Homocysteine as Graves eye disease biomarker and application thereof

Abstract

The invention discloses homocysteine as a Graves eye disease biomarker and application thereof, the biomarker is homocysteine (Hcy), application of a reagent for detecting the biomarker in preparation of a product for diagnosing Graves eye diseases, and the reagent is used for detecting the level of Hcy in sample serum. By detecting the level of homocysteine (Hcy) in serum, an objective and quantitative biomarker is provided for diagnosis of Graves eye diseases (GO), the diagnosis accuracy is remarkably improved, and the defects that existing clinical activity scores are high in subjectivity and insensitive to latent patients are overcome; the Hcy level is in positive correlation with GO activity, and can be used for monitoring disease progression and treatment effect, optimizing disease management, reducing serious complications and improving life quality of patients.

Description

Technical Field

[0001] The present invention relates to the field of medical technologies, and particularly relates to homocysteine as a biomarker for Graves' ophthalmopathy and its applications. Background Art

[0002] Graves' ophthalmopathy (GO) is an autoimmune disease associated with thyroid dysfunction, usually occurring in patients with hyperthyroidism. Approximately 25%-50% of patients with Graves' disease (GD) will develop GO. This disease is divided into an active phase and an inactive phase. The active phase is characterized by inflammation and vasodilation, resulting in red, swollen, and painful eyes; the inactive phase is mainly characterized by fibrosis, causing painless restriction of eye movement. In severe cases, GO can lead to compressive optic neuropathy or corneal ulcer, and even vision loss. Currently, although the clinical activity score (CAS) is widely used to evaluate the activity of GO, it is highly subjective and lacks sensitivity to subclinical patients. The treatment of GO mainly focuses on symptom control, lacks effective therapies targeting the etiology, and early identification of active and severe GO remains a clinical challenge.

[0003] Studies have shown that orbital fibroblasts (OFs) are key cells in the pathogenesis of GO, and their overactivation can lead to proliferation, increased secretion of inflammatory mediators, and excessive production of extracellular matrix. Multiple cytokines and growth factors can activate OFs, and among them, the Akt signaling pathway plays an important role in OFs-mediated adipogenesis, but the specific mechanism by which it regulates the proliferation and migration of OFs in GO is still unclear.

[0004] Homocysteine (Hcy) is a sulfur-containing amino acid, which is related to cardiovascular diseases, inflammation, and autoimmune diseases. In hypothyroidism, the level of thyroid peroxidase antibody is related to the level of Hcy. In addition, polymorphisms of methylenetetrahydrofolate reductase (MTHFR) that affect Hcy metabolism are associated with the susceptibility to GD and GO. Hcy can affect multiple signaling pathways, including the PI3K / Akt pathway, and studies have found that Hcy promotes the proliferation and migration of multiple cells by activating the Akt signaling pathway. However, the role of Hcy in GO has not been clarified.

[0005] Based on the above research, the present application proposes that Hcy may activate OFs through the Akt signaling pathway, thereby promoting the progression of GO. This study aims to compare the Hcy levels in the sera of GD patients and GO patients, analyze the correlation between Hcy levels and CAS, and explore the effect of Hcy on the activation of OFs and its potential mechanism, in order to provide new ideas and methods for the diagnosis and treatment of GO. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide homocysteine as a biomarker for Graves' ophthalmopathy and its application, to solve the problems of inaccurate diagnosis and lack of effective treatment means for Graves' ophthalmopathy (GO), and to provide an objective diagnosis method and a new treatment strategy for reducing Hcy levels by discovering the potential of homocysteine (Hcy) as a biomarker.

[0007] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0008] Use of a reagent for detecting a biomarker in the preparation of a product for diagnosing Graves' ophthalmopathy (GO), wherein the biomarker is homocysteine (Hcy).

[0009] Preferably, in the above technical solution, the reagent is used to detect the level of Hcy in a sample serum.

[0010] Preferably, in the above technical solution, the product includes a kit, a test strip, and a protein chip.

[0011] Preferably, in the above technical solution, the detection method is enzyme-linked immunosorbent assay (ELISA) or other quantitative detection methods.

[0012] A kit for detecting the level of homocysteine (Hcy) in serum, the kit including an antibody or antigen for detecting the Hcy level and a detection reagent.

[0013] Preferably, in the above technical solution, the detection reagent is an enzyme-linked immunosorbent assay (ELISA) reagent.

