title: Leukocytes from diabetic patients kill retinal endothelial cells: effects of berberine.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Eye Hospital, The First Affiliated Hospital, Harbin Medical University, Harbin, China.
authors.forename: Pei
authors.initials: P
authors.lastname: Tian
authors.forename: Hongyan
authors.initials: H
authors.lastname: Ge
authors.forename: Haitao
authors.initials: H
authors.lastname: Liu
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Kern
authors.forename: Lingling
authors.initials: L
authors.lastname: Du
authors.forename: Linan
authors.initials: L
authors.lastname: Guan
authors.forename: Sheng
authors.initials: S
authors.lastname: Su
authors.forename: Ping
authors.initials: P
authors.lastname: Liu
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001599:Berberine / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*; D018821:CD18 Antigens / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D003921:Diabetes Mellitus, Experimental / Q000473:pathology*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D005260:Female; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D016328:NF-kappa B / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D012160:Retina / Q000473:pathology*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D018821:CD18 Antigens; D016328:NF-kappa B; D001599:Berberine; D018799:Intercellular Adhesion Molecule-1; D005947:Glucose