targeted prevention and treatment of arsenic exposure so as to protect human health. Such work may also have implications for patients with immune inflammation caused by other diseases.

CD4⁺CD25⁺ regulatory T cells (Tregs) are subsets of cells that differentiate from naive leukocyte differentiation antigen 4-positive T lymphocytes (CD4⁺T). CD4⁺CD25⁺Foxp3⁺Tregs are the main type of Tregs in the body. They are very important immunosuppressive cells that play a crucial role in maintaining immune homeostasis (Qiu et al., 2020). Forkhead box P3 (Foxp3) is a specific marker molecule for CD4⁺CD25⁺Foxp3⁺Tregs; it plays a key role in the development and differentiation of Tregs and is essential for Tregs to play an immunosuppressive role (Valencia and Lipsky, 2007).

Nuclear factor-κb (NF-κB) is an important transcription factor in cells. Under normal physiological conditions, NF-κB is inactivated and mainly distributed in the cytoplasm. After being activated by external stimuli, NF-κB mediates the transcription of multiple signalling pathway-related genes through NF-κB p65 transfer into the nucleus and participates in crucial life processes such as cell proliferation, differentiation, and apoptosis. NF-κB plays a key role in the body's immune inflammation (DiDonato et al., 2012; Hayden and Ghosh, 2011). Research indicates that NF-κB is the upstream regulatory gene of Foxp3; it can regulate Foxp3 by promoting Foxp3 methylation and inhibiting Foxp3 mRNA expression (Polansky, et al., 2010). Moreover, NF-κB can directly bind to and regulate Foxp3(Ferrandino, et al., 2018). Interleukin-2 (IL-2) is a cell growth factor secreted by T lymphocytes; it maintains the differentiation and proliferation of T lymphocytes, which play a key role in the body's immune regulatory network (Abbas et al., 2018). Additionally, IL-2 induces and maintains Foxp3 expression. Numerous studies have shown that IL-2 promotes Foxp3 expression, contributes to the maturation and functional development of Tregs, and plays an essential role in immune tolerance (Dikiy et al., 2021).

Rosa roxburghii Tratt (RRT) is a perennial deciduous shrub of Rosaceae. It grows naturally throughout China, including in Guizhou, Sichuan, and Yunnan provinces. Because RRT is abundant in vitamin C, superoxide dismutase (SOD), polyphenols, flavones, polysaccharides, triterpenes, amino acids, and trace elements, it has excellent immunity, anti-oxidation, and anti-cancer effects, among others; its functional activities are unmatched by other fruits (Jiang et al., 2022; N. Li et al., 2022a, 2022b). Therefore, as an edible medicinal plant, RRT was included in the Quality Standard of Traditional Chinese Medicinal Materials and Ethnic Medicinal Materials of Guizhou Province (2003), and the following year, was listed as a new food resource for general food management by the Ministry of Health of China (Announcement No. 17, 2004) (Wang et al., 2021). Research suggests that the flavonoids in RRT enhance radioprotection by regulating TNF-α/NF-κB and IL-1α/IL-6 to reduce immune inflammation. Moreover, the polysaccharide (RTFP) extracted from RRT fruit can be used as a natural anti-inflammatory agent to reduce chronic obesity-induced colitis (Wang et al., 2022; Xu et al., 2018). Our previous studies discovered that RRTJ can alleviates the arsenic-induced Tregs imbalance and complement abnormalities, and improves the body's immune function; however, the underlying mechanism is not fully understood (Dong et al., 2022).

In this study, the mRNA level of Foxp3, the CD4⁺CD25⁺Foxp3⁺Tregs percentage, the NF-κB p65 and NF-κB p50 mRNA expression levels, and the content of IL-2 in the peripheral blood of arseniasis patients were first evaluated. Then, RRT juice (RRTJ) was applied as an intervention for patients with arseniasis, and the changes in the above indicators after the intervention were compared and analysed. Finally, the possible mechanism underlying the effect of RRTJ on immune inflammation in patients with arseniasis was preliminarily explored. This study contributes to our understanding of the possible mechanisms underlying immune inflammation caused by a Tregs imbalance in arseniasis patients. Moreover, these findings highlight the potential application of an edible medicinal plant in the prevention and control of immune inflammation caused by arsenic or other diseases.

