Chonnam Med J.  2011 Aug;47(2):116-121. 10.4068/cmj.2011.47.2.116.

Effects of Epigallocatechin-3-Gallate on the Expression of TGF-beta1, PKC alpha/betaII, and NF-kappaB in High-Glucose-Stimulated Glomerular Epithelial Cells

• Park SJ
• Jeong JM
• Jeong HS
• Park JS
• Kim NH

• KMID: 2048795
• DOI: http://doi.org/10.4068/cmj.2011.47.2.116

Abstract

Epigallocatechin-3-gallate (EGCG) is the most potent antioxidant polyphenol in green tea. In the present study, we investigated whether EGCG plays a role in the expression of transforming growth factor-beta1 (TGF-beta1), protein kinase C (PKC) alpha/betaII, and nuclear factor-kappaB (NF-kappaB) in glomerular epithelial cells (GECs) against high-glucose injury. Treatment with high glucose (30 mM) increased reactive oxygen species (ROS)/lipid peroxidation (LPO) and decreased glutathione (GSH) in GECs. Pretreatment with 100 microM EGCG attenuated the increase in ROS/LPO and restored the levels of GSH, whereas ROS, LPO, and GSH levels were not affected by treatment with 30 mM mannitol as an osmotic control. Interestingly, high-glucose treatment affected 3 separate signal transduction pathways in GECs. It increased the expression of TGF-beta1, PKC alpha/betaII, and NF-kappaB in GECs, respectively. EGCG (1, 10, 100 microM) pretreatment significantly decreased the expression of TGF-beta1 induced by high glucose in a dose-dependent manner. In addition, EGCG (100 microM) inhibited the phosphorylation of PKC alpha/betaII caused by glucose at 30 mM. Moreover, EGCG (1, 10, 100 microM) pretreatment significantly decreased the transcriptional activity of NF-kappaB induced by high glucose in a dose-dependent manner. These data suggest that EGCG could be a useful factor in modulating the injury to GECs caused by high glucose.

Keyword

Glomerular epithelial cell; Epigallocatechin-3-gallate; Transforming growth factor-beta1; Protein kinase C alpha/betaII; Nuclear factor-kappaB

MeSH Terms

Catechin
Epithelial Cells
Glucose
Glutathione
Mannitol
NF-kappa B
Phosphorylation
Protein Kinase C
Reactive Oxygen Species
Signal Transduction
Tea
Transforming Growth Factor beta1
Catechin
Glucose
Glutathione
Mannitol
NF-kappa B
Protein Kinase C
Reactive Oxygen Species
Tea
Transforming Growth Factor beta1

Figure

• 

  FIG. 1 Effects of EGCG on ROS production, content of GSH, and lipid peroxidation in glucose-treated GECs. (A) EGCG inhibited the ROS production by 30 mM glucose in GECs. Cells (1×104) were incubated in DMEM-serum-free media for 24 hours. Then the cells were treated with EGCG for 2 hours before adding glucose (30 mM). ROS production was assessed by using DCF-DA, as described in Materials and Methods. (B) EGCG inhibited the GSH suppression by 30 mM glucose in GECs. Cells (6×105) were treated with 30 mM glucose for 24 hours. GSH production was assessed by using DTNB reagent as described in Materials and Methods. (C) EGCG suppressed the LPO production by 30 mM glucose in GECs. LPO production was assessed by using TBA as described in Materials and Methods. *p<0.0001 compared with the control group. †p<0.0001, ‡p<0.0005, compared with the 30 mM glucose-treated group. Values are expressed as means±S.E. of three independent experiments.

• 

  FIG. 2 Effects of EGCG on the mRNA expression of TGF-β1. (A) GECs were pretreated with EGCG (100 µM), glucose (30 mM), or a combination of the two for 24 hours. Cellular RNAs were extracted and the mRNA expression of TGF-β1 and GAPDH was analyzed by RT-PCR. (B) The mRNA expression of TGF-β1 was measured by real-time RT-PCR as described in Materials and Methods. *p<0.0001 compared with the control group. †p<0.001, ‡p<0.0001, