High glucose stimulates TNFα and MCP-1 expression in rat microglia via ROS and NF-κB pathways

Abstract

Aim: To investigate whether high glucose stimulates the expression of inflammatory cytokines and the possible mechanisms involved.

Methods: ELISA and real-time PCR were used to determine the expression of the inflammatory factors, and a chemiluminescence assay was used to measure the production of reactive oxygen species (ROS).

Results: Compared to low glucose (10 mmol/L), treatment with high glucose (35 mmol/L) increased the secretion of tumor necrosis factor (TNF)α and monocyte chemotactic protein-1 (MCP-1), but not interleukin (IL)-1β and IL-6, in a time-dependent manner in primary cultured rat microglia. The mRNA expression of TNFα and MCP-1 also increased in response to high glucose. This upregulation was specific to high glucose because it was not observed in the osmotic control. High-glucose treatment stimulated the formation of ROS. Furthermore, treatment with the ROS scavenger NAC significantly reduced the high glucose-induced TNFα and MCP-1 secretion. In addition, the nuclear factor kappa B (NF-κB) inhibitors MG132 and PDTC completely blocked the high glucose-induced TNFα and MCP-1 secretion.

Conclusion: We found that high glucose induces TNFα and MCP-1 secretion as well as mRNA expression in rat microglia in vitro, and this effect is mediated by the ROS and NF-κB pathways.

Figure 1

High glucose induces an inflammatory reaction in rat microglia. (A) Time course of TNFα secretion stimulated by high-glucose treatment. Rat microglia were cultured under low glucose (LG; 10 mmol/L) or high glucose (HG; 35 mmol/L), and the secretion of TNFα in the supernatant was measured with ELISA at the indicated time points. (B) The high-glucose treatment increased TNFα secretion independent of osmotic pressure. Rat microglia were cultured for 24 h in LG, HG, or mannitol (25 mmol/L+10 mmol/L glucose). (C) Time course of MCP-1 secretion stimulated by the high-glucose treatment. (D) The high-glucose treatment increased MCP-1 secretion independent of osmotic pressure. (E, F) High glucose had no effect on IL-1β and IL-6 secretion. The data represent the mean±SD of three independent experiments. ^bP<0.05 vs LG.

Figure 2

High-glucose treatment promotes the expression of TNFα and MCP-1 mRNA in rat microglia. (A) Real-time PCR analysis of the upregulation of TNFα mRNA by HG at different time points. ^bP<0.05 vs 0 h. (B) Treatment with high glucose elevated TNFα mRNA independent of osmotic pressure. Rat microglia was treated with LG, HG or mannitol for 3 h. ^bP<0.05 vs LG. (C) Real-time PCR analysis of the upregulation of MCP-1 mRNA by HG at different time points. ^bP<0.05 vs 0 h. (D) Treatment with high glucose increased MCP-1 mRNA independent of osmotic pressure. Rat microglia was treated with LG, HG or mannitol for 6 h. The data represent the mean±SD of three individual experiments. ^bP<0.05 vs LG.

Figure 3

ROS play a key role in the high glucose-induced secretion of TNFα and MCP-1 in rat microglia. (A) Effect of LG and HG on cellular ROS level. Microglia was stimulated with LG and HG for 20 min followed by a luminol-derived chemiluminescent assay. Cellular ROS levels were quantified. (B) The role of ROS in high glucose-induced TNFα secretion. (C) The role of ROS in high glucose-induced MCP-1 secretion. After treatment with 2 mmol/L of the ROS scavenger NAC for 1 h, microglia was cultured in LG or HG for 24 h. The medium was collected and the levels of TNFα and MCP-1 were analyzed by ELISA. Data represent the mean±SD of three experiments. ^bP<0.05 vs LG. ^eP<0.05 vs HG.

Figure 4

NF-κB is essential for high glucose-induced TNFα and MCP-1 secretion in rat microglia. After treatment with 10 μmol/L MG132 or 5 μmol/L PDTC for 1 h, microglia was cultured in LG or HG for 24 h. The medium was collected and the secretion of TNFα and MCP-1 was measured by ELISA. (A) Inhibition of the NF-κB pathway blocked high glucose-mediated TNFα secretion. (B) Inhibition of the NF-κB pathway blocked high glucose-mediated MCP-1 secretion. Data represent the mean±SD of three experiments. ^bP<0.05 vs control. ^eP<0.05 vs HG.