Materials and methods

Subjects and study design

A total of 250 subjects were recruited for this study. Among them, 209 were residents from the arseniasis area of Xingren County, Guizhou Province, and were clinically diagnosed with arseniasis according to the Diagnostic Criteria for Endemic Arseniasis (WS/T 211-2015). Based on the presence of skin lesions, the patients were divided into a mild arseniasis group (palmo plantar with skin keratosis of grade I or trunk with skin pigmentation of grade I and depigmentation of grade I simultaneously) (63 cases), an intermediate arseniasis group (palmo plantar with skin keratosis or trunk with skin pigmentation of grade II or depigmentation of grade II) (73 cases), and a severe arseniasis group (palmo plantar with skin keratosis or trunk with skin pigmentation of grade III or depigmentation of grade III) (73 cases). In addition, 41 healthy residents from a non-arsenic-exposed village took part in this study; all healthy residents had no abnormal clinical findings and similar living habits, economic level, and education level to those in the arsenic-poisoned area. These healthy residents served as the control group. All subjects were 18–65 years of age and had no previous occupational arsenic exposure, immunodeficiency or autoimmune diseases, infection within one month, recent history of consumption of seafood or drugs that affect immune regulation or arsenic metabolism, or long-term exposure to environments that affect the immune system.

To assess the potential application of RRTJ in arsenic-induced immune inflammation, the above arseniasis patients were matched according to age and sex and signed an informed consent form agreeing to participate in the intervention study, and were randomly assigned to the RRTJ (53 cases) or placebo group (53 cases) in a double-blind manner. Patients received 20 mL of either RRTJ (purchased from Sinopharm Group Guizhou Health Industry Development Co., LTD., National Health Commission, PRC Health Food License No. [2002]0004) or placebo (no RRTJ active ingredients, colour and taste consistent with RRTJ) per day (according to the recommended dose in the health food instructions). The treatments were orally administered for three months. A total of 89 arseniasis patients completed the intervention survey as required: 46 in the RRTJ group and 43 in the placebo group (Fig. 1).

Fig. 1.

Grouping and study-flow diagram

Early morning urine was collected from all subjects to detect urinary arsenic levels and early morning fasting peripheral blood was collected from all subjects and the intervention subjects before and after the intervention to detect the IL-2 content, NF-κB p65, NF-κB p50 and Foxp3 mRNA expression levels, and CD4⁺CD25⁺Foxp3⁺Tregs percentage. After giving informed consent, the subjects underwent a physical examination, urine and blood sample collection, and indicator testing.

Detection of arsenic content in urine

Urine was digested in a microwave with 6 ml of concentrated nitric acid and 2 ml of hydrogen peroxide. Then, 2% nitric acid was added to the digested sample to a total volume of 25 ml, and the urinary arsenic content was detected by inductively-coupled plasma mass spectrometry (ICP-MS) (XSeries2, Thermo Fisher Scientific, USA). The results were normalized to the urinary creatinine content.

Quantitation of NF-κB p65, NF-κB p50, and Foxp3 mRNA expression levels

TRIzol (Invitrogen, USA) was used to extract the total RNA from the peripheral blood sample and a Reverse Transcription Kit (Thermo Fisher Scientific, USA) was used to reverse transcribe to cDNA. The NF-κB p65, NF-κB p50 and Foxp3 mRNA expression levels were detected by Real-time fluorescence quantitative PCR (RT-qPCR) (Bio-Rad, USA). GAPDH was used as the internal reference, and the NF-κB p65, NF-κB p50 and Foxp3 mRNA expression levels were calculated by the 2^−△△Ct method. The gene primers and PCR reaction conditions are provided in Supplementary Tables 1 and 2.

Detection of the IL-2 content

The peripheral blood IL-2 content was detected by enzyme-linked immunosorbent assay (ELISA) (BD Biosciences, USA).

