Inhibition And Its Mechanism Of Methylglyoxal-induce Cytotoxicity By Catechin In Human Vascular Endothelial Cells

Methylglyoxal(MGO) is a reactive carbonyl species (RCS) that have cytotoxicity. MGOis also a dicarbonyl compound formed by the fragmentation of triose phosphates,intermediates of glycolysis, and the metabolism of acetone, the intermediates ofgluconeogenesis. In addition, MGO is also formed by microbial flora that located in rumen ofruminant animals under the condition of hyperglycemia. It is known that MG inducescarbonyl stress and oxidative stress, leading to metabolic disorders and dysfunction ofepithelial cells. MGO can be seen as the precursor of the advanced glycosylation end products(AGE), therefor, the mechanism that MGO plays during the development of diabetescomplications and alzheimer’s disease, become a research hotspot. Now it is acknowledgedthat the disorder of glycometabolism leads to increaseing of glycosylation of proteins, thedenaturation and dysfunction of proteins. While the gradually accumulation of AGEs in targetorgans further promotes carbonyl stress and oxidative stress, leading to inflammation and thedevelopment of pathological. MGO induces cell toxicity though mang ways, includingpromoting protein–protein crosslinks, protein-nucleic acid crosslinks, the formation of freeradical, and the inhibition of glycolysis et. It was reported that MGO-induced apoptosis ismediated by the mitochondria, However, the mechanisms governing mitochondrial damage byMGO are unclear. Therefore our study attempts to explore the mechanism from oxidativestress, mitochondrial membrane potential (MMP), the formation of AGEs, the change ofmitochondrial morphology and hexokinase, etc, to investigate the mechanism of MGOcytotoxicity. Meanwhile, catechins, a natural composition of tea， can improve diabetickidney, and also has a protective effect on insulin sensitivity, shows a potential treatment fordiabetic complications. In addtion, the vitro experiments show that catechins can effectivelycapture the carbonyl of glyoxal (GO), methyl glyoxal (MGO) in incubation conditions, thusinhibiting the carbonylation reaction. Therefore, in this experiment we make a preliminarystudy of the mechanism that catechin suppress the cytotoxicity of MGO.1．For examine the cytotoxic and its mechanism of MGO on endotheial cells, wedetected the cell viability and mortality by MTT and trypan blue, respectively, and evaluat cytotoxic. The IC50that MGO on ECV304cell is1.3±0.1mM; the IC50of Hy.926cells is2.4±0.1mM. The effects of MGO can be inhibiied by Catechins, aminoguanidine andN-acetyl cysteine. Meanwhile the results are consistent with the result of the cell survival rateexamed by trypan blue.2. The oxidative stress was detected by DCFH-DA probe method and FOX method;MGO promoted the increase of ROS generation in EA.hy926cells, as well as hydrogenperoxide, but had no significant promoting effect on ECV304cell. Aminoguanidine canreduce oxidative stress induced by MGO, however catechins further strengthen the oxidativestress induced by MGO instead. It is showed that ROS is not the event of MGO-inducedcytotoxicity, and the antioxidant effect of catechins is not the mechanism that catechins’protection.3. Using rhodamine123to detect MMP and mitochondrial morphological changes.MGO induced the increase of MMP dependenting on concentration, after rising, MPPmaintained new equilibrium and mitochondria swelling occurred at the same time.Aminoguanidine inhibited the effect of MGO on MMP. Catechin intervention group wascompletely inhibitied the increase of MMP. On the mitochondrial morphology, AG andcatechins inhibited mitochondrial swelling in different extent.4. The activation of Caspase-3occurs when MGO-induced apoptosis which mediated bymitochondria. The activation of Caspase-3induced by MGO was inhibited by Catechineffectively. The results of flow cytometry also proved that catechin has an effect on theinhibition of apoptosis caused by MGO, and superior to the same concentration of AG.Above all, the damage of MGO on cells is mainly due to the apoptosis pathwaysmediated by mitochondria, according to the analysis of existing data, this rusult is in line withthe model of apoptosis mediated by the close of voltage dependent anion channel (VDAC).At the same time, catechins significantiy inhibitied this way. ROS and AGEs are limitedcontribution in the cytotoxicity caused by MGO with short term (24h).