[0014] Preferably, in the above technical solution, the kit further includes a standard product for calibrating the detection result and an operation manual.

[0015] A method for detecting the level of homocysteine (Hcy) in serum using the above kit, comprising the following steps:

[0016] (1) Collect a serum sample of the patient;

[0017] (2) Use the kit to detect the level of Hcy in the serum;

[0018] (3) Compare the detected Hcy level with the normal reference value or the serum Hcy level of patients with Graves' disease (GD). If the Hcy level is significantly higher than the normal reference value or the serum Hcy level of GD patients, it indicates that the patient may have Graves' ophthalmopathy (GO) or GO is in the active stage.

[0019] A pharmaceutical composition for treating Graves' ophthalmopathy (GO), the pharmaceutical composition contains an active ingredient capable of reducing the level of homocysteine (Hcy) in the serum.

[0020] The above technical solution of the present invention has the following beneficial effects:

[0021] (1) Improved diagnostic accuracy: By detecting the level of homocysteine (Hcy) in the serum, it provides an objective and quantitative biomarker for the diagnosis of Graves' ophthalmopathy (GO), making up for the deficiencies of the existing clinical activity score (CAS) being highly subjective and insensitive to subclinical patients, and helping to more accurately identify GO patients, especially those in the active stage.

[0022] (2) Optimization of disease monitoring and management: The Hcy level is positively correlated with the activity of GO, which can be used to monitor the disease progression and treatment effect, providing a basis for clinicians to adjust the treatment plan, thereby optimizing the management of GO, reducing the occurrence of serious complications, and improving the quality of life of patients.

[0023] (3) Provision of new treatment targets: It reveals the role of Hcy in the pathogenesis of GO, providing new targets for the development of therapeutic drugs to reduce the Hcy level, and is expected to fill the gap that the existing treatments mainly focus on symptom control and lack effective therapies for the cause, bringing a breakthrough in the treatment of GO.

[0024] (4) Standardization of detection tools: The provided kits and detection methods are standardized and easy to operate, and are easy to promote and use in clinics and laboratories, helping to achieve early diagnosis and standardized treatment of GO, and having good application prospects and potential economic value. Brief Description of the Drawings

[0025] The drawings incorporated in and forming a part of the specification illustrate embodiments of the present invention and, together with the description, are used to explain the principles of the present invention.

[0026] Figure 1.Significant changes in serum homocysteine (Hcy) levels in patients with Graves' ophthalmopathy (GO). A total of 63 patients with Graves' disease (GD) and 68 patients with Graves' ophthalmopathy (including 28 patients with active GO and 48 patients with inactive GO) were included in this study. A, Serum Hcy levels in patients with GD (n = 63) and patients with GO (n = 68). Data are expressed as mean ± standard deviation. Compared with the GD group, ****P < 0.0001. GD: Graves' disease; GO: Graves' ophthalmopathy; Hcy: homocysteine. B, Comparison of Hcy concentrations in patients with active (n = 28) and inactive (n = 40) GO. Data are expressed as mean ± standard deviation. Compared with the GD group, ****P < 0.0001.

[0027] Figure 2 .The serum Hcy level in patients with GO is positively correlated with the clinical activity score (CAS). Correlation between the serum Hcy level and CAS in patients with GO (n = 68). Spearman correlation analysis (two-sided) was used to evaluate the statistical significance. P < 0.05 was considered statistically significant. Abbreviations: Hcy: homocysteine; CAS: clinical activity score; r: Spearman rank correlation coefficient; GO: Graves' ophthalmopathy.

[0028] Figure 3 .Under both physiological and inflammatory conditions, Hcy promotes the proliferation of orbital fibroblasts (OFs). A, As described in the Materials and Methods section, human OFs were treated with different concentrations of Hcy or an equal volume of solvent (water) for 24 hours (h). Cell proliferation was evaluated using the CCK-8 method. The effect of Hcy on proliferation was expressed as relative cell viability. n = 5. Data are expressed as mean ± standard error. Compared with control group cells, **P < 0.01, ****P < 0.0001. B, Human OFs were treated with 20 ng / ml recombinant human tumor necrosis factor α (rHu TNFα) or an equal volume of solvent, and simultaneously combined with 2 mM Hcy or an equal volume of solvent for 24 hours. Cell proliferation was evaluated using the CCK-8 method. The effect of Hcy on TNFα-induced OFs proliferation was expressed as relative cell viability. n = 5. Data are expressed as mean ± standard error. Compared with control group + control group cells, ****P < 0.0001. Compared with rHu TNFα + control group cells, ##P < 0.01.