Measurement of CD4⁺CD25⁺Foxp3⁺Tregs percentage

Flow cytometry (BD GolgiPlug™, BD Biosciences, USA) was used to detect the CD4⁺CD25⁺Foxp3⁺Tregs percentage. Separation of peripheral lymphocytes (PBMC) was performed by the density gradient method, and 2 × 10⁶ PBMC cells were cultured in vitro for 4–6 h with 4 μl BD Leukocyte Activation Cocktail (BD Biosciences, USA). The cells were then resuspended in PBS and added to 10 μl anti-CD4-FITC (BD Biosciences, USA), 5 μl anti-CD25-PE-cy7 (BD Biosciences, USA), and corresponding isotype control, respectively. The cells were then incubated in the dark at 4 °C for 30 min. Next, 1 ml Fixation/Permeabilization Buffer (BD Biosciences, USA) was added and the cells were again incubated in the dark at 4 °C for 40 min. After removing the Fixation/Permeabilization Buffer, 10 μl Foxp3-Aleax Flour (BD Biosciences, USA) and corresponding isotype control were added, and the cells were again incubated in the dark at 4 °C for 45 min. Finally, the supernatant was removed and 300 μl PBS was added to the resuspended cells. Flow cytometry was performed to detect the CD4⁺CD25⁺Foxp3⁺Tregs percentage.

Statistical analysis

SPSS22.0 statistical software was used to perform the statistical analyses. Quantitative data were tested for normality and homogeneity of variance. Age was normally distributed, and thus, one-way analysis of variance was used for comparisons among multiple groups, with data expressed as the mean ± SD. The remaining indicators exhibited skewed distributions, and thus, the Kruskal–Wallis test followed by the Mann–Whitney U-test with Bonferroni correction was used for comparisons between multiple groups, with the data expressed as the median (M) and interquartile range (P₂₅ ~ P₇₅). Sex differences between groups were evaluated using the Chi-square test. The relationships between the variables were evaluated using the Spearman correlation analysis. To compare the indicators before and after the intervention, paired sample t-tests were performed. P < 0.05 was considered statistically significant.

Results and discussion

Demographic characteristics of the subjects

A total of 209 arseniasis patients took part in this study, including 63 mild cases, 73 intermediate cases, and 73 severe cases; there were 41 cases in the control group. As shown in Table 1, there were differences in age and sex between the control group and the arseniasis group (F = 8.190, χ² = 33.106, P < 0.001). The urinary arsenic content of patients with different degrees of arseniasis was significantly higher than that of the control group (H = 11.196, P < 0.05). There were no significant differences in age and sex between the RRTJ and placebo groups (P > 0.05), as shown in Supplementary Table 3.

Table 1.

Demographic characteristics of the subjects

Variable	Control subjects (n = 41)	Arseniasis subjects			Statistical value	P value
		Mild (n = 63)	Intermediate (n = 73)	Severe (n = 73)
Age (mean ± SD, years)	45.76 ± 7.83	51.05 ± 8.41	52.30 ± 7.66	52.55 ± 6.69	8.190	< 0.001a
Sex (N, %)
Male	12 (29.27)	27 (42.86)	35 (47.95)	58 (79.45)	33.106	< 0.001b
Female	29 (70.73)	36 (57.14)	38 (52.05)	15 (20.55)
Urinary As (M, P25-P75, μg/g Cr)	20.20 (11.01 ~ 28.20)	27.82 (19.75 ~ 41.47)	27.56 (15.80 ~ 43.77)	26.64 (17.58 ~ 40.90)	11.196	< 0.05c

^aOne-way analysis, the statistical value is F value

^bChi-square test, the statistical value is χ² value

^cKruskal–Wallis test and Mann–Whitney U test, the statistic-value is H value

Arsenic inhibits Foxp3, reducing the CD4⁺CD25⁺Foxp3⁺Tregs percentage and causing an inflammatory imbalance

