Background. Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes mellitus (DM). A recent in vitro study found that the suppressor of cytokine signaling 6 (SOCS6) plays a protective role in DR and DM. However, to date, no clinical studies have focused on the role of SOSC6 in DR development.
Objectives. The present study aimed to investigate the expression and clinical significance of serum SOCS6 in DR.
Materials and methods. A total of 159 DR patients were enrolled in the study. Additionally, 156 type 2 DM (T2DM) patients without DR were recruited as controls. Serum levels of SOCS6, C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), vascular endothelial growth factor (VEGF), and angiopoietin-2 (ANG-2) were measured using enzyme-linked immunosorbent assay (ELISA). Demographic and clinical data were collected.
Results. Age, the course of DM, systolic blood pressure (SBP), diastolic blood pressure (DBP), and the levels of low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) were significantly higher in proliferative DR (PDR) patients. Serum SOCS6 levels in PDR patients were remarkably lower than in non-PDR patients or non-DR T2DM patients. The Pearson’s analysis showed that SOCS6 was negatively correlated with CRP, IL-6, TNF-α, IL-1β, VEGF, and ANG-2. The serum levels of CRP, IL-6, TNF-α, IL-1β, VEGF, and ANG-2 in the SOCS6 low expression group were significantly increased compared to patients with high SOCS6 levels. Receiver operating characteristic (ROC) curves showed that SOCS6 could be a potential diagnostic biomarker for DR. For logistic regression, 3 models were used. It was found that SOCS6, the course of DM, SBP and DBP in model 1, IL-1β and TNF-α in model 2, and VEGF and ANG-2 in model 3 were risk factors for DR.
Conclusions. The SOCS6 is decreased in DR patients and is related to severity and clinical outcomes, including inflammatory and angiogenic factors.
Key words: inflammatory factors, angiogenic factors, SOCS6, diabetic retinopathy
Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia. Type 2 DM (T2DM) is currently the most common type of the disease, accounting for 95% of total DM cases.1, 2 In 2021, DM was estimated to affect 10.5% (536.6 million) of individuals aged 20–79 years old, worldwide.3 China is the country with the greatest number of DM cases in the world. Over the past 30 years, the prevalence of DM in China has increased by about 10 times.4, 5
Diabetic retinopathy (DR) is one of the most common microvascular complications of DM, with a global prevalence rate of about 27%.6 Diabetic retinopathy is divided into 2 types, namely nonproliferative DR (NPDR) and proliferative DR (PDR), which are classified from low to high, according to severity.7 Diabetic retinopathy is one of the main causes of visual impairment and blindness in adults, with studies reporting that DR accounts for 4.8% of cases of blindness around the world.8 Many risk factors are reported to be associated with DR, including DM, increased blood pressure, elevated blood glucose levels, and impaired kidney function.9 However, deeper insights are still needed to predict DR onset and clinical outcomes.
As a member of the suppressor of cytokine family of proteins, suppressor of cytokine signaling 6 (SOCS6) is generally considered to regulate bacterial infection-induced inflammation.10 Studies have suggested that SOCS6 can regulate a variety of physiological and pathological processes, including inflammation, cell proliferation, apoptosis, and angiogenesis.11, 12, 13 In the pathogenesis of DR, it is thought that neuroinflammation, oxidative stress and vascular-related risk factors play central roles.14, 15, 16 A recent in vitro study found that SOCS6 plays a protective role in DR and DM.12 However, to date, no clinical studies have focused on the role of SOSC6 in the development of DR.
In this prospective observational study, we aimed to explore the expression of SOCS6 in DR patients and to examine its correlation with clinical results. The study may reveal the clinical significance of SOCS6 in patients with DR and provide novel targets for DR treatment.