[0029] Figure 4. Under physiological and inflammatory conditions, Hcy promotes the migration of OFs. A - B, Human OFs were treated with Hcy or an equal volume of solvent (water) for 24 hours. The cell migration of OFs was evaluated using a scratch - wound healing assay. (A) Representative images of scratch - wound healing, (B) Quantitative analysis of migration activity. n = 3. Data are presented as mean ± SEM. Compared with control cells, **P < 0.01. C - D, Human OFs were treated with 20 ng / ml rHu TNFα or an equal volume of solvent, and simultaneously combined with 2 mM Hcy or an equal volume of solvent for 24 hours. The cell migration was evaluated using a scratch - wound healing assay. (C) Representative images of scratch - wound healing, (D) Analysis of migration activity. n = 3. Data are presented as mean ± SEM. Compared with control group + control group cells, ****P < 0.0001. Compared with rHu TNFα + control group cells, ##P < 0.01.

[0030] Figure 5 . Under physiological and inflammatory conditions, Hcy activates the Akt signaling pathway in human OFs. A, Human OFs were treated with Hcy or an equal volume of solvent (water) for 24 hours. The phosphorylation level of Akt was detected by Western blot (WB) assay. The upper figure is a representative WB image, and the lower figure is the quantitative analysis of the pAkt / Akt level. n = 3. Data are presented as mean ± SEM. Compared with control cells, *P < 0.05. B, Human OFs were treated with 20 ng / ml rHu TNFα or an equal volume of solvent, and simultaneously combined with 2 mM Hcy or an equal volume of solvent for 24 hours. The phosphorylation level of Akt was detected by WB assay. The upper figure is a representative WB image, and the lower figure is the quantitative analysis of the pAkt / Akt level. n = 3. Data are presented as mean ± SEM. Compared with control group + control group cells, ***P < 0.001, ****P < 0.0001. Compared with rHu TNFα + control group cells, ##P < 0.01. Detailed implementation manners

[0031] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that: Unless otherwise specifically stated, the relative arrangements of components and steps, numerical expressions, and numerical values set forth in these embodiments do not limit the scope of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials and reagents used, unless otherwise specified, can all be obtained from commercial sources. The equipment used in the experiments, unless otherwise specified, is well - known to those skilled in the art.

[0033] Example 1

[0034] (I) Materials and methods

[0035] (1) Ethical approval statement

[0036] This study was approved by the Ethics Committee of Beijing Tongren Hospital, Capital Medical University (Ethical number: TRECKY2016-003, TREC2020-XJS02). All patients and volunteers signed written informed consent forms.

[0037] (2) Study design and clinical sample collection

[0038] The diagnosis of GO was determined according to the guidelines of the European Group on Graves Orbitopathy (EUGOGO). Orbital CT or MRI examinations were performed to exclude any orbital space-occupying diseases. CAS is the most effective and widely used tool for evaluating the activity of GO. It consists of seven criteria, and each present item scores 1 point. When the total score ≥ 3 / 7, GO is classified as the active phase; when the total score < 3 / 7, it is the inactive phase. According to the 2016 guidelines of the American Thyroid Association (ATA), combined with diffuse goiter, elevated serum thyroxine (T4), and decreased thyroid-stimulating hormone (TSH) levels, and performing TRAb detection, GD was diagnosed clinically. All included GD and GO patients had hyperthyroidism and were initially treated with the antithyroid drug methimazole (Tapazole, Merck KGaA, Germany). Clinical and demographic data were collected, including gender, age, thyroid function, thyroid autoantibodies, and medical history. Exclusion criteria included age < 18 years or > 65 years, systemic diseases (diabetes, stroke, heart disease, renal or liver dysfunction, and cancer), history of gastrointestinal surgery, pregnancy and lactation, and alcohol or drug addiction.

[0039] In the ELISA study, a total of 131 serum samples were analyzed, including 63 from GD patients and 68 from GO patients (40 non-active patients and 28 active patients). Orbital connective tissue explants were taken from the surgical waste of patients who underwent orbital decompression surgery due to severe GO at Beijing Tongren Hospital, Capital Medical University (n = 4). Control orbital tissues (n = 5) were taken from the surgical waste of patients without thyroid or inflammatory diseases who underwent cosmetic surgery. This study was approved by the Ethics Committee of Beijing Tongren Hospital, Capital Medical University.

