目的

糖尿病微血管并发症是糖尿病最常见且致残性强的并发症，可引发视网膜病变、肾病及神经病变等一系列严重后果。然而，目前缺乏糖尿病人群，特别是从糖耐量受损(impaired glucose tolerance, IGT)开始的微血管并发症的长期观察随访数据，且糖尿病微血管并发症发病的分子机制仍未完全清楚。本研究旨在调查分析，1986年来自大庆地区新诊断糖尿病(newly diagnosis diabetes, NDD)、糖耐量受损和糖耐量正常(normal glucose tolerance, NGT)人群，34年随访期间严重微血管并发症的发生率和发生风险及不同性别人群严重微血管并发症发病特点；并探讨高血糖引起微血管并发症的潜在分子机制。

方法

本研究纳入来自大庆地区的1631名受试者，于1986年首次诊断其血糖状态，包括598名NDD、540名IGT和493名NGT受试者，并随访34年。所有受试者均随访至死亡、失访或至2020年12月31日。严重糖尿病微血管并发症定义为复合严重糖尿病视网膜病变、严重糖尿病肾病和严重糖尿病性神经病变。采用Kaplan-Meier方法计算出NDD、IGT和NGT组严重微血管并发症的累积发病率，并通过Cox比例风险模型及时依协变量Cox回归模型对相关危险因素进行了调整，评估不同组别之间并发症发生率差异的风险比(hazard ratio, HR)。并且，通过对性别分层，分析不同血糖水平的男性和女性人群各自严重微血管并发症的发病特点。

此外，对糖尿病微血管并发症进行了相关分子机制探讨。分别用5.5mM/L和30mM/L两种浓度葡萄糖处理培养好的脐静脉内皮细胞36小时(hour, h)后，进行转录组测序，利用高通量测序技术获取并分析内皮细胞在高糖和正常糖培养条件下基因表达的差异，探索糖尿病导致微血管并发症的过程中可能的分子机理。

结果

在34年随访中，NDD、IGT和NGT组严重微血管并发症的累计发病率分别为65.03%(95% CI: 58.90%-70.48%)、32.9%(95% CI: 28.19%-37.81%)和16.18% (95% CI: 12.64%-20.11%)。调整年龄、性别、身体质量指数、血压、吸烟状态等影响因素后，与NGT人群比较，NDD人群的严重微血管并发症发生风险为6.25倍(95% CI: 4.65-8.38, P<0.0001)，IGT人群为2.16倍(95% CI: 1.58-2.95, P<0.0001)。当糖尿病被作为时间依存协变量处理时，在调整了相关混杂因素后，在NDD联合IGT人群中，已有糖尿病及随访期间进展为糖尿病的IGT受试者发生严重微血管并发症的风险是在随访期间保持非糖尿病状态受试者的8.89倍(95% CI: 4.40-17.98, P<0.001)，单纯IGT人群中进展为糖尿病的风险是未进展为糖尿病的4.67倍(95% CI: 2.25-9.69, P <0.001)。按性别分层分析结果显示，男性NDD人群34年严重微血管并发症累积发病率为68.02%(95% CI: 57.27%-76.61%)，略高于女性(63.37%, 95% CI: 55.69%-70.09%)；男性IGT人群与女性IGT人群严重微血管并发症累积发病率基本相同(32.33% vs. 33.17%)。与NGT人群相比，无论NDD或IGT人群，男性人群严重微血管并发症发病风险(NDD: HRs: 7.07, 95% CI: 4.58-10.93; IGT: HRs: 2.49, 95% CI: 1.58-3.93)均高于女性人群(NDD: HRs: 5.98, 95% CI: 4.00-8.94; IGT: HRs: 2.00, 95% CI: 1.30-3.01)。

转录组测序中的功能富集分析结果表明，显著性差异基因多在细胞因子受体相互作用通路中富集，包括肿瘤坏死因子(tumor necrosis factor, TNF)信号通路、白介素-17信号通路(interleukin-17, IL-17)、核苷酸结合寡聚化结构域(nucleotide-binding oligomerization domain, NOD)样受体信号通路和Toll样受体信号通路，提示免疫反应尤其是先天性免疫反应在糖尿病微血管并发症的发生发展中的促进作用。

