A retrospective analysis and clinical application value of the expression levels of high mobility group protein B1, β1 globulin and Toll-like receptor 4 in serum of children with rheumatoid arthritis

doi:10.21203/rs.3.rs-2192963/v1

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Research Article

A retrospective analysis and clinical application value of the expression levels of high mobility group protein B1, β1 globulin and Toll-like receptor 4 in serum of children with rheumatoid arthritis

https://doi.org/10.21203/rs.3.rs-2192963/v1

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Methods

50 healthy children are selected as the healthy control group during the same period. The molecular levels of serum HMGB family proteins in the children are detected by Enzyma-Linked Immunosorbent Assay (ELISA), and flow cytometry is used to detect the levels of serum Toll-like receptors and their expression levels.

Results

The classification and expression levels of serum globulin are detected by automatic biochemical analyzer. By comparison of the expression levels of serum HMGB1, β1 globulin and TLR-4, the correlation between the expression levels of the above factors and the incidence of disease is analyzed. The experimental results show that expression levels of serum HMGB1, β1 globulin and TLR-4 in the study group are significantly higher than those in the control group (P < 0.05).

Conclusion

It is clearly evident that Serum HMGB1, β1 globulin and TLR-4 are highly expressed in the serum of children with JIA, and are positively correlated with the occurrence of the disease.

As one of the diseases with a high disability rate in childhood, the common clinical symptoms of JIA include fever, rash and symmetric swelling. Few researches have been done to elucidate its pathogenesis [9]. Most studies speculated that the pathogenesis is closely related to the body's abnormal immune function, and rubella virus is isolated from the joint fluid cells of most patients, and adenovirus or coxsackie virus infection is isolated from the joint fluid of a few patients [10]. Other studies have shown that heredity is also one of the important factors causing such diseases. Most studies have demonstrated that Major Histocompatibility Complex genes (MHC) determine whether immune defects occur in the body. It also determines the type and degree of immune deficiency [11]. More research and points out that the immunological factor is the influence of the important causes of JIA, JIA children at different time can detect the different serum T cell types, especially CD4 + cells, the most common classification of different T cell can by activating interleukins, tumor necrosis factor thus mediated joints to prevent injuries, Thus causing JIA[12]. At present, the clinical factors of JIA mainly include IL-6, IL-8, GM-CSF, etc., and there are relatively few studies on serum HMGB1, β1 globulin, TLR-4, but some studies show abnormal expression of these serum factors in patients with rheumatoid arthritis, so the author speculated that there is a certain connection between JIA and the onset of children. Therefore, through the detection and further analysis of serum HMGB1, β1 globulin and TLR-4 in children with JIA, the relationship between the expression level and the incidence of JIA and its clinical application value are discussed, in order to provide a new way for the early diagnosis and improvement of prognosis of this kind of disease.

Some results suggest that the levels of HMGB1, β1 globulin and TLR-4 are positively correlated with the incidence of JIA. Xu et al. [13] pointed out that HMGB1, as a binding protein widely distributed in the body nucleus, is involved in the movement process of important cells in human body and can be secreted from the nucleus to the extracellular area under the induction of inflammatory response. In addition, HMGB1 is secreted out of the nucleus and can also bind to a variety of receptors on the cell surface, which can maintain or even aggravate the inflammatory response of the body [14]. TLR4 as one of HMGB1 receptor, is a kind of exist in the cell surface used for signaling transmembrane receptor, has in the immune response to accurately identify the important role of pathogens and activate the immune response, and can induce secreted cytokines and regulate the immune system, so the author speculates in children with JIA TLR4 main reason for the increased in serum as TLR participates in the recognition of HMGB1. Some studies have shown that HMGB1 is positively correlated with TLR4, HMGB1 is synthesized in large numbers and released into the blood under the stimulation of some factors, thus the expression level of TLR4 also increases to participate in the signal mediated process of HMGB1 [15]. Globulin, as a serum protein with immune function in human body, has a variety of different types. β1 globulin is mainly used in clinical detection of liver lesions [16]. When the body is infected, the immune system will secrete a variety of globulin to resist external invasion, resulting in increased expression level of β1 globulin [17]. Therefore, it can also confirm most of the above speculations about immunological factors leading to the occurrence of JIA [18] .