The immune system performs essential immune functions in the body and plays a crucial role in maintaining health. Dysfunction of the immune system is the basis of most diseases, including infection, inflammation, aging, tumours, etc. In recent years, numerous studies have shown that arsenic exposure can cause damage to the immune system. Our previous report suggested that arseniasis patients have an immune-inflammatory imbalance, with elevated IL-6 and IL-17A levels and a decreased IL-10 level in the peripheral blood as well as decreased levels of IFN-γ and IL-2 in the skin tissue (Fang et al., 2022; Xia et al., 2020). We also found abnormal secretion of cytokines such as IL-6, IL-17A, IL-10, and TGF-β1 in the peripheral blood, liver, kidneys, and lungs of arsenic-exposed rats, confirming an imbalance in the immune response with arsenic exposure (Dong et al., 2021; Wang et al., 2020). Studies by other researchers have also revealed an arsenic-induced immune imbalance. For example, studies have demonstrated that arsenic exposure can cause abnormal levels of TNF-α, IFN-γ, IL-1β, IL-6, and IL-12 in the lungs and spleens of mice in a time-dependent manner (Jamal et al., 2020). The effect of arsenic on the immune system is the basis of arsenic-induced damage to multiple organs and systems. Therefore, it is of great practical significance to explore the possible mechanism and targets of arsenic-induced immune system damage and carry out targeted interventions or treatments to protect the health of arsenic-exposed people.

Regarding the possible mechanisms and targets of arsenic-induced immune system damage, our previous studies demonstrated that Foxp3 methylation levels are increased and mRNA expression levels are decreased in the peripheral blood lymphocytes of people with arseniasis, and Foxp3 methylation has great potential for identifying the risk of arseniasis (Fang et al., 2022; Ma et al., 2022). Foxp3 is specifically expressed in Tregs; it is recognized as a specific biomarker and is required for the differentiation, development, and maturation of Tregs (Hori et al., 2003). CD4⁺CD25⁺Foxp3⁺Tregs are a subset of T cells with immunosuppressive functions; they play an important role in the negative regulation of the immune response and autoimmune tolerance. Tregs were first reported in 1995 by the Japanese scientist Sakaguchi (Sakaguchi et al., 1995). Tregs are present in the spleen and peripheral blood of humans and mice. They play an immunomodulatory role in thymus differentiation, development, and maturation and are released into the peripheral blood to achieve a negative immunomodulatory effect mainly by inhibiting the activation and proliferation of CD4⁺ and CD8⁺ T cells. Thus, they play a crucial role in the occurrence of inflammatory diseases in multiple organs or systems. In this study, the Foxp3 mRNA expression level (Fig. 2A) and the CD4⁺CD25⁺Foxp3⁺Tregs percentage (Fig. 2B) in the peripheral blood of arseniasis patients gradually decreased with increasing degree of arseniasis, with overall lower levels than those in the control group (χ² = 45.316, 74.705, P < 0.001), these results suggest that arsenic-induced immune system injury is related to the inhibition of Foxp3 and Tregs. In addition, we also observed that the decrease of Foxp3 and Tregs in the intermediate and severe arseniasis group was greater than that in the mild arseniasis group, indicating that the decrease of Foxp3 and Tregs was related to the severity of the disease, further indicating that arsenic could increase its toxicity by inhibiting Foxp3 and Tregs. It has been reported that fatal autoimmune syndromes observed in Foxp3-mutated and Foxp3-deficient mice are caused by Treg defects. Tregs inhibit disease progression when transplanted into neonatal Foxp3-deficient mice (Fontenot et al., 2003). In adult mice with Foxp3 deletion, the immunosuppressive function of Treg cells is decreased and inflammatory cytokines are increased, thus promoting the occurrence and development of tissue inflammation (Hu et al., 2018). In the same way, decreased Foxp3 expression, a reduced CD4⁺CD25⁺Foxp3⁺Tregs percentage, and increased inflammatory responses have been observed in patients or animal models with inflammation of the liver, kidney, lungs, and other organs, as well as in asthma and other diseases. We further conducted correlation analysis and indicated that the CD4⁺CD25⁺Foxp3⁺Tregs percentage was positively correlated with the Foxp3 mRNA expression level (r = 0.216, P < 0.001), this result suggest that the decreased percentage of Tregs in arseniasis patients is caused by the inhibition of Foxp3 expression.

Fig. 2.

