高血糖通过糖基化己糖激酶降低红细胞变形能力的机制研究

The decreased red blood cell deformability (RCD) is an important pathogenetic basis of diabetic microangiopathy. Previous studies mainly focused on the increase in intracellular fluid viscosity and the decrease in membrane fluidity and deformability, which were caused by non-enzymatic glycosylation of hemoglobin, membrane proteins, and membrane skeleton. However, RCD is also closely related to intracellular ATP levels. Pilot experimental results showed that high glucose can reduce the ATP levels in RBCs during storage, and the activity of hexokinase (HK), the first key enzyme of glycolysis, decreases in diabetic microangiopathy patients’ RBCs. Since protein glycosylation modification is very common in diabetic patients, we hypothesize that high glucose might reduce RCD though glycosylation of HK, which could further inhibit the RBC energy metabolism. This study aims to determine the deformability, glycolytic flux，intermediate metabolic products, as well as HK activity and glycosylation modification on high-glucose cultured RBCs firstly by hemorheological, metabolomic, Seahorse XF extracellular flux and glycosylation analyses. Then, the correlation between HK glycosylation and RCD in diabetic microangiopathy patients will be analyzed to further elucidate the molecular mechanisms of decreased RCD in diabetic microangiopathy patients, which will provide new ideas for the diagnosis, treatment and prevention of diabetic microangioathy patients.

红细胞变形能力（red blood cell deformability，RCD）降低是糖尿病微血管病变的重要发病基础。以往研究主要集中于血红蛋白、膜蛋白及膜骨架非酶糖基化导致的细胞内液粘度增高及膜流动性和变形能力下降。然而，RCD与细胞内ATP水平密切相关，前期预实验发现高糖降低储存期RBC内ATP水平，且糖尿病微血管病变患者RBC内己糖激酶（hexokinase，HK）活性下降。由于糖尿病患者体内蛋白糖基化修饰非常普遍，因此推测高糖可能通过糖基化修饰HK影响RBC能量代谢进而降低RCD。本研究首先通过血流变、代谢组、细胞能量代谢分析及糖基化检测等明确高糖对体外培养RBC变形能力、糖酵解通量和中间产物以及HK活性和糖基化的影响，然后进一步分析微血管病变患者RBC内HK糖基化修饰与RCD的相关性，阐明微血管病变RCD下降的分子机制，为微血管病变的诊断、治疗和预防提供新的思路。

糖尿病(Diabetes mellitus，DM)是以慢性高血糖为特征的终身代谢性疾病，发病率逐年增高，成为严重威胁人类健康的全球性疾病。本项目利用多功能酶标仪、血球检测仪、流式细胞仪、Seahorse能量代谢仪、代谢组和糖基化蛋白质组学等检测手段，通过临床病例对照和体外高糖孵育实验研究糖尿病患者红细胞葡萄糖摄取率、糖酵解能力、ATP含量、糖酵解中间代谢产物以及己糖激酶(Hexokinase，HK)、丙酮酸激酶(Pyruvate kinase，PK)和磷酸果糖激酶(Phosphofructokinase，PFK)等糖酵解关键酶活性的改变及其与红细胞变形能力(red cell deformability，RCD)的相关性。结果显示，与正常对照组相比，糖尿病微血管病变患者红细胞的红细胞刚性指数(rigidity index, IR)、红细胞分布宽度(red blood cell distribution width, RDW)、平均红细胞体积(mean corpuscular volume, MCV)、网织红细胞计数(reticulocyte count, RET)、葡萄糖摄取率、基础糖酵解、糖酵解能力均增高，但葡萄糖反应性、衰亡率及变形指数(deformation index, TK)下降；同时红细胞内ATP含量、PFK和PK活性、糖酵解中间产物增高，HK活性降低。与无微血管合并症患者相比，糖尿病微血管病变患者红细胞内ATP含量降低，差异具有显著性；糖尿病微血管病变患者TK与HbA1c和TC浓度呈正相关，与ATP含量和HK活性呈负相关；IR与HbA1c、Non-LDL-C及TC呈正相关，与HK、PK值呈负相关。综上，红细胞在血糖调节过程中也发挥重要作用，可通过加强对血液中葡萄糖的摄取、提高葡萄糖反应性和糖酵解关键酶活性来增强糖酵解能力，加强对血液中葡萄糖的摄取和清除，防止和减少糖基化终末产物(advanced glycation end products，AGEs)对其它重要组织细胞的损伤；然而糖尿病患者长期高血糖也将损伤红细胞引起红细胞衰亡增多、变形能力下降、刚性增强。当红细胞损伤严重超过骨髓造血能力时，糖酵解关键酶HK活性下降，以限制红细胞对葡萄糖的摄取。因此HK活性可能作为糖尿病及糖尿病微血管病变的早期监测指标，辅助糖尿病患者进行病情监控，预防微血管并发症。