3.1 General Information

82 children with JIA admitted to our hospital from February 2020 to March 2021 are included in the study group, and 50 healthy children in the same period are selected as the healthy control group. The study group included 38 girls and 44 boys, aged from 6 to 14 years, with an average age of (9.45 ± 4.26) years, and the course of disease is 9–32 months. The control group included 23 girls and 27 boys, aged from 6 to 13 years, with an average of (9.02 ± 4.18) years. There is no significant difference in age, gender and other general information between the two groups (P > 0.05), indicating comparability. In the study group, 82 children are treated with the same method for 6 months. According to the prognosis, they are divided into good group (n = 39) and bad group (n = 43).

All patients enrolled in the study signed informed consent, the specific provisions include: (1) the examination method used in this study is safe, and the examination measures adopted are safe methods that have been used in clinical practice; (2) The treatment period is 6 months, during which the serum factors of patients in the study group are regularly detected; (3) The original data (including the test sheet) in the process of this study belong to the research group, but we will protect your privacy, no matter when your name will not appear in the public publications, if relevant departments need to use these data, we have the right; (4) Your participation is entirely voluntary. You have the right to choose not to participate in this study or to withdraw at any time without affecting the normal treatment of your disease. However, I hope to complete this study as far as possible without any special reasons. In any case, please inform your physician.

Inclusion criteria include the following: (1) signed informed consent; (2) Meet the clinical diagnostic criteria for JIA; (3) Complete clinical data; (4) Age ≤ 15 years old. Exclusion criteria include the following: (1) patients with poor treatment compliance that affected the study results; (2) persons with immune dysfunction; (3) complicated with major organ or blood diseases; (4) Those who took drugs without permission during the treatment had influence on the study results.

3.2 Proposed methods

3.2.1 Enzyme-Linked Immunosorbent Assay (ELISA) is used to detect serum HMGB1

5ml of venous blood is taken from each group in fasting state in the morning, and put into a centrifuge (produced in Beijing Medical Centrifuge Factory). The centrifuge parameters are set at 3500r/min, the centrifuge radius is 15cm, and the centrifuge duration is 10min. After centrifugation, the upper serum is separated and stored at -20℃ for future use. According to the instructions of ELISA kit, the double-antibody sandwich method is used for detection, TMB color display, and the ENZYme-plate meter (produced by Bio-RAD Company in the United States) is used to detect THE OD value at 450nm. CurveExpert software is used for analysis, and the actual concentration of HMGB1 is calculated according to the linear regression equation.

3.2.2 TlR-4 expression is detected by flow cytometry

Add 100ul of anticoagulant whole blood into each test tube, take one tube as the homotype control group and add cd14-fitc20ul, mouse-igg1-apc10ul and mouse-igg1-pe20ul into it, add cd14-fitc20ul, tlr4-pe5ul into the other test tube, Shake them evenly and store them at 4℃ for 20min. 2ml of cell lysate is added to each tube, and then fully mixed and placed at room temperature for another 10min. 10min later, the tubes are put into the centrifuge, and the centrifuge parameters are set to 1500r/min, the centrifuge radius is set to 15cm, and the centrifuge lasted for 7min. After centrifugation, supernatant is discarded and 2ml PBS ishing solution is added, and supernatant is discarded again by same-velocity centrifugation; PBS200ul is added into each test tube for measurement, and BD FACSAria flow cytometry (produced by BD Company in the United States) is used for detection. Before detection, the solution in each test tube is evenly oscillated. BD FACSDiva software is used for analysis. The relative content of TLR4 protein is expressed by Mean Fluorescence Index (MFI), and it can be defined as: MFI= (mean fluorescence intensity of protein expression in the sample - mean fluorescence intensity of protein expression in the homotype control sample)/mean fluorescence intensity of normal control sample.

3.2.3 Detection of serum β1 globulin

Serum samples to be measured and the expression level of β1 globulin in serum is detected by automatic biochemical analyzer.

3.2.4 Prognosis assessment and grouping

All the children in the study group are treated for 6 months. At the end of the treatment, the expression levels of serum HMGB1, β1 globulin and TLR-4 are detected, problems occurred during the follow-up are recorded and professional guidance is given. The prognosis is determined according to clinical normal indicators, and the children are divided into good group and bad group.

