Tian Pei, Ge Hongyan, Liu Haitao, Kern Timothy S, Du Lingling, Guan Linan, Su Sheng, Liu Ping
Eye Hospital, The First Affiliated Hospital, Harbin Medical University, Harbin, China.
Mol Vis. 2013 Oct 2;19:2092-105. eCollection 2013.
Accumulating evidence in animals suggests that leukocytes are involved in the pathogenesis of diabetic retinopathy. The present study was designed to investigate whether leukocytes from diabetic patients could kill retinal endothelial cells and whether that cytotoxicity could be inhibited in vivo by administration of berberine.
Human retinal endothelial cells (HRECs) were cocultured (24 h) with leukocytes freshly isolated from nondiabetic and diabetic patients, and leukocyte-mediated death of HRECs was analyzed with flow cytometry. HRECs or leukocytes were incubated with antibodies against intercellular adhesion molecule-1(ICAM-1) or integrin beta-2, or with various concentrations of berberine. The protein expression levels of inflammatory factors were investigated using western blots, and activities of antioxidant enzymes and malondialdehyde content were examined as markers of oxidative stress. In addition, leukocytes were isolated from 28 diabetic patients with retinopathy and nondiabetic patients before and after 1 month in vivo therapy with berberine. The effects of the berberine on leukocyte-mediated killing of endothelial cells was again assessed.
Leukocytes from diabetic patients induced more apoptosis of HRECs in a coculture system than did cells from nondiabetic patients, and this killing occurred primarily via direct cell-cell contact. Berberine inhibited the leukocyte-mediated killing of HRECs in vitro, the decrease in antioxidant enzyme activities, the nuclear translocation of nuclear factor kappa B, and the increase in intercellular adhesion molecule-1 and inducible nitric oxide synthase expression and malondialdehyde content in HRECs cultured in high glucose. Berberine also decreased integrin beta-2 expression of leukocytes in vitro and in vivo. Oral consumption of berberine for 1 month likewise inhibited the diabetes-induced increase in leukocyte-mediated killing of HRECs.
Our findings suggest that leukocytes from diabetic patients kill retinal endothelial cells, and that berberine can inhibit this leukocyte-mediated killing of vascular endothelium. Coculture of leukocytes with HRECs might serve as a biomarker to study the role of leukocytes in the development of diabetic retinopathy, and the data are consistent with berberine being a therapy against diabetic retinopathy.
动物研究中越来越多的证据表明白细胞参与糖尿病视网膜病变的发病机制。本研究旨在探讨糖尿病患者的白细胞是否能杀死视网膜内皮细胞，以及黄连素在体内给药是否能抑制这种细胞毒性。
将人视网膜内皮细胞（HRECs）与从非糖尿病和糖尿病患者新鲜分离的白细胞共培养（24小时），并用流式细胞术分析白细胞介导的HRECs死亡情况。HRECs或白细胞与抗细胞间黏附分子-1（ICAM-1）或整合素β-2抗体，或与不同浓度的黄连素一起孵育。使用蛋白质印迹法研究炎症因子的蛋白表达水平，并检测抗氧化酶活性和丙二醛含量作为氧化应激的标志物。此外，从28例糖尿病视网膜病变患者和非糖尿病患者中分离白细胞，在体内用黄连素治疗1个月前后进行比较。再次评估黄连素对白细胞介导的内皮细胞杀伤作用的影响。
在共培养系统中，糖尿病患者的白细胞比非糖尿病患者的细胞诱导更多的HRECs凋亡，并且这种杀伤主要通过直接细胞间接触发生。黄连素在体外抑制白细胞介导的HRECs杀伤、抗氧化酶活性的降低、核因子κB的核转位以及高糖培养的HRECs中细胞间黏附分子-1和诱导型一氧化氮合酶表达及丙二醛含量的增加。黄连素在体外和体内也降低了白细胞的整合素β-2表达。口服黄连素1个月同样抑制了糖尿病诱导的白细胞介导的HRECs杀伤增加。
我们的研究结果表明，糖尿病患者的白细胞可杀死视网膜内皮细胞，并且黄连素可抑制这种白细胞介导的血管内皮杀伤作用。白细胞与HRECs共培养可能作为一种生物标志物来研究白细胞在糖尿病视网膜病变发展中的作用，并且这些数据与黄连素作为治疗糖尿病视网膜病变的药物一致。
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