Luteolin alleviates methylglyoxal-induced cytotoxicity in osteoblastic MC3T3-E1 cells

Abstract

Methylglyoxal (MG), a reactive sugar-derived metabolite, exerts harmful effects by inducing oxidative stress, which aggravates a series of diabetic complications, including osteoporosis. The present study was performed to examine the effects of luteolin, a dietary polyphenolic flavonoid, on MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells. Pretreatment of MC3T3-E1 osteoblastic cells with luteolin prevented MG-induced cell death and production of tumor necrosis factor-alpha, intracellular reactive oxygen species, mitochondrial superoxide, and cardiolipin peroxidation. In addition, luteolin increased the levels of glutathione and nuclear factor erythroid 2-related factor 2 (Nrf2) and decreased the inhibition of heme oxygenase-1 activity by MG. Pretreatment with luteolin prior to MG exposure reduced MG-induced mitochondrial dysfunction and increased the peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and nitric oxide levels, suggesting that luteolin may induce mitochondrial biogenesis. Taken together, these observations indicated that luteolin has potential as a preventive agent against the development of diabetic osteopathy related to MG-induced oxidative stress in diabetes.

Keywords: Glutathione; Mitochondrial function; Nitric oxide; Osteoblasts; Reactive oxygen species.

Fig. 1

Effects of luteolin on the viability of osteoblasts. Osteoblasts were treated with luteolin (Lut) or aminoguanidine (AG) 1 h before exposure to 400 μM MG for 48 h. ^# P < 0.05, compared to untreated cells; *P < 0.05, compared to cells treated with MG alone

Fig. 2

Effects of luteolin on TNF-α level in MG-treated MC3T3-E1 cells. Osteoblasts were treated with luteolin (Lut) or aminoguanidine (AG) 1 h before exposure to 400 μM MG for 48 h. ^# P < 0.05, compared to untreated cells; *P < 0.05, compared to cells treated with MG alone

Fig. 3

Effects of luteolin on RAGE and sRAGE levels in MG-treated cells. Osteoblasts were treated with luteolin (Lut) or aminoguanidine (AG) 1 h before exposure to 400 μM MG for 48 h. ^# P < 0.05, compared to untreated cells; *P < 0.05, compared to cells treated with MG alone

Fig. 4

Inhibitory effect of luteolin on MG-induced oxidative stress in MC3T3-E1 cells. Osteoblasts were treated with luteolin (Lut) or aminoguanidine (AG) 1 h before exposure to 400 μM MG for 48 h. a The data show changes in levels of ROS, which was measured by the DCF fluorescence method. b Mitochondrial superoxide levels were detected using MitoSOX™ Red mitochondrial superoxide indicator. c Cardiolipin oxidation was measured using 5 μM NAO. ^# P < 0.05, compared to untreated cells; *P < 0.05, compared to cells treated with MG alone

Fig. 5

Effects of luteolin on reduced glutathione (GSH), HO-1, and Nrf2 levels in osteoblastic MC3T3-E1 cells. Osteoblasts were treated with luteolin (Lut) or aminoguanidine (AG) 1 h before exposure to 400 μM MG for 48 h. *P < 0.05, compared to cells treated with MG alone

Fig. 6

Effects of luteolin on the MG-induced mitochondrial dysfunction in osteoblastic MC3T3-E1 cells. Osteoblasts were treated with luteolin (Lut) or aminoguanidine (AG) 1 h before exposure to 400 μM MG for 48 h. (A) Changes in mitochondrial membrane potential (MMP) were monitored by loading with the fluorescent probe JC-1. (B) The ATP concentrations were determined by luciferase reaction. (C) Intracellular PGC-1α levels were measured using ELISA. (D) Cells were loaded with DAF-FM-DA as an indicator of nitric oxide. ^# P < 0.05, compared to untreated cells; *P < 0.05, compared to cells treated with MG alone