Authors: Tang Huan, Luo Guanghua, Yao Shuang, Wang Min, Pan Lili, Yu Miaomei, Yu Yang, Liu Yao
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To observe the expressing changes of apolipoprotein M (ApoM), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) in human retinal vascular endothelial cells (HRECs) under the high glucose culture condition and investigate the inhibitory effects of ApoM overepression on the expressions of TNF-α and MCP-1.
HRECs were cultured in DMEM containing 10% fetal bovine serum and 5.5 mmol/L D-glucose and assigned to 6 groups.The cells in the normal control group were cultured in above culture medium; the cells in the high glucose group were treated using the DMEM with 30 mmol/L D-glucose; ApoM was transfected into the cells using lentiviral vector in the ApoM transfected group; lentiviral vector without ApoM sequence was transfected in the empty vector group; the cells transfected by empty vector were cultured in high glucose culture medium in the empty vector+ high glucose group; the cells in the ApoM transfection+ high glucose group were treated by ApoM sequence transfection and high glucose incubation.The relative expression of ApoM, TNF-α and MCP-1 mRNA was detected using real-time quantitative PCR, and the relative expression of ApoM protein was evaluated using Western blot assay.
Compared with the normal control group, the mRNA expression levels of ApoM, TNF-α and MCP-1 in the high glucose group were significantly increased (t=5.517, 3.295, 2.555; all P<0.05). HRECs grew well after infected with lentivirus.The relative expression level of ApoM mRNA in the ApoM transfected group was 236.400±39.270, which was significantly higher than 1.000±0.153 in the empty vector group (t=5.995, P<0.01). An enhanced protein band of ApoM was seen in the ApoM transfected group, and the protein band was absent in the empty vector group.The relative expression band in the ApoM transfected group was 1.000±0.249 and 2.978±0.285 in the cells cultured with normal culture medium or high glucose culture medium, respectively, with a significant difference between them (t=5.056, P<0.01). The relative expressions of TNF-α and MCP-1 in the mRNA levels were significantly different among the empty vector group, empty vector+ high glucose group, ApoM transfected group and ApoM transfection+ high glucose group (F=5.966, P=0.026; F=14.410, P=0.002). Compared with the empty vector+ high glucose group, the relative expressions of TNF-α and MCP-1 mRNA were considerably reduced in the ApoM transfection+ high glucose group (P=0.017, 0.004).
High glucose environment up-regulates the expression of ApoM, MCP-1 and TNF-α in HRECs.Overexpression of ApoM inhibits the up-regulation of MCP-1 and TNF-α expression induced by high glucose.