[0040] (3) ELISA detection of serum Hcy

[0041] Serum samples were collected and stored at -80 °C until analysis. The levels of Hcy were measured using a commercially available enzyme-linked immunosorbent assays (ELISA) kit according to the manufacturer's instructions. The levels of Hcy were measured using a human Hcy (Cat. No.: CED984Ge) ELISA kit (Cloud-Clone Corp, Houston, TX, USA). The detection limit was <40.22 ng / ml. This kit was designed to detect human serum or plasma samples, and no cross-reactivity was reported.

[0042] (4) Cell culture and reagents

[0043] A human OFs culture system was established from orbital connective tissue explants according to the literature method. Briefly, the orbital connective tissue explants were washed three times with phosphate-buffered saline (PBS), immediately cut into small pieces after collection, and directly placed into a culture dish. After adherence, the explants were immersed in DMEM / F12 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 20% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% penicillin / streptomycin (Thermo Fisher Scientific, USA). Cells were cultured in a humidified environment of 37 °C, 95% air and 5% CO 2 . OFs usually migrated out from the explants within about 4 days and reached confluence within about 10 days. Then the cells were passaged with 0.25% trypsin / EDTA (Gibco Laboratories, New York, USA). After centrifugation at 300 × g for 5 minutes at room temperature, the supernatant was discarded, and the cell pellet was resuspended in 10 mL of DMEM / F12 medium containing 10% FBS and 1% penicillin / streptomycin. The cell suspension was filtered through a 70-μm cell strainer to establish a cell line. After the first passage, OFs were cultured in DMEM / F12 medium containing 10% FBS and 1% penicillin / streptomycin, and the medium was changed every 2 - 3 days. The 3rd - 6th passage cells were used in the experiment. Hcy (Cat. No.: GC60991) was purchased from GlpBio, California, USA. Human recombinant protein TNFα (rHu TNFα) was purchased from Sangon Biotech Co., Ltd., Shanghai, China.

[0044] (5) Cell proliferation assay

[0045] Using the method described previously, the proliferation activity of OFs was detected using a Cell Counting Kit-8 (CCK-8; TransGen Biotech Co., Ltd., Beijing, China)

[25] . Briefly, OFs were seeded into 96-well plates, and when the cells reached 80% confluence, they were treated with different concentrations of Hcy or an equal volume of solvent (H 2 O) for 24 hours (h). The working concentrations of Hcy were 1 mM, 2 mM, and 4 mM. Cells treated with an equal volume of solvent served as the control group. To evaluate the effect of Hcy on proliferation under inflammatory conditions, OFs were treated with 20 ng / ml rHu TNFα and Hcy (2 mM) or an equal volume of solvent simultaneously for 24 h. At 0.5 - 1 h before the end of incubation, a CCK-8 solution with a final concentration of 10% (v / v) was added to each well of the 96-well plate and gently mixed. Then the plate was immediately returned to the incubator. After incubation for 0.5 - 1 h, the absorbance (A) was measured at 450 nm using the above-mentioned multifunctional reader, with a reference wavelength of 630 nm. The proliferation activity of OFs was expressed as relative cell viability, and the calculation formula was as follows: relative cell viability = (experimental group A450 / A630) / (control group A450 / A630). The experiment was repeated five times and at least three times. The Hcy stock solution was prepared with H 2 O and diluted to the required final concentration in the medium.

[0046] (6) Western blot assay

[0047] OFs were seeded into 6-well plates, and when the cells reached 80% confluence, they were treated with Hcy (2 mM) or an equal volume of solvent (H 2 O) for 24 h. To evaluate the effect of Hcy on the Akt signaling pathway under inflammatory conditions, OFs were treated with 20 ng / ml rHu TNFα and Hcy (2 mM) or an equal volume of solvent simultaneously for 24 h. After treatment and final incubation, the cells were washed twice with PBS and then harvested using RIPA lysis buffer. Western blot assays were performed according to the standard method described previously [26 - 28]. Anti-pAkt (4060S) and anti-Akt (9272S) were purchased from Cell Signaling Technology, Inc., USA. Anti-β-actin was purchased from Sigma-Aldrich, Inc., USA (A5316). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to quantitatively analyze the image intensity of each band. In the experiment, β-actin was used as a normalization control.