The mRNA expression level of Foxp3 (A) and the CD4⁺CD25⁺Foxp3⁺Tregs percentage (B) in the peripheral blood of arseniasis patients decreased. C Representative flow cytometry (FCM) images of the CD4⁺CD25⁺Foxp3⁺Tregs percentage in the peripheral blood of the subjects; Abscissa axis: content of CD4⁺CD25⁺, Vertical axis: content of Foxp3⁺, Q1-1: CD4⁻CD25⁻FoxP3⁺ T cells, Q2-1: CD4⁺CD25⁺Foxp3⁺Tregs, Q3-1: negative cells, Q4-1: CD4⁺CD25⁺Foxp3⁻ T cells. The data are shown as the median (M) and interquartile range (P25 ~ P75). ^*P < 0.05, ^***P < 0.001

Arsenic activated the inflammatory regulatory genes NF-κB p65 and NF-κB p50 and inhibited IL-2 in the peripheral blood

However, it is still unclear how Foxp3 is regulated in arseniasis patients. It has been reported that activation of NF-κB signalling can directly regulate Foxp3. NF-κB is an essential intracellular transcription factor. By activating NF-κB p65 and binding with NF-κB p50 to form heterodimers, NF-κB plays a key role in inflammatory and immune responses through its effects on a variety of biological processes such as cell proliferation, differentiation, and apoptosis (Oh et al., 2017). In this study, compared with the control group, the NF-κB p65 (Fig. 3A) and NF-κB p50 (Fig. 3B) mRNA expression levels in the peripheral blood of arseniasis patients were increased (χ² = 11.450, P < 0.01; χ² = 19.767, P < 0.001). In studies of diseases such as arthritis, elevated expression of NF-κB p65 leads to decreased Foxp3, which, in turn, triggers an inflammatory response. Several studies have demonstrated high expression of NF-κB p65 or NF-κB p50 in animal models of organ damage, such as liver and kidney damage induced by arsenic, and in arsenic-induced cell injury (Xu et al., 2021), however, its relationship with Foxp3 remains unclear. The results of this study show that Foxp3 was a negative correlation with the NF-κB p65 mRNA expression level (r = − 0.135, P < 0.05), and suggests that arsenic-induced activation of the NF-κB pathway may be one of the mechanisms underlying the reduction in the peripheral blood Foxp3 mRNA expression level and CD4⁺CD25⁺Foxp3⁺Tregs percentage in arseniasis patients, and thus, the observed immune inflammation.

Fig. 3.

The mRNA expression levels of NF-κB p65 (A) and NF-κB p50 (B) increased, and the content of IL-2 (C) decreased in the peripheral blood of arseniasis patients. The data are shown as the median (M) and interquartile range (P25 ~ P75). **P < 0.01, ***P < 0.001

In this study, we also found that the content of IL-2 (Fig. 3C) in the peripheral blood of arseniasis patients was significantly lower than that of the controls (χ² = 101.823, P < 0.001). IL-2 is a soluble glycoprotein produced by lymphocytes and other cells after antigen or mitogen activation and can regulate the directed differentiation and functional activity of immune cells and has a wide range of biological activities. The decrease of IL-2 level in arsenic poisoning patients may be one of the reasons for arsenic-induced immunotoxicity. Just as important, some studies have pointed out that IL-2 is another important regulator of Foxp3. It has been found that IL-2 promotes Foxp3 expression in the development of CD4⁺T cells in the thymus and contributes to the maturation and functional development of Tregs (Cheng et al., 2013). In addition, some studies have reported that IL-2 mainly promotes the expression of Foxp3 by inducing the activation of STAT5(Dikiy et al., 2021). Mice lacking IL-2 or its receptors and mice lacking downstream signalling molecules of IL-2 do not develop Tregs and exhibit autoimmune diseases characterized by autoantibody production and inflammatory bowel disease (Popmihajlov et al., 2012). In this study, we also found that the Foxp3 mRNA expression level in peripheral blood of arseniasis patients was positively correlated with the content of IL-2(r = 0.186, P < 0.01), and suggests that the decrease in IL-2 content induced by arsenic may be another mechanism underlying the inhibition of Foxp3 in the peripheral blood of arseniasis patients. However, further exploration of the regulatory mechanism between IL-2 and Foxp3 in arsenic-induced toxicity is still needed.

Although we did not observe significant differences in NF-κB p65 and NF-κB p50 mRNA expression and IL-2 content among patients with different degrees of arseniasis, correlation analysis has shown that there was a significant correlation between Foxp3 mRNA expression with NF-κB p65 mRNA expression and IL-2 content.

