Building on our previous study, the present study innovatively developed a DR risk prediction model that integrates five key variables: Neutrophil, 25(OH)D3, Duration of T2DM, HbA1c and ApoA1. The model not only demonstrated excellent predictive ability, but also filled an important gap in previous studies through external validation. During the development and external validation phases of our study, the model achieved AUC values of 0.834 and 0.851, respectively, which is a significant advantage over previous models (0.709 and 704, respectively)20. The calibration curve and Hosmer–Lemeshow (H–L) test results further confirmed the high accuracy of the model in adjusting the actual and predicted probabilities of outcome events. In addition, decision curve analysis (DCA) demonstrated that implementation of the model in clinical practice yields significant net benefits, thus highlighting its practical relevance in the field of medical research.Our study reveals a significant positive correlation between ApoA1 and the severity of DR. Previous studies addressing the effect of ApoA1 on DR are scarce. However, recent studies have shown a positive correlation between high-density lipoprotein (HDL) and DR. Given that ApoA1 is the major structural protein of HDL, this finding provides some support for the hypothesis that ApoA1 may have an effect on DR. In a pivotal case–control study, Simo et al.21 compared ApoA1 expression in the retinas of diabetic and nondiabetic patients. It was found that overexpression of ApoA1 is an early phenomenon in the retinas of diabetic patients, which may be closely related to the pathophysiologic process of DR. In contrast, the study by Vinodhini et al.22 did not manage to establish a direct link between ApoA1 levels and DR severity. The widespread use of statins in the study and control groups may have contributed to this finding. In another study, Ankit and his team23 found that diabetic patients had higher levels of ApoA1 in vitreous fluid and retinal pigment epithelial cells compared to nondiabetic patients, which may increase the risk of developing diabetes. ApoA1 has an antifibrinolytic role in the atherosclerotic process and the plasma fibrinolytic system, and high levels of ApoA1 may lead to small-vessel occlusion24, which could promote the development of DR, which is consistent with the results of our study.In addition, we noted differences in the composition of ApoE and HDL. Apolipoproteins are not only structural proteins, but also have enzymatic activity and receptor binding. The cholesterol in HDL, on the other hand, actually refers only to the cholesterol lipid content in it. Past studies have often focused only on HDL and ignored the role of other important lipoproteins, which may have led to less comprehensive and rigorous scientific studies. Furthermore, the metabolic environment of diabetic patients is significantly different from that of healthy individuals. In the setting of diabetes, lipoproteins undergo non-enzymatic glycation, oxidation, and modification of advanced glycation end products, and these changes may have an impact on test results. In addition, the accuracy of lipoprotein measurements may be variable, with sample handling, non-enzymatic glycation and oxidation having varying effects on different assays.It is worth mentioning that the level of ApoA1 is more stable than the level of blood lipids, especially in the group of diabetes patients, and its level will not be significantly affected by the pre meal state. Therefore, compared with high-density lipoprotein and lipid indicators, apolipoprotein A1 may be a more accurate and reliable biomarker of diabetes retinopathy (DR).Vitamin D, a fat soluble vitamin, exists in various forms in the human body. Among them, serum 25 hydroxyvitamin D3 [25(OH)D3] is its main circulating form and is widely considered a key indicator for evaluating vitamin D levels. In recent years, the relationship between vitamin D and microvascular complications of type 2 diabetes has gradually received extensive attention from scholars at home and abroad. Among them, diabetes retinopathy (DR), as one of the major microvascular complications of diabetes, is particularly worth studying. In a meta-analysis of 17,664 type 2 diabetes patients12, the study found that the risk of DR was significantly increased in patients with vitamin D deficiency. In a cross-sectional study, Millen et al.25 divided 15,792 subjects into three groups based on their serum 25(OH)D3 levels: vitamin D sufficient, vitamin D insufficient, and vitamin D deficient. The results showed that the incidence of DR was higher in vitamin D deficient subjects, consistent with the results of this study, further demonstrating the correlation between serum vitamin D status and DR risk. This suggests