Mechanism Of Methylglyoxal Induced Mitochondrial Membrane Permeability Transition Pore Opening In EA.hy926 Cell

Methylglyoxal(MGO), a kind of endogenous dicarbonyl compound, the active carbonyl of MGO easily reacts with amino acid residues forming advanced glycation end products(AGEs). Intracellular accumulation of MGO can induce carbonyl and oxidative stress, thereby causing cell damage.The accumulation of MGO and AGEs involve in the development of vascular complications in diabetes, kidney disease, therefore, inhibiting the cytotoxicity induced by MGO for the prevention and control of the above disease is particularly important. At present, the main mechanisms of cytotoxicity induced by MGO include AGEs- RAGE, promoting reactive oxygen species(ROS) generation, inducing mitochondrial damage and cell apoptosis, but the mechanism of apoptosis is not demonstrated clearly. The mitochondrial membrane permeability transition pore(mPTP) plays a key role in this pathway, but whether MGO induced mPTP opening or the mechanism of induced by MGO is unclear. Glycogen synthase kinase 3β(GSK3β) is the key regulator protein of mPTP. Hexokinase II was disrupted from mitochondria when GSK3β was activated, leading to the opening of mPTP. Therefore, in the model of EA.hy926, this study focuses on mPTP to investigate the mechanism of cytotoxity induced by MGO from the angle of oxidative stress and AGEs-RAGE.The experimental results are as follows:1. To research the mechanism of cytotoxity induced by MGO, 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide(MTT) and trypan blue staining were used to detected cytotoxic effect of MGO on EA.hy926 cells. Rhodamine 123 was used to detect the change of mitochondrial membrane potential(MMP), DCFH-DA probe was used to detect ROS induced by MGO, mitochondrial matrix swelling and cell apoptosis were examed. Results showed that the cell viability of EA.hy926 cells reduced, cytotoxity enhanced, MMP reduced, the formation of ROS increased, mitochondrial matrix swelling and apoptosis increased as the concentration of MGO increased. The carbonyl capture agent aminoguanidine(AG), 4-Hydroxy-TEMPO(TEMPOL), an inhibitor of free radical, and SB216763, LiCl inhibitors of GSK3β can significantly inhibit the effects of MGO.2. The protein expression of none-fluorescence AGEs-CML, RAGE, the 9 serine phosphorylation of GSK3β kinase and the mitochondrial HKII were tested by Western blot. Results showed that as MGO increased, CML and RAGE protein expression increased significantly, and those increase were significantly inhibited by AG and TEMPOL. However, as MGO increased, the phosphorylation of 9 serine of GSK3β kinase decreased, and this decrease was significantly inhibited by AG, TEMPOL and SB216763; the expression of HKII in mitochondria decreased when EA.hy926 incubated with 0.5 and 1 mM MGO. This decrease was significantly inhibited by AG, TEMPOL, SB216763 and LiCl.In summary, MGO induced cytotoxity in EA.hy926, and the mechanism may be associated with mitochondrial injury and apoptosis. Further experiments proved that MGO caused EA hy926 apoptosis associated with mPTP, and the possible mechanism is that(1) MGO activated GSK3β through CML- RAGE pathway,(2) MGO activated GSK3β through oxidative stress, which induced mPTP changes,(3) MGO induced oxidative stress to regulated mPTP directiy.