Effect of Imperatorin on High Glucose Induced Retinal Endothelial Cell Injury by Regulating the HMGB1-RAGE-NF-κB Signaling Pathway
MA Jing1, WANG Qi2, CHEN Cailing3, TAN Dandan3, LIU Yandong4, WANG Weiwei3*
This study investigated the effect of IMP (imperatorin) on high glucose induced retinal endothelial cell injury by regulating the HMGB1 (high-mobility group protein box 1)-RAGE (receptor for advanced glycation end products)-NF-κB (nuclear factor kappa-B) signaling pathway. High glucose induced hRECs in retinal endothelial cells were treated with IMP at a concentration of 10-320 μmol/L to screen for the optimal drug concentration. hRECs (human retinal endothelial cells) were separated into normal glucose group (NG group, 5.5 mmol/L glucose), high glucose group (HG group, 25 mmol/L glucose), low concentration imperatorin group (IMP-L group, 25 mmol/L glucose+20 μmol/L IMP), medium concentration imperatorin group (IMP-M group, 25 mmol/L glucose+40 μmol/L IMP), high concentration imperatorin group (IMP-H group, 25 mmol/L glucose+80 μmol/L IMP), and high concentration imperatorin+recombinant HMGB1 protein group (IMP-H+rHMGB1 group, 25 mmol/L glucose+80 μmol/L IMP+200 μg/L rHMGB1). CCK-8 assay kit was applied to detect the activity of hRECs cells. Flow cytometry was applied to detect the apoptosis of hRECs cells. The cell angiogenesis was detected by the lumen formation test. ELISA (enzyme linked immunosorbent assay) was applied to detect levels of IL-6, TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β), MDA, and SOD in cells. Western blot was applied to detect the expression of proteins related to the HMGB1-RAGE-NFκB signaling pathway. The results showed that IMP was able to promote the proliferation of high glucose induced hRECs in a concentration dependent manner, subsequently, IMP concentrations of 20, 40, and 80 μmol/L were selected for subsequent experiments. Compared with the NG group, the cell survival rate and SOD activity in the HG group were decreased; the apoptosis rate, the number of lumen formation, the levels of IL-6, TNF-α, IL1β, MDA, and the expression of HMGB1, RAGE, NF-κB p65 proteins were increased (P<0.05). Compared with the HG group, the cell survival rate and SOD activity were increased in the IMP-L group, IMP-M group, and IMP-H group; the apoptosis rate, the number of lumen formation, the levels of IL-6, TNF-α, IL-1β, MDA, and the expression of HMGB1, RAGE, NF-κB p65 proteins were decreased and in a concentration dependent manner (P<0.05). Compared with the IMP-H group, the cell survival rate and SOD activity in the IMP-H+rHMGB1 group were significantly reduced; the apoptosis rate, the number of lumen formation, the levels of IL-6, TNF-α, IL-1β, MDA, and the expression of HMGB1, RAGE, NF-κB p65 proteins were significantly increased (P<0.05). The results suggest that IMP may reduce inflammatory response and cell apoptosis by inhibiting the HMGB1- RAGE-NF-κB signaling pathway, thereby alleviating high glucose induced injury to retinal endothelial cells
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