[0048] (7) Cell migration assay

[0049] Using the method described previously, the migratory activity of OFs was detected using a scratch wound healing assay

[29] . Briefly, OFs were seeded into 6-well plates and starved for 12 h in DMEM / F12 medium without FBS when the cells reached 95% confluence. A scratch was made in each well with a flat P200 micropipette tip, and then washed with 1×PBS to remove any debris. Bright-field images were taken at a specific position at time 0 as the baseline for analysis. Then the cells were treated with Hcy (2 mM) or an equal volume of solvent (H 2 O) for 24 h. To evaluate the effect of Hcy on cell migration under inflammatory conditions, OFs were treated with 20 ng / ml rHu TNFα and Hcy (2 mM) or an equal volume of solvent simultaneously for 24 h. After treatment and final incubation, images were taken at the same position at 24 h. Wound area analysis was performed using ImageJ software, and the area at each time point was normalized to the baseline area at the initial scratch time 0.

[0050] (8) Statistical analysis

[0051] IBM SPSS 27.0 software and GraphPad Prism 8.0.1 software were used to analyze demographic, clinical, and laboratory data. Continuous variables with a normal distribution were expressed as the mean ± standard error of the mean (SEM), while non-parametric variables were expressed as the median (interquartile range Q1, Q3). For comparisons between different groups, Student's t-test was used for two-group comparisons, and one-way ANOVA and Kruskal-Wallis tests were used for multi-group comparisons, respectively. P < 0.05 was considered statistically significant. For differential impact analysis, the Benjamini and Hochberg false discovery rate (BH) correction method was used to correct a large number of analyses to control the false positive rate. The correlations between all variables were determined by Spearman rank correlation and multivariate linear regression analysis.

[0052] (II) Results

[0053] (1) Serum homocysteine (Hcy) levels were significantly altered in patients with Graves ophthalmopathy (GO) compared with the Graves disease (GD) group

[0054] In this study, we detected the serum Hcy levels of 131 hyperthyroid patients, including 63 GD patients and 68 GO patients, using an enzyme-linked immunosorbent assay (ELISA) method.

[0055] The characteristics of the participants in this study are summarized in Table 1. There were no significant differences between the two groups in terms of free triiodothyronine (FT3), free thyroxine (FT4), and thyroid-stimulating hormone (TSH) levels. However, there were differences between the GD group and the GO group in terms of gender, age, and thyroid-stimulating hormone receptor antibody (TRAb) levels. ELISA results showed that the Hcy levels in patients with GO were significantly higher than those in the GD group ( Figure 1 Panel A). In addition, to explore whether there were differences in serum Hcy levels between active and inactive GO patients, we divided GO patients into an inactive group (n = 40) and an active group (n = 28) according to the clinical activity score (CAS) (using a CAS value of 3 as the cut-off, CAS < 3 for the inactive group, and CAS ≥ 3 for the active group). The research results showed that the serum Hcy levels in active GO patients were significantly higher than those in inactive patients ( Figure 1 Panel B). These results suggest that serum Hcy may be related to the progression of GO.

[0056] Table 1. Characteristics of participants at baseline [mean ± standard deviation, n(%), median (interquartile range)]

[0057]

[0058] In Table 1, abbreviations: GD: Graves' disease; GO: Graves' ophthalmopathy; FT3: free triiodothyronine; FT4: free thyroxine; TRAb: thyroid-stimulating hormone receptor antibody. Superscript letter a: significantly different from GD.

[0059] (2) The serum Hcy level in GO patients was positively correlated with the CAS of GO patients

[0060] CAS is the most commonly used index for staging the activity of GO based on typical inflammatory manifestations. To evaluate the association between Hcy levels and GO activity, we performed a Spearman correlation analysis on serum Hcy levels and CAS scores.

[0061] It is worth noting that there was a significant positive correlation between the serum Hcy level and the CAS value in GO patients ( Figure 2 ). In addition, multiple linear regression analysis showed that after adjusting for age and gender factors, Hcy was still an independent factor affecting CAS (Table 2). Taken together, these research results suggest that Hcy is positively correlated with disease severity, highlighting the importance of monitoring Hcy levels in GO patients.

[0062] Table 2. Multiple linear regression analysis of influencing factors of Hcy in GO patients

[0063]

[0064] In Table 2, abbreviations: Hcy: homocysteine; CAS: clinical activity score. Note: ***P < 0.001.

