The Role And Significance Of OX40 Ligand(OX40L) In Patients With T2DM: Association With Atherosclerotic Macrovascular Diseases

With the improvement of living standards, westernized lifestyle and aging problem of society, threats of chronic disease are increasing, of which diabetes and its chronic complications are the ruthless killers of health hazards. Recent studies suggest that type 2 diabetes(T2DM) is an immune-related chronic inflammatory disease. Immune dysregulation may play a crucial role in the developing process of T2 DM and its complications. OX40 L is a member of the TNF superfamily. Combining with its receptor OX40, the pair of costimulatory molecules can provide a second signal for the long-term activation of T, B cells[1]. In this study, we use immunofluorescence labeling and flow cytometry technology to analyze membrane OX40 L on monocytes of the patients, and the serum level of s OX40 L were detected with ELISA method. The biological effects of both membrane-type and soluble OX40 L were analyzed in the development of diabetic vascular disease.Part I The expression and clinical significance of OX40/OX40 L in patients with type 2 diabetes mellitus and macrovascular complicationsObjective To investigate the expression of OX40/OX40 L on the peripheral immune cells and serum soluble OX40L(s OX40L) in patients with diabetes and diabetic macrovascular complications.Methods This experiment divided into four groups: group A(42 diabetes without carotid intimal thickening or plaques) and group B(31 diabetes with carotid plaques), group C(25 diabetes with acute myocardial infarction), group D(33 Healthy controls). The expression of OX40 on CD4+, CD8+ T lymphocyte cell and OX40 L on CD14+ monocyte of the four groups were analyzed using immunofluorescence labeling and flowcytometry technology, and the serum level of s OX40 L were detected with ELISA method.Results The expression of OX40 on CD4+, CD8+ T lymphocyte cell between four groups were no difference(P>0.05); Compared with group D, expression of OX40 L on CD14+ monocyte was up-regulated in group A, B and C(P<0.01); The expression of OX40 L on CD14+ monocyte was up-regulated in group C as compared with group A, B(P<0.01, P<0.05). Compared with group D, the expression of serum s OX40 L was up-regulated in group A、B and C(P<0.01); And it was up-regulated in group C as compared with group A, B(P<0.01, P < 0.05).Conclusion The expression of OX40 L on CD14+ monocyte and serum soluble OX40 L was abnormally increased in T2 DM, which were closely related to the severity of vascular disease.Part II The impact and biological significance of high glucose on OX40 L expression on monocyte cells and soluble OX40 L levelsObjective To observe the impact of high glucose on the expression of OX40 L on CD14+monocyte and the soluble OX40 L levels in the culture supernatant. And also to explore the biological significance of high glucose on monocytes.Methods(1) Peripheral blood were collected from healthy people, then lymphocyte separation medium(Ficoll) were used to isolate the peripheral blood mononuclear cells(PBMC). Cells were allowed to adhere for 1-2h, finally supernatant suspension cells were abandoned, and adherent cells(CD14+ monocyte cells) were collected(purity of about 90%);(2) The experimental groups were divided into four groups depending on the different glucose concentrations(5.5,11,22,33mmol/L) for 12 h, 24 h, 36 h, 48 h. Treatment cells and the supernatant were collected respectively;(3) The expression of OX40 L on CD14+ monocyte were analyzed by flow cytometry technology;(4) And the level of s OX40 L and TNF-α in the culture supernatant were detected with ELISA method.Results(1) The flow cytometry results showed that, the expression of m OX40 L on the CD14+ monocytes in high glucose groups were significantly up-regulated as compared to the control group in a concentrate- and time-dependent manner(P<0.05).(2) ELISA tests showed, s OX40 L level in the culture supernatants were elevated in high glucose groups,and there is significant difference between the groups(P<0.05); and TNF-α were also elevated.Conclusion High glucose upregulated OX40 L on the CD14+ monocytes and increased s OX40 L levels in the cell culture supernatant, and it is related to the inflammatory factor. It suggests that high glucose may enhance the inflammation effect through costimulatory molecules expressed by monocytes.