3.3 Observation indicators

The observation indicators include: (1) The expression levels of serum HMGB1, β1 globulin and TLR-4 in the study group and the control group are compared; (2) The correlation between the expression levels of serum HMGB1, β1 globulin and TLR-4 and the incidence of JIA is analyzed; (3) Draw receiver operating characteristic curve (ROC) to evaluate the clinical diagnostic value of single test and combined test for such diseases; (4) The expression levels of serum HMGB1, β1 globulin and TLR-4 in the good group and the bad group are compared; (5) ROC curve is drawn to evaluate the prognostic value of single test and combined test for such patients.

3.4 Statistical Processing

SPSS 25.0 statistical software is used for data analysis. Normality test is performed on the data first. If the data followed normal distribution and homogeneity of variance, it is represented by mean ± standard deviation. Paired sample T is used for testing within the group, and variance comparison is used between groups. Data that did not follow the normal distribution are represented by median and quartile spacing, the rank-sum test of two related samples in the nonparametric test is used within the group, and the rank-sum test is used for comparison between the groups. Measurement data are expressed as mean ± standard deviation (‾x ± s). Descriptive statistical analysis is conducted by percentage, and x2 test is performed. (3) Spearman correlation coefficient is used to analyze the correlation between the expression levels of serum HMGB1, β1 globulin and TLR-4 and the incidence of JIA. All the above results show significant difference with P < 0.05.

4.1 Analysis of serum indexes of JIA children

The results show that HMGB1 had the most significant change in HMGB family (P < 0.01). In toll-like receptor classification, TLR2, TLR4 and TLR9 had significant differences (P < 0.01), but TLR4 had more significant differences. In the serum globulin family, the serum β 1-globulin levels of JIA children are significantly different from normal values (P < 0.01), as shown in Table 1.

Table 1

Analysis of serum indexes of JIA children.
	Normal value	JIA	t	P
HMBG
HMBG1	13.62 ± 0.27	28.79 ± 1.18	-71.799	< 0.001
HMGB2	10.25 ± 0.24	13.20 ± 0.52	-37.699	< 0.001
HMGB3	11.26 ± 0.37	14.29 ± 0.41	-28.613	< 0.001
toll-like receptor
TLR2	0.98 ± 0.10	1.14 ± 0.11	-8.385	0.006
TLR3	0.89 ± 0.03	0.93 ± 0.04	-6.099	0.001
TLR4	1.00 ± 0.08	1.17 ± 0.12	-8.357	< 0.001
TLR5	0.96 ± 0.09	0.98 ± 0.07	-1.426	0.156
TLR9	1.01 ± 0.12	1.08 ± 0.10	-3.613	< 0.001
globulin
α1globulin	2.00 ± 1.00	2.17 ± 1.04	-0.990	0.324
α2globulin	2.87 ± 1.58	3.01 ± 1.53	-0.504	0.615
β1globulin	2.26 ± 0.43	3.33 ± 0.79	-8.806	< 0.001
γglobulin	2.04 ± 0.72	2.38 ± 0.69	-2.701	0.008

4.2 Comparison of serum factor levels between study group and control group

The results show that the expression levels of serum HMGB1, β1 globulin and TLR-4 in the study group are significantly higher than those in the control group (P < 0.05), as shown in Table 2 and Fig. 1.

Table 2

Comparison of serum factor levels.
Indicators	Control group(n = 50)	Study group(n = 82)	t	P
HMGB1(ng/ml)	16.06 ± 0.49	28.79 ± 1.18	-71.799	< 0.001
β1 globulin(mg/L)	2.26 ± 0.43	3.33 ± 0.79	-8.806	< 0.001
TLR-4	1.00 ± 0.08	1.17 ± 0.12	-8.357	< 0.001

4.3 Spearman correlation coefficient analysis of the correlation between serum factor level and the incidence of JIA

Spearman correlation coefficient shows that the expression levels of SERUM HMGB1, β1 globulin and TLR-4 are significantly positively correlated with the incidence of JIA ( P < 0.05), that is, the higher the detection factor level, the higher the incidence of the disease, as shown in Table 3.