[0065] (3) Under both physiological and inflammatory conditions, Hcy stimulates the proliferation and migration of orbital fibroblasts (OFs).

[0066] Given the significant changes in serum Hcy levels in patients with GO, it was hypothesized that Hcy might play a key role in the pathogenesis of GO and could be a potential therapeutic target. To test this hypothesis, we performed a series of in vitro experiments using primary cultured human OFs. Existing studies have confirmed that there are no significant differences in the morphology and function of OFs after in vitro passage culture of orbital connective tissues from GO patients and healthy controls.

[0067] In this study, we further found that there were no significant differences in the proliferation and inflammatory responses of OFs after in vitro passage culture of orbital connective tissues from GO patients and healthy controls (data not shown). Based on the above findings, OFs derived from orbital connective tissues of healthy individuals were used in subsequent experiments of this study.

[0068] Existing studies have shown that Hcy can significantly promote the differentiation, proliferation and migration of primary cardiac fibroblasts, and induce the proliferation and phenotypic transformation of vascular smooth muscle cells (VSMCs). Given that cell proliferation and migration are key characteristics of OFs in GO patients, we first used the CCK-8 method to evaluate the effect of Hcy on cell proliferation. Under physiological conditions, the CCK-8 assay results showed that compared with the control group, cell proliferation increased in a dose-dependent manner after Hcy treatment ( Figure 3 Figure A). Further experiments showed that recombinant human tumor necrosis factor α (rHu TNFα) could significantly induce the proliferation of OFs ( Figure 3 Figure B). Notably, Hcy could significantly exacerbate the rHu TNFα-induced proliferation of OFs ( Figure 3 Figure B).

[0069] Subsequently, the scratch assay results showed that under physiological conditions, compared with the control group, cell migration increased significantly after Hcy treatment ( Figure 4 Figure A). Further experiments showed that rHu TNFα could significantly induce the migration of OFs ( Figure 4 Figure B). Interestingly, Hcy could significantly exacerbate the rHu TNFα-induced migration of OFs ( Figure 4 Figure B). These research results indicate that targeting Hcy may be a promising strategy for the treatment of GO by improving the proliferation and migration of OFs.

[0070] (4) Under physiological and inflammatory conditions, Hcy activates the Akt signaling pathway in human OFs

[0071] The Akt signaling pathway has been demonstrated to play a key role in adipogenesis, cell migration, proliferation, and inflammation in GO. Studies have shown that phosphorylation of Akt at serine 473 (phospho-Akt, pAkt), a key marker of PI3K / Akt activation, is elevated in OFs from GO patients and is considered an important factor in the pathogenesis of GO. It has been reported that Hcy can activate the Akt signaling pathway in various cell types. Therefore, to explore the potential mechanism by which Hcy induces OFs proliferation and migration, we investigated its effect on the Akt signaling pathway. Notably, Hcy treatment significantly promoted the activation of the Akt pathway, as evidenced by an increase in the level of pAkt ( Figure 5 Panel A). In addition, Hcy enhanced TNFα-induced Akt phosphorylation ( Figure 5 Panel B). These findings indicate that the Akt signaling pathway plays an important role in the pathogenic effect of Hcy on OFs from GO patients.

[0072] (III) Discussion

[0073] It should be emphasized that elevated serum Hcy levels may serve as a new biomarker for differentiating GD patients with and without GO, even when their thyroid functions are matched. In addition, this study showed that serum Hcy levels were elevated in active GO patients compared with those in inactive GO patients, suggesting a possible link between Hcy and orbital inflammatory activity. Moreover, under physiological and inflammatory conditions, Hcy stimulates OFs proliferation and migration, indicating that it may play a role in the pathogenesis of GO. From a mechanistic perspective, under these conditions, Hcy activates the Akt signaling pathway in OFs, further supporting the view that it is involved in the progression of GO.

