Authors: Han Jing, Yan Xiaolong, Xiaoxi Qiao
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Studies showed that inflammatory process participates in the pathogenesis and development of diabetic retinopathy targeting retinal vascular endothelial cells (RVECs). A growing body of evidence revealed that metformin reduces the risk of micro- and macro-vascular complications by protecting blood-brain barrier, however, whether it plays a protective effect on human retinal vascular by similar mechanism is still unelucidated.
This study was to investigate the effects of metformin on the proliferation, migration and secreting monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) of human retinal vascular endothelial cells (RVECs) under the stimulation of tumor necrosis factor-alpha (TNF-α).
RVECs were cultured and divided into normal control group, metformin (5 mmol/L) group, TNF-α 2.5 ng/ml group, and TNF-α+ metformin (5, 10, 20 and 40 mmol/L, respectively) groups.Corresponding drugs were added into medium according to grouping for 24 hours.Cell numbers were calculated before and after treatment.The metabolic activity (absorbancy) of RVECs was measured with MTS assay.Cell migration of RVECs was assessed with transwell migration assay.The MCP-1 and IL-8 concentrations in the cell supernatant were detected by ELISA assay.
The number of the cells was significantly different among the normal control group, metformin group, TNF-α group, and TNF-α+ metformin (5, 10, 20 and 40 mmol/L, respectively) groups (F=189.31, P<0.01). The metabolic activities of RVECs were 0.32±0.02, 0.32±0.03, 0.97±0.02, 0.90±0.05, 0.76±0.15, 0.74±0.05 and 0.41±0.03; migrated cell numbers were (1 214±49), (1 200±45), (1 648±43), (1 309±48), (1 279±73), (961±60) and (942±106)/field; the concentrations of MCP-1 were (0.385±0.050), (0.362±0.060), (2.285±0.200), (1.131±0.180), (0.622±0.120), (0.537±0.090) and (0.492±0.130) μg/ml, and those of IL-8 were (0.385±0.080), (0.390±0.120), (1.123±0.130), (0.899±0.180), (0.680±0.060), (0.417±0.090) and (0.335±0.100) μg/ml in the normal control group, metformin group, TNF-α group, and TNF-α+ metformin (5, 10, 20 and 40 mmol/L, respectively) groups, showing significant differences among the groups (F=73.31, 103.89, 150.92, 268.32, all at P<0.01). The cell number, cell metabolic activity, migrated cell number, and MCP-1 and IL-8 levels in the cell supernatant were evidently increased in the TNF-α group compared with the normal control group, and those in the TNF-α+ 10 mmol/L metformin group, TNF-α+ 20 mmol/L metformin group and TNF-α+ 40 mmol/L metformin group were significantly decreased in comparison with the TNF-α group (all at P<0.05).
Metformin can inhibit TNF-α-induced proliferation, migration and MCP-1 and IL-8 secretion of the cells, and therefore plays a protective role on RVECs in the inflammatory environment.