Table 3

Spearman correlation coefficient.
Indicators	JIA
Indicators	r	P
HMGB1(ng/ml)	0.712	< 0.001
β1 globulin(mg/L)	0.743	< 0.001
TLR-4	0.691	< 0.001

4.4 ROC curve analysis of the diagnostic value of single and combined tests for JIA

The diagnostic efficacy of each indicator is shown in Table 3. ROC curve shows that compared with the single detection, the combined detection of HMGB1, β1 globulin and TLR-4 had higher diagnostic efficacy (all P < 0.05), as shown in Table 4 and Fig. 2.

Table 4

Diagnostic efficacy of each index for JIA.
indicators	95%CI	P	sensitivity(%)	specificity(%)	AUC	Cutoff value
joint detection	0.786 ~ 0.928	< 0.001	85.40%	86.00%	0.857	0.836
HMGB1	0.712 ~ 0.886	< 0.001	81.70%	82.00%	0.799	0.786
β1 globulin	0.767 ~ 0.908	< 0.001	74.40%	78.00%	0.838	0.795
TLR-4	0.734 ~ 0.891	< 0.001	78.90%	79.00%	0.813	0.783

4.5 Comparison of the serum factor levels between good group and the bad group

The results show that the levels of all indicators in the good group are significantly lower than those in the bad group (P < 0.05), as shown in Table 5 and Fig. 3.

Table 5

Comparison of serum factors in children with different prognostic outcomes.
	Good group(n = 39)	Bad group(n = 43)	t	P
HMGB1(ng/ml)	18.31 ± 1.04	25.89 ± 1.52	-26.155	< 0.001
β1 globulin(mg/L)	2.71 ± 0.83	3.11 ± 1.03	-2.164	0.033
TLR-4	1.05 ± 0.07	1.10 ± 0.09	-3.345	0.001

4.6 ROC curve analysis of the prognostic value of single and combined tests for JIA

The diagnostic efficacy of each indicator is shown in Table 5. ROC curve shows that compared with single detection, the combined detection of HMGB1, β1 globulin and TLR-4 had higher diagnostic efficacy for prognosis assessment (all P < 0.05), as shown in Table 6 and Fig. 4.

Table 6

Diagnostic efficacy of each index for JIA prognosis.
Indicators	95%ci	P	Sensitivity(%)	Specificity(%)	Auc	Cutoff value
joint detection	0.780 ~ 0.952	< 0.001	86.00%	87.20%	0.866	0.839
HMGB1	0.609 ~ 0.837	0.001	81.40%	75.90%	0.723	0.804
β1 globulin	0.508 ~ 0.750	0.044	69.80%	67.00%	0.629	0.618
TLR-4	0.531 ~ 0.771	0.019	68.50%	66.80%	0.651	0.632

In this study, the expression levels of high mobility group protein B1 (HMGB1), β1 globulin and Toll-like receptor 4 (TLR-4) in serum of children with rheumatoid arthritis (JIA) are investigated. All the children in the study group are treated in the same way and followed up for 6 months. At the end of the follow-up, the serum factors are detected again, and the results show that the levels of each factor in the poor group are significantly higher than that in the good group (P < 0.05). Moreover, ROC curve shows that HMGB1 combined with β1 globulin and TLR-4 level detection have high diagnostic efficacy in evaluating the prognosis of children with JIA. The immune system gradually reduces the secretion of globulin. Thus, the expression of β1 globulin, HMGB1, TLR 1–4 in children with JIA expression level in the serum are significantly higher than those of healthy children. Obviously, joint detection has high application value for early diagnosis of suspected JIA children. Meanwhile, the prognosis of these diseases can be evaluated by detecting the levels of the above factors, which is of great significance for the diagnosis and improvement of the prognosis of children. Also, there are still some shortcomings. Firstly, the sample size is too small, which should be further expanded in the next study to make the research results more representative. In addition, there are relatively few studies on the level of HMGB1 combined with β1 globulin and TLR-4 in children with JIA at the present stage. Thus, it needs to be further verified by other scholars to make the results more rigorous.

Declarations

Ethics approval and consent to participate

All methods were carried out in accordance with relevant guidelines and regulations. The study was approved by the Capital Institute of Pediatrics’s ethics committee.

Declarations

Consent to publish

Not applicable.