结论

NDD人群严重微血管并发症的34年累积发病率是IGT人群的两倍，IGT人群严重微血管并发症的34年累积发病率是NGT人群的两倍。无论男性女性，NDD人群和IGT人群严重微血管并发症风险均显著高于NGT人群。因此，糖尿病人群应在早期严格控制血糖和其他危险因素，进而预防严重微血管并发症的发生。糖尿病致微血管并发症的分子机制与TNF信号通路、IL-17信号通路、NOD样受体信号通路和Toll样受体信号通路有关。

Aims

Microvascular disease is a kind of common and specific complication of diabetes. However, there is a lack of over 30 years follow-up data on microvascular disease in population with diabetes, especially in people with impaired glucose tolerance (IGT), and the molecular mechanism of the onset of microvascular disease is still unclear. This study aimed to investigate the incidence of severe microvascular disease in adults with newly diagnosis diabetes (NDD), IGT and normal glucose tolerance (NGT) and the characteristics of severe microvascular disease in different gender population during the 34 years from 1986 to 2020; and explore the potential molecular mechanisms of microvascular disease caused by hyperglycemia.

Methods

This study selected 1631 participants, including 598 NDD participants, 540 IGT participants, and 493 NGT participants, who were initially identified in 1986 and followed up to 34 years. All participants were followed until either the date of death, loss to follow-up, or December 31, 2020. Sever microvascular disease was defined as a composite outcome of sever retinopathy, sever nephropathy and sever neuropathy. We calculated the cumulative incidence rate of sever microvascular disease in NGT, IGT and NDD groups by Kaplan Meier method, and adjusted for related risk factors by Cox proportional hazards models and time-dependent Cox proportional hazards models to derived the hazard ratios (HR) between different glucose status groups. Moreover, we stratified the participants by sex and analyzed the characteristics of severe microvascular disease in both male and female populations. In addition, we performed transcriptome sequencing after treating human umbilical vein endothelial cells with 5.5 mM/L and 30 mM/L glucose concentrations respectively 36 hours. We used high-throughput sequencing technology to obtain and analyze the differences in gene expression of endothelial cells under high glucose and normal glucose culture conditions, and explore the possible molecular mechanism in the process of microvascular disease caused by diabetes.

Results

During 34-year follow-up, in NDD, IGT and NGT groups, the cumulative incidence of sever microvascular disease was 65.03%(95% CI: 58.90%-70.48%), 32.9%(95% CI: 28.19%-37.81%) and 16.18% (95% CI: 12.64%-20.11%), respectively. After adjusting for related risk factors, including age, sex, smoking, BMI and BP, the risk of severe microvascular disease in the NDD population was 6.25 times (95% CI: 4.65-8.38, P<0.0001) than that of the NGT population , and the risk of severe microvascular disease in IGT population was 2.16 times (95% CI: 1.58-2.95, P<0.001) than that of the NGT population. When diabetes was treated as a time-dependent variable, after adjusting for relevant confounders, in the combined population of participants with IGT and NDD at baseline, the risk of developing sever microvascular disease was 8.89 times (95% CI 4.40-17.98, P<0.001) higher in participants with diabetes (prevalent and incident diabetes) than in those who remained non-diabetic during follow-up. Stratified analysis by sex showed that the cumulative incidence of severe microvascular disease in the male NDD population was 68.02% (95% CI: 57.27%-76.61%) over 34 years, slightly higher than that in the female NDD population (CI: 63.37%, 95% CI: 55.69%-70.09%); the cumulative incidence in the male IGT population was similar to that in the female IGT population (32.33% vs. 33.17%). Regardless of the NDD or IGT population, males (NDD: HRs=7.07, IGT: HRs=2.49) have a higher risk of developing severe microvascular diseases than females (NDD: HRs=5.98, IGT: HRs=2.00).The results of functional enrichment analysis showed that the significantly different genes were mostly enriched in the cytokine-cytokine receptor interaction pathway, including the TNF signaling pathway, IL-17 signaling pathway, NOD-like receptor signaling pathway and Toll-like receptor signaling pathway, indicating the role of immune response, especially innate immune response, in the occurrence and development of diabetic microvascular disease.

Conclusions

The incidence and risk of severe microvascular disease in diabetes and impaired glucose tolerance groups are significantly higher than those in normal glucose tolerance groups. The effective management of optimal glycemic control in the early stage of diabetes, and intensive lifestyle interventions to delay the progression to diabetes in IGT population are crucial to prevent microvascular disease. The molecular mechanism of microvascular disease is related to the TNF signaling pathway, IL-17 signaling pathway, NOD-like receptor signaling pathway and Toll-like receptor signaling pathway.