that 25(OH)D3 may serve as an important serum marker for predicting DR, which can aid in early screening of DR patients. In addition, Jamali et al.14 found in vitro experiments on rats that mice carrying vitamin D receptors performed better in promoting pericellular density arrest and endothelial cell survival compared to mice without vitamin D receptors, and were able to inhibit ischemia mediated retinal neovascularization through vitamin D. Lazzara et al.13 also found in vitro experiments on human cells that supplementing vitamin D can rebuild the integrity of the blood retinal internal barrier by increasing the expression of VE cadherin and ZO-1 proteins, protecting human retinal endothelial cells from high glucose induced damage. These in vitro studies provide new insights into the role of vitamin D in retinal protection.Therefore, in the future health management of DR patients, attention should be paid to monitoring serum 25(OH)D3, and appropriate supplementation of vitamin D should be considered as one of the possible treatment strategies for preventing or improving DR.Diabetes duration is an important independent risk factor in DR prediction models and is recognized as a major diabetic complication risk factor. Diabetes duration is strongly associated with the development of a variety of diabetes-related macrovascular and microvascular complications. Liu YY et al.26 in a 16-year cohort study of patients with T2DM, found that the prevalence of DR was 25% at 10 years of disease duration, and increased dramatically to 50% at 15 years of disease duration. Joanne et al.27 also found that DR was a significant risk factor in the prediction model of DR, and was recognized as a major risk factor for diabetic complications. A pooled analysis of 35 epidemiologic studies also found that the prevalence of DR increased significantly with disease duration, ranging from 21.1% in patients with a disease duration of less than 10 years to 76.3% when the disease duration was more than 20 years. Al Rubeaan et al.28 showed that diabetes with a disease duration of more than 10 years and older age of the patient were significant risk factors for DR. There was a 50-fold increase in the prevalence of DR between the youngest group with the shortest Duration of T2DM and the oldest group with the longest duration27. A study by Alramadan et al.29 also found that increasing age was significantly associated with the onset of DR. The prevalence of DR in patients ≤ 60, 61–70 and ≥ 71 years of age was 38.5%, 46.8% and 55.4%, respectively, and these patients generally had a long duration of type 2 diabetes. The baseline analysis of this study showed that the Duration of T2DM in DR patients was significantly longer than that in non-DR patients, and the risk of DR increased significantly with longer disease duration, which is consistent with national and international literature. Therefore, the frequency of screening should be increased in patients with longer disease duration in order to prevent or slow down the development of DR at an early stage.It is speculated that the Duration of T2DM may be an indicator reflecting the overall level of blood glucose control and the exposure period of risk factors. Calderon et al.30 and Solomon SD et al.31 believe that blood glucose indicators are closely related to the severity of diabetes retinopathy (DR). For patients with T2DM, the higher the level of HbA1C, the higher the incidence of DR; On the contrary, when the HbA1C level decreases, the risk of DR occurrence will also decrease accordingly. In a cross-sectional study32 involving about 127,000 diabetes patients in northern Jiangsu, China, high HbA1C levels were confirmed to be an important risk factor for DR.In this study, the data in Table 1 clearly shows that the levels of HbA1C in the DR patient population are significantly higher than those in the non DR patient population, and there is a statistically significant difference between the two. This further confirms the role of HbA1C as an independent risk factor for DR patients. HbA1C is a product formed by the combination of blood glucose and hemoglobin, usually used to reflect the patient’s blood glucose control status over the past 8 to 12 weeks. Although the production process of glycosylated hemoglobin is relatively slow, it is not easy to decompose once it is formed, so it becomes an important indicator to monitor the blood sugar level of diabetes patients. Long term hyperglycemia may trigger irreversible vascular changes, but glycated hemoglobin, as an important indicator of blood glucose levels, is of great importance33. The level of glycosylated hemoglobin not only reflects the blood sugar content, but also is closely related to the complications related to diabetes, such as microvascular and