Declarations

Competing interests

The authors declare that they have no competing interests.

Funding

Not applicable.

Authors Contribution

Wei Wang: contributed to the conception of the study, Yuanyuan Li: performed the experiment, Feng He: performed the data analyses and wrote the manuscript. All authors reviewed the manuscript.

Acknowledgement

None.

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1. Background

Rheumatoid arthritis in children, also known as JIA, is a connective tissue disease experienced by children in childhood. The disease is mainly characterized by chronic arthritis, which is often accompanied by multiple organ damage [1]. According to the data, the global incidence rate of JIA is about 0.007%~0.401%. In terms of the location of the disease, the disease can be divided into systemic type, multi joint type and mild arthritis type, which is easy to cause disability in children. Therefore, early prevention and management of such diseases is of great significance to the healthy growth of children [2]. However, there is no study to clarify the specific pathogenesis of JIA. According to most studies, different types and expression levels of serum factors in children with JIA affect the occurrence and type of disease [3]. As a highly conserved nuclear protein, HMGB1 widely exists in mammalian cells and plays an important role in the pathogenesis of sepsis, tumors and other diseases. As a kind of serum protein with immune function, globulin has many types. When the immune system is damaged, β1 Globulin can secrete a large amount of globulin and resist invasion by stimulating the lymphatic system [4, 5]. As a ligand of HMGB1, TLR-4 can activate immune cells and carry out immune inflammatory reaction in vivo [6]. At present, the commonly used factors to measure JIA include Tumor Necrosis Factor α(TNF-α)ཤ Interleukin (IL), etc. However, HMGB1, β1 globulin and TLR-4 had relatively little effect on JIA. Therefore, this study detected the above-mentioned serum factors in children with JIA to evaluate their impact and relationship on the development of such diseases, and provided a new method for the diagnosis of JIA, which will be reported as follows [7].

The diagnosis of rheumatoid arthritis depends on the comprehensive results of many aspects. The rheumatoid arthritis classification standard revised by the American rheumatology society is based on the following clinical symptoms [8]. If there are 4 or more clinical symptoms, and the clinical manifestations of other arthritis are excluded, rheumatoid arthritis can be diagnosed. These clinical manifestations are: (1) Morning stiffness is at least 1 hour, and the continuous time span is ≥ 6 weeks; (2) If 3 or more joints are involved, the time should be ≥ 6 weeks; (3) Wrist, metacarpophalangeal joint or proximal interphalangeal joint involvement ≥ 6 weeks; (4) The duration of symmetrical arthritis was ≥ 6 weeks; (5) There are rheumatoid subcutaneous nodules; (6) X-ray changes; (7) The serum rheumatoid factor was positive.

This paper is organized as follows: Section 2 discusses the related work, and Section 3 presents the proposed methods and observation indicators. In section 4, the comparative results and analysis is proposed. Finally, in Section 5, some concluding remarks are made.

We confirmed that we obtained informed consent from all subjects and/or their legal guardian(s).

No competing interests reported.

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Version 1

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A retrospective analysis and clinical application value of the expression levels of high mobility group protein B1, β1 globulin and Toll-like receptor 4 in serum of children with rheumatoid arthritis

Status:

Version 1

Abstract

Methods

Results

Conclusion

2. Related work

3. Proposed methods and observation indicators

3.1 General Information

3.2 Proposed methods

3.2.1 Enzyme-Linked Immunosorbent Assay (ELISA) is used to detect serum HMGB1

3.2.2 TlR-4 expression is detected by flow cytometry

3.2.3 Detection of serum β1 globulin

3.2.4 Prognosis assessment and grouping

3.3 Observation indicators

3.4 Statistical Processing

4. Comparative results and analysis

4.1 Analysis of serum indexes of JIA children

4.2 Comparison of serum factor levels between study group and control group

4.4 ROC curve analysis of the diagnostic value of single and combined tests for JIA

4.5 Comparison of the serum factor levels between good group and the bad group

4.6 ROC curve analysis of the prognostic value of single and combined tests for JIA

5. Conclusions

Declarations

Declarations

Declarations

Declarations

Funding

Authors Contribution

Acknowledgement

References

Unsectioned Paragraphs

Additional Declarations

Status:

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