RRTJ inhibits NF-κB and increases IL-2 to reduce the Foxp3-mediated CD4⁺CD25⁺Foxp3⁺Tregs imbalance in the peripheral blood of arseniasis patients

RRT is an edible medicinal plant with high nutritional and medicinal properties. Our previous study confirmed that RRTJ modulates cytokine levels in the peripheral blood of arseniasis patients and alleviates inflammatory responses (Dong et al., 2022). In order to clarify whether the anti-inflammatory mechanism of RRT in patients with arseniasis is related to the expression of Foxp3 mediated by NF-κB and IL-2, in this study, an RRTJ intervention was performed on arseniasis patients. The results indicated that, after taking RRTJ, the peripheral blood NF-κB p65 (Fig. 4A) and NF-κB p50 (Fig. 4B) mRNA expression levels of the arseniasis patients were significantly decreased (t = 2.282, P < 0.05; t = 3.330, P < 0.01), the IL-2 content (Fig. 4C) increased (t = -5.333, P < 0.001), and the downstream Foxp3 mRNA expression level (Fig. 5A) and CD4⁺CD25⁺Foxp3⁺Tregs percentage (Fig. 5B) were significantly increased (t = − 2.602, P < 0.05; t = − 3.098, P < 0.01). These may be related to the high content of ascorbic acid, polysaccharides, flavonoids, terpenoids, superoxide dismutase, polyphenols, multivitamins and trace elements in RRT (Li et al., 2022a, 2022b; Ni et al., 2022), which are beneficial to increase the antioxidant capacity of the body, reduce the stress level of the body, and thus alleviate the immune damage induced by arsenic. These results provide an intervention and therapeutic target for immune inflammation in arseniasis patients and provide some evidence for the application of RRTJ to inhibit immune inflammation in arseniasis patients.

Fig. 4.

RRTJ can inhibit the mRNA expression levels of NF-κB p65 (A) and NF-κB p50 (B) and increase the content of IL-2 (C) in the peripheral blood of arseniasis patients. The data are shown as the median (M) and interquartile range (P25 ~ P75). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Fig. 5.

RRTJ can increase the mRNA expression level of Foxp3 (A) and the CD4⁺CD25⁺Foxp3⁺Tregs percentage (B) in the peripheral blood of arseniasis patients. C Representative flow cytometry (FCM) images of the CD4⁺CD25⁺Foxp3⁺Tregs percentage in the peripheral blood of the subjects; Abscissa axis: content of CD4⁺CD25⁺, Vertical axis: content of Foxp3⁺, Q1-1: CD4⁻CD25⁻FoxP3⁺ T cells, Q2-1: CD4⁺CD25⁺Foxp3⁺Tregs, Q3-1: negative cells, Q4-1: CD4⁺CD25⁺Foxp3⁻ T cells. The data are shown as the median (M) and interquartile range (P25 ~ P75). *P < 0.05, **P < 0.01

There are several limitations of this study that should be noted. Importantly, due to the limited types of biological samples and blood volumes obtained in this study, the mechanism underlying the observed effects could not be investigated in depth. Future comprehensive in vitro and in vivo studies will need to be performed to further investigate the underlying mechanisms.

In summary, the imbalance in Tregs in the peripheral blood of arseniasis patients may be related to the activation of NFκB and the inhibition of IL-2, which results in down-regulation of the expression of Foxp3. RRTJ can inhibits NF-κB and increases IL-2, and alleviates the Foxp3-mediated Tregs imbalance in the peripheral blood of arseniasis patients. These findings provide an intervention and therapeutic target for immune inflammation in arseniasis patients and offer preliminary evidence indicating that RRTJ inhibits immune inflammation.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

QW: Formal analysis, writing—original draft, writing—review & editing. XF: Investigation, validation, formal analysis. BS: Investigation. KZ: Investigation. MY: Investigation. SW: Investigation. AZ: Conceptualization, methodology, investigation, resources, data Curation, writing—review & editing, supervision, project administration, funding acquisition. All authors have read and approved the final manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (No. 81430077).

Declarations

Conflict of interest

The authors declare no conflicts of interest.

Ethical approval

This study was approved by the Ethics Committee of Guizhou Medical University (No. 201403001) and all subjects gave written informed consent. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. Informed consent was obtained from all patients for inclusion in the study.