macrovascular diseases34. Recent studies35 have shown that as HbA1c levels increase, it may affect the ability of hemoglobin to carry oxygen, thereby affecting retinal function, while a decrease in HbA1c levels helps to reduce the risk of DR30. Therefore, for patients with type 2 diabetes, strict blood glucose control and reduction of HbA1c level are still the key to diabetes management and prevention or mitigation of DR disease progress.In the present study, we establish for the first time neutrophils as a protective factor in DR, a finding that has rarely been mentioned in previous studies. As the most abundant leukocyte type in the human body, accounting for up to 65% of all neutrophils, neutrophils, in addition to their well-known phagocytosis, have a unique mechanism of suicidal attack, neutrophil net trapping death (NETosis). This process culminates in the formation of neutrophil extracellular traps (NETs)36, and a large number of recent studies37,38,39 have demonstrated that NETs play an important role in the development and progression of DR. This is mainly because the hyperglycemic environment stimulates neutrophils to produce superoxide and a variety of cytokines that promote NETosis.In an experimental study40, researchers found that serum levels of tumor necrosis factor-alpha (TNF-α) were elevated in diabetic patients, a change that stimulated the formation of NETs in neutrophils and led to the release of intracellular serine proteases, including neutrophil elastase (NE), which, in turn, led to an increase in the levels of NE and enzyme activity detected in the serum of diabetic patients. In turn, the increase in NE promotes the aggregation of neutrophils toward sites of inflammation and creates a negative feedback regulatory mechanism, which may further exacerbate the development of autoimmune diabetes. Thus, in diabetic patients, a decrease in the number of neutrophils and an increase in NETs are clearly key factors in this complex process. During the formation of NETs, neutrophils release negatively charged free DNA that activates coagulation factor XII, which triggers a series of reactions, including activation of kinin-releasing enzyme and production of bradykinin. Bradykinin binds to G protein-coupled bradykinin B1 and B2 receptors, which are highly expressed in the retina, and thus participates in both acute and chronic inflammatory injury processes in the DR41. Neutrophils, which originate mainly from the bone marrow and are released into the peripheral blood42, play an important role in innate immunity, acting as the first line of cellular defense. Therefore, a decrease in neutrophil numbers may signal an increase in NETs in the body, thereby promoting the onset and progression of DR, which provides an important early warning signal for diabetic patients. Few current predictive models involve neutrophils, making this study innovative in its exploration of this area.In this study, an innovative predictive model was developed for assessing the risk of DR based on the analysis of risk factors associated with DR. Through external validation, the model showed excellent diagnostic efficacy and goodness of fit, providing an effective tool for early prediction of DR. Considering the large population of diabetic patients and the uneven distribution of healthcare resources in China, especially in remote areas, screening for DR is extremely challenging. The novel prediction model developed in this study, based on existing research, will greatly improve the current screening status quo and provide an efficient and practical tool. By utilizing simple and easily accessible data for the initial assessment of diabetic patients, this model not only provides a scientific assessment basis for primary and general practitioners, but also helps to guide further diagnostic and therapeutic decisions, thus effectively reducing the incidence of DR. In addition, despite the results of this study, as a retrospective study, its conclusions still need to be validated and strengthened by future prospective studies. This not only provides a more reliable clinical basis for the prevention and treatment of DR, but also points the way for future research directions.To summarize, this predictive model serves as an effective tool for assessing the risk of DR, incorporating five key clinical features. It is not only applicable to areas with uneven distribution of healthcare resources, but can also play an important role in primary care settings where fundus examination is not available. The application of this model will help clinicians to intervene at an early stage, thus effectively reducing the morbidity and mortality of DR in the future, which will have far-reaching implications for improving the long-term health prognosis of diabetic patients.
