This study was designed to investigate whether EGCG plays a cytoprotective role in glomerular epithelial cells (GECs) under high glucose conditions. MTT assays showed that treatment with a high concentration of glucose (30 mM) significantly increased ...

This study was designed to investigate whether EGCG plays a cytoprotective role in glomerular epithelial cells (GECs) under high glucose conditions. MTT assays showed that treatment with a high concentration of glucose (30 mM) significantly increased apoptosis among cultured GECs as compared with that observed for a normal glucose concentration (5 mM). Pretreatment with EGCG (1-100 μM) significantly increased cell viability under the high glucose condition in a dose-dependent manner. DNA fragments was induced by glucose at 30 mM for 24 hr, whereas pretreatment for 2 hr with EGCG (1-100 μM) conferred significant protection against this glucose-induced DNA fragmentation. Moreover, DAPI staining assays showed that treatment with a high concentration of glucose (30 mM) increased DNA fragmentation / condensation in GECs as compared with that observed for a normal glucose concentration (5 mM). Pretreatment of GECs with EGCG (1-100 μM) significantly increased cell viability under the high glucose condition in a dose-dependent manner. Mechanism of reactive oxygen species (ROS)-mediated apoptosis induced by glucose (30 mM) was examined. It was found that glucose at 30 mM rapidly stimulated the generation of intracellular ROS, whereas pretreatment with EGCG (1-100 μM) significantly decreased generation of ROS under the high glucose condition in a dose-dependent manner. As well as, treatment of high glucose (30 mM) increased lipid peroxidation (LPO) and decreased glutathione (GSH) in GECs. Pretreatment with 100 μM EGCG attenuated the increase of LPO and restored the activity of GSH. These results suggest that high glucose-induced oxidative stress is a critical role in GECs apoptosis. Next, the role of EGCG in relation of p38 MAPK and ERK1/2 was investigated in GECs at high glucose conditions. It was found that glucose at 30 mM rapidly increased the phosphorylations of p38 MAPK and ERK1/2, whereas pretreatment with EGCG (1-100 μM) significantly prevented these phosphorylations in glucose concentration at 30 mM in a dose-dependent manner. Expression of apoptotic factors revealed that EGCG prevented the expression of the pro-apoptotic factors, such as Bad, caspase-3 and -9 by glucose at 30 mM, whereas EGCG pretreatment increased the expressions the anti-apoptotic factors, such as Bcl-2 and Bcl-xL in GECs. These data indicate that EGCG protects against apoptotic cell death by stimulating phospho-p38 MAPK and phospho-ERK1/2 and up-regulation of antiapoptotic factors, the other side down-regulation of proapoptotic factors in high glucose-conditioned GECs. Next, it was investigated the role of transforming growth factor-beta1 (TGF-β1), protein kinase C (PKC) α/βII and nuclear factor-kappaB (NF-κB) in GECs against high glucose injury. Glucose at 30 mM rapidly increased the expressions of mRNA and protein of TGF-β1, the other side, pretreatment with EGCG (1-100 μM) significantly prevented expressions of TGF-β1 in glucose concentration at 30 mM in a dose-dependent manner. In addition, EGCG (100 μM) inhibited phospho-PKC α/βII activation caused by glucose at 30 mM and pretreatment with EGCG (1-100 μM) significantly decreased the expression of transcriptional activity of NF-κB induced by glucose at 30 mM in a dose-dependent manner. These data indicate that 1) the protective mechanism of EGCG against high glucose-induced apoptotic cell death includes stimulation of phospho-p38 MAPK and phospho-ERK1/2 and modulation of apoptosis-regulating factors, 2) EGCG prevents high glucose-induced expression of TGF-β1, phospho-PKC α/βII and NF-κB.

When cells were subjected to glucose at 30 mM for 24 hr, it was observed early apoptotic changes. MTT assays showed that treatment with a high concentration of glucose (30 mM) significantly increased apoptosis among cultured GECs as compared with that ...

When cells were subjected to glucose at 30 mM for 24 hr, it was observed early apoptotic changes. MTT assays showed that treatment with a high concentration of glucose (30 mM) significantly increased apoptosis among cultured GECs as compared with that observed for a normal glucose concentration (5 mM). Pretreatment with EGCG (1-100 μM) significantly increased cell viability under the high glucose condition in a dose-dependent manner. DNA fragments was induced by glucose at 30 mM for 24 hr, whereas pretreatment for 2 hr with EGCG (1-100 μM) conferred significant protection against this glucose-induced DNA fragmentation. Moreover, DAPI staining assays showed that treatment with a high concentration of glucose (30 mM) increased DNA fragmentation / condensation in GECs as compared with that observed for a normal glucose concentration (5 mM). Pretreatment of GECs with EGCG (1-100 μM) significantly increased cell viability under the high glucose condition in a dose-dependent manner. Mechanism of reactive oxygen species (ROS)-mediated apoptosis induced by glucose (30 mM) was examined. It was found that glucose at 30 mM rapidly stimulated the generation of intracellular ROS, whereas pretreatment with EGCG (1-100 μM) significantly decreased generation of ROS under the high glucose condition in a dose-dependent manner. As well as, treatment of high glucose (30 mM) increased lipid peroxidation (LPO) and decreased glutathione (GSH) in GECs. Pretreatment with 100 μM EGCG attenuated the increase of LPO and restored the activity of GSH. These results suggest that high glucose-induced oxidative stress is a critical role in GECs apoptosis. Next, the role of EGCG in relation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) was investigated in GECs at high glucose conditions. It was found that glucose at 30 mM rapidly increased the phosphorylations of p38 MAPK and ERK1/2, whereas pretreatment with EGCG (1-100 μM) significantly prevented these phosphorylations in glucose concentration at 30 mM in a dose-dependent manner. Subsequently, it was examined the role of EGCG in the expression of apoptotic factors. Expression of apoptotic factors revealed that EGCG prevented the expression of the pro-apoptotic factors, such as Bad, caspase-3 and -9 by glucose at 30 mM, whereas EGCG pretreatment increased the expressions the anti-apoptotic factors, such as Bcl-2 and Bcl-xL in GECs. These data indicate that EGCG protects against apoptotic cell death by stimulating phospho-p38 MAPK and phospho-ERK1/2 and up-regulation of antiapoptotic factors, the other side down-regulation of proapoptotic factors in high glucose-conditioned GECs. Next, it was investigated the role of transforming growth factor-beta1 (TGF-β1), protein kinase C (PKC) α/βII and nuclear factor-kappaB (NF-κB) in GECs against high glucose injury. Glucose at 30 mM rapidly increased the expressions of mRNA and protein of TGF-β1, the other side, pretreatment with EGCG (1-100 μM) significantly prevented expressions of TGF-β1 in glucose concentration at 30 mM in a dose-dependent manner. In addition, EGCG (100 μM) inhibited phospho-PKC α/βII activation caused by glucose at 30 mM and pretreatment with EGCG (1-100 μM) significantly decreased the expression of transcriptional activity of NF-κB induced by glucose at 30 mM in a dose-dependent manner. These data indicate that 1) the protective mechanism of EGCG against high glucose-induced apoptotic cell death includes stimulation of phospho-p38 MAPK and phospho-ERK1/2 and modulation of apoptosis-regulating factors, 2) EGCG prevents high glucose-induced expression of TGF-β1, phospho-PKC α/βII and NF-κB. Thus, EGCG could be a useful factor modulating the GECs injury by high glucose. Furthermore, EGCG might be a useful candidate for controlling diabetic nephropathy effectively.