[0074] GO is a multi-factorial autoimmune disease with a range of symptoms and signs, and about 2-3% of GO patients will develop severe disease. In such cases, vision loss may occur due to corneal ulcer or optic neuropathy, and orbital decompression surgery is usually required. Therefore, GO has a significant negative impact on the quality of life of patients. Preventive strategies, including the elimination of modifiable risk factors, may help reduce the prevalence of clinically significant GO and potentially shift the disease burden towards the milder end described above. Previous studies have identified several factors associated with the development and progression of GO in GD patients, including CAS, TRAb levels, duration of hyperthyroidism, and smoking. Although CAS is widely used for disease assessment, it has limitations. CAS includes two subjective components, spontaneous pain and gaze-induced pain, making it less objective, and it is evaluated in a binary manner (present or absent) without a grading standard. Therefore, there is still a lack of a more sensitive and objective biomarker for assessing different stages of GO. Previous studies reported that Hcy levels were lower in GD patients, and there was a positive and independent association between Hcy and thyroid peroxidase antibody in GD patients. However, no study has elucidated the changes in Hcy in GO patients, especially in different disease stages. This study shows for the first time that in the case of hyperthyroidism, the serum Hcy level in GO patients is significantly higher than that in GD patients. More interestingly, the serum Hcy level in active GO patients is significantly higher than that in inactive patients. In addition, our analysis shows a significant positive correlation between Hcy levels and CAS, indicating that elevated Hcy levels may be a potential risk factor for the progression of active GO.

[0075] Hcy has been shown to be crucial in maintaining redox homeostasis and is involved in many physiological and pathological processes. Recent studies have shown that Hcy treatment promotes the differentiation, proliferation, and migration of various cell types. In this study, we revealed for the first time that Hcy stimulates the proliferation and migration of OFs under both physiological and inflammatory conditions. These findings support the view that targeting Hcy may be a new key strategy for treating GO.

[0076] The Akt signaling pathway has been confirmed to play a key role in adipogenesis, cell migration, proliferation, and inflammation in GO. In addition, Akt is abnormally activated in OFs from GO patients, which is considered an important factor in the pathogenesis of GO. However, the underlying mechanism of Akt activation remains incompletely understood. In this study, we confirmed for the first time that Hcy activates the Akt signaling pathway in human OFs under both physiological and inflammatory conditions. Importantly, our data provide strong evidence that Akt activation promotes the progression of GO disease. Our results complement the view that blocking the Akt signaling pathway may have therapeutic benefits for GO.

[0077] In summary, our study demonstrated for the first time that the serum Hcy level in GO patients was significantly higher than that in GD patients without GO. Notably, the Hcy concentration in active GO patients was significantly higher than that in inactive patients. Our analysis revealed a significant positive correlation between circulating Hcy levels and CAS. In addition, Hcy promoted the proliferation and migration of OFs by activating the Akt signaling pathway. Taken together, these findings suggest that elevated Hcy levels may be a new potential risk factor for the progression of active GO, and targeting Hcy may be a promising strategy for the treatment of GO.

[0078] Although the present invention has been disclosed above by way of examples, it is not intended to limit the present invention. Any person skilled in the art can make various different selections and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is defined by the claims and their equivalent forms.

Claims

1. Use of a reagent for detecting a biomarker in the preparation of a product for diagnosing Graves' ophthalmopathy (GO), characterized in that: The biomarker is homocysteine ​​(Hcy).

2. The use according to claim 1, characterized in that: The reagent is used to detect the level of Hcy in sample serum.

3. The use according to claim 1, characterized in that: The products include test kits, test strips, and protein chips.

4. The use according to claim 1, characterized in that: The detection method is enzyme-linked immunosorbent assay (ELISA) or other quantitative detection methods.

5. A kit for detecting homocysteine ​​(Hcy) levels in serum, characterized in that: The kit comprises an antibody or antigen for detecting the Hcy level and a detection reagent.

6. The kit according to claim 5, characterized in that The detection reagent is an enzyme-linked immunosorbent assay (ELISA) reagent.

7. The kit according to claim 5, characterized in that The kit also includes a standard substance for calibrating the test results and an operating instruction.

8. A method for detecting homocysteine ​​(Hcy) levels in serum using the kit according to any one of claims 5 to 7, characterized in that: The following steps are involved: (1) Collect serum samples from patients; (2) using the kit to detect the level of Hcy in serum; (3) The detected Hcy level is compared with the normal reference value or the serum Hcy level of Graves' disease (GD) patients. If the Hcy level is significantly higher than the normal reference value or the serum Hcy level of GD patients, it indicates that the patient may have Graves' ophthalmopathy (GO) or GO is in the active progressive stage.

9. A pharmaceutical composition for treating Graves' ophthalmopathy (GO), characterized in that: The pharmaceutical composition comprises an active ingredient capable of reducing the homocysteine ​​(Hcy) level in serum.

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