Materials and methods
A total of 159 DR patients who were admitted to Jiangxi Provincial People’s Hospital between March 2019 and December 2021 were enrolled in the study. The diagnosis of T2DM was in line with the guidelines for the prevention and treatment of T2DM in China (2017 edition),17 including 1) typical diabetes symptoms (polydipsia, polyuria, hyperphagia, unexplained weight loss) and 2) fasting plasma glucose concentration higher than 7.0 mmol/L, plasma glucose concentration higher than 11.1 mmol/L, or oral glucose tolerance test 2-hour plasma glucose concentration higher than 11.1 mmol/L. Diabetic retinopathy was diagnosed with fundus photography with dilated pupils using a retinograph (CR-2 AF Digital Retinal Camera; Canon Inc., Tokyo, Japan), and all patients were diagnosed and classified according to the international clinical DR severity scales.18
The criteria for inclusion were as follows: 1) patients diagnosed with DR according to the above guidelines; and 2) patients ≥18 years old. The exclusion criteria were: 1) diagnosis of type 1 DM, gestational DM or another special type of DM; 2) presence of diabetic ketoacidosis, hyperosmolar nonketotic diabetic coma or other acute DM complications; 3) presence of cataracts, glaucoma, uveitis, retinal detachment, optic nerve disease, high myopia, and other diseases significantly affecting the fundus examination; and 4) presence of serious infection, malignancy, or severe liver, renal or cardiovascular dysfunction. All patients who met the above criteria were consecutively enrolled.
The DR patients were further divided into: 1) patients with no fundus abnormalities; 2) NPDR patients, including patients with microaneurysms, hard exudates, retinal hemorrhages, and intraretinal microvascular abnormalities; and 3) PDR patients, including patients with neovascularization, fibrous tissue or hemorrhages in the vitreous body. Additionally, 156 T2DM patients without retinopathy were recruited during the same period. The inclusion and exclusion criteria for T2DM patients were the same as the diagnostic criteria for T2DM listed above.
For sample size calculations, the following formula2 was used1, 2 (Equation 1):
According to previous studies, δ = 2, σ = 7 and Z1−α/2 = 1.96 (δ – tolerance error; σ – standard deviation, 1–α – confidence level), and the minimal sample size was 48. All patients signed a written informed consent form. The study was approved by the Ethical Committee of Jiangxi Provincial People’s Hospital (approval No. 2021007).
Blood sampling and ELISA
Serum levels of SOCS6, C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), vascular endothelial growth factor (VEGF), and angiopoietin-2 (ANG-2) were measured using enzyme-linked immunosorbent assay (ELISA). Briefly, fasting cubital venous blood (5 mL) was collected from all patients within 24 h of admission. The blood samples were centrifuged at 2000 g for 15 min. After centrifugation, the levels of SOCS6, CRP, IL-6, TNF-α, IL-1β, VEGF, and ANG-2 were measured using commercially available ELISA kits (SOCS6: MBS2610988 (MyBioSource, San Diego, USA), detection range 0.156–10 ng/mL; CRP: EK1316, detection range 312–20000 pg/mL; IL-6: EK0410, detection range 4.69–300 pg/mL; TNF-α: EK0525, detection range 15.6–1000 pg/mL; IL-1β: EK0392, detection range 3.9–250 pg/mL; VEGF: EK0539, detection range 31.2–2000 pg/mL; ANG-2: EK0657, detection range 156–10000 pg/mL), according to the manufacturer’s (unless stated otherwise: Boster Bio-Engineering, Wuhan, China) instructions.
Patient data collection
Demographic and clinical data were collected from all patients, including age, body mass index (BMI), sex, the course of DM, systolic blood pressure (SBP), diastolic blood pressure (DBP), use of tobacco, etc. Whole blood testing was performed using an automatic biochemical analyzer (Hitachi 7600; Hitachi Corp., Tokyo, Japan), and the levels of fasting plasma glucose (FPG), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were recorded.
Continuous data are presented as mean ± standard deviation (M ±SD) for normally-distributed data or median (minimum to maximum) for non-normally-distributed data (confirmed with Kolmogorov–Smirnov test). For non-normally distributed data, Mann–Whitney U test was used for comparisons between the 2 groups. For normally distributed data, comparisons between the 2 groups were conducted using Student’s t-test. The χ2 test was used to analyze the rates. The correlations among SOCS6, inflammatory factors and angiogenic factors were determined with Pearson’s rank correlation analysis. Receiver operating characteristic (ROC) curves were used for the analysis of SOCS6 in DR patients. The logistic regression was conducted for DR risk factors. The Hosmer–Lemeshow test and the Nagelkerke’s R2 were used for the goodness-of-fit analysis. A value of p < 0.05 was considered statistically significant. The SPSS v. 18.0 software (SPSS Inc., Chicago, USA) was used for all statistical analyses.
Basic patient characteristics
This study included 159 DR patients – 81 NPDR patients and 78 PDR patients. The course of DM was significantly longer, and the age, SBP, DBP, and the levels of LDL-C and TC were significantly higher in PDR patients compared to the NPDR patients (p < 0.05). Compared to the T2DM patients without retinopathy (n = 156), the course of DM was significantly longer, and the age, SBP, DBP, ratio of smokers, and the levels of LDL-C and TC were markedly higher in the DR patients (Table 1). No other significant differences were found.
Expression of SOCS6
Next, the expression levels of serum SOCS6 were determined. The DR patients showed significantly lower SOCS6 levels compared to the T2DM patients without retinopathy (Figure 1). In addition, the serum SOCS6 levels in PDR patients were remarkably lower than in NPDR patients, suggesting that SOCS6 is associated with the severity of DR.
Correlations between SOCS6 and angiogenic and inflammatory factors
The IL-6, TNF-α, IL-1β, VEGF, and ANG-2 levels in PDR patients were remarkably higher than in NPDR patients (Figure 2). Comparisons with T2DM patients without retinopathy showed that the CRP, IL-6, TNF-α, IL-1β, VEGF, and ANG-2 levels in DR patients were also pronouncedly higher. The Pearson’s analyses showed that SOCS6 levels correlated negatively with CRP, IL-6, TNF-α, IL-1β, VEGF, and ANG-2 (Table 2).
Correlations between serum SOCS6 levels and T2DM patients’ clinical outcomes
Next, all patients were divided into SOCS6 high-expression and low-expression groups according to the mean value (4.15 ng/mL), and the clinical characteristics between these groups were compared. As summarized in Table 3, the serum levels of IL-6, TNF-α, IL-1β, VEGF, and ANG-2 in the SOCS6 low-expression group were significantly higher compared to those in the SOCS6 high-expression group (p < 0.05). In addition, compared to the SOCS6 high-expression group, the course of DM was significantly prolonged, and the age SBP, DBP, as well as the levels of LDL-C and TC were significantly increased in the SOCS6 low-expression group. These results suggest that SOCS6 is associated with clinical outcomes and severity in T2DM patients.
Diagnostic value of SOCS6 and angiogenic and inflammatory factors for DR
The ROC curves were constructed to evaluate the diagnostic value of SOCS6 in DR patients. The results showed that SOCS6 could be a potential diagnostic biomarker for DR (Figure 3). The area under the curve (AUC) for SOCS6 was 0.871 and the cutoff value was 4.61 ng/mL, with a sensitivity of 85% and a specificity of 69%. The SOCS6 could also be used as a biomarker for NPDR or PDR patients. As shown in Figure 3, the AUC for SOCS6 was 0.825, with a cutoff value of 3.66 ng/mL, a sensitivity of 79%, and a specificity of 64%.
Logistic regression for risk factors for DR
Finally, we used the logistic regression to identify the risk factors for DR. For logistic regression, we used 3 models for the entry method. In model 1 (age, BMI, course of DM, SBP, DBP, TC, TG, HDL-C, LDL-C, and SOCS6), the results of the Hosmer–Lemeshow test (p = 0.139) and the Nagelkerke’s R2 (0.844) showed adequate goodness-of-fit, with SOCS6, the course of DM, SBP, and DBP identified as risk factors for DR (Table 4). In model 2 (inflammatory factors CRP, IL-6, TNF-α, and IL-1β), the Hosmer–Lemeshow test (p = 0.999) and the Nagelkerke’s R2 (0.981) showed adequate goodness-of-fit, and IL-1β and TNF-α were identified as risk factors for DR. In model 3 (angiogenesis factors VEGF and ANG-2), the Hosmer–Lemeshow test (p = 1.000) and the Nagelkerke’s R2 (0.981) showed adequate goodness-of-fit, with both VEGF and ANG-2 identified as risk factors for DR.
Globally, it is estimated that the number of DR cases will grow to 191 million by 2030, with vision-threatening DR patients numbering over 56 million.19 Despite the development of DR diagnostic and treatment strategies, DR is still the leading cause of blindness in working-age population. However, at present, DR is diagnosed using fundus photography with dilated pupils20 and lacks specific biomarkers. Thus, it is urgent to develop new biomarkers and comprehensive approaches to reduce the risk of vision loss by prompt diagnosis and early treatment of DR. In the present study, we showed that serum SOCS6 levels were decreased in DR patients and were associated with clinical outcomes and severity.
A number of studies have indicated that cytokines, angiogenic markers and some clinical biomarker changes may cause DR. For example, a prospective study by Muni et al. found that serum high-sensitivity CRP is significantly correlated with the risk for DR.21 In addition, a case-control study by Churchill et al. confirmed that VEGF is associated with the severity of DR.22 In the current study, we found that the inflammatory factors CRP, IL-6, IL-1β, and TNF-α were elevated in DR patients. These results are consistent with several previous studies.23, 24, 25 Changes in several clinical biomarkers are also associated with DR. For example, a 3-year prospective study in Taiwan showed that serum uric acid levels are associated with DR.9 Gao et al. reported that there may be a significant correlation between serum thyroglobulin antibody levels and the severity of DR.26 However, at present, there are no specific biomarkers for the diagnosis of DR.
The SOCS6 is an anti-inflammatory factor in many diseases, including DM. It has been previously reported that the inhibition of SOCS6 causes a significant increase in cell permeability and inflammation, and that overexpressing SOCS6 reverses cell permeability and the inflammatory response.27 Chen et al. found that the upregulation of lncRNA FGD5-AS1 could protect against periodontitis through enhancing the levels of SOCS6.11 Meng et al. showed that the knockdown of miR-16-5p could suppress cell proliferation and accelerate the apoptosis of fibroblast-like synoviocytes through targeting SOCS6.28 In addition, Xue et al. reported that miR-494-3p facilitated high glucose-induced renal fibrosis through the promotion of cell apoptosis and epithelial–mesenchymal transformation via targeting SOCS6.29 The SOCS6 is also correlated with angiogenic markers. Yuan et al. confirmed that the upregulation of SOCS6 inhibits angiogenesis and tumor xenograft growth, and is significantly associated with the prognosis of human prostate cancer.30
The SOCS proteins improve glucose metabolism, reduce the deleterious effects of inflammation and promote neuroprotection. Most of the known SOCS proteins are involved in the regulation of insulin resistance, β-cell failure and the eventual development of DM.31 Several studies have also focused on the molecular mechanistic effects of SOCS6 in DM. Luo et al. confirmed that SOCS6 could regulate inflammation, oxidative stress and apoptosis in retinal epithelial cells, and attenuate DR in a rat model.32 In addition, Liu et al. suggested that the constitutive expression of the SOCS6 protein in retinal neurons improves glucose metabolism in vivo and in vitro.33 Xiao et al. confirmed that MEG3 targets the miR-19b/SOCS6 axis in order to inhibit high glucose-induced human retinal microvascular endothelial cells apoptosis, while increasing the expression of SOCS6 and benefiting DR.12
As inflammation and vascular risk factors play important roles in the pathogenesis of DR, we inferred that SOCS6 may also influence DR development through the regulation of inflammation and angiogenesis. However, there is a lack of relevant reports regarding the role of SOCS6 in DR. In the present study, we found that the expression of serum SOCS6 is decreased in DR and DM patients, and is correlated with the severity of multiple clinical features, including inflammatory and angiogenic factors.
The present study has some limitations. First, we only included a small sample of the study population. In addition, we examined a relatively small number of inflammatory and angiogenic factors. Finally, the molecular mechanisms of SOCS6 that influence DR development were not examined.
This study showed that the serum levels of SOCS6 are decreased in DR patients. Serum SOCS6 levels were also related to DR severity and clinical outcomes, including inflammatory and angiogenic factors. Thus, this study provides more evidence for the role of SOCS6 in DR.
All data can be obtained from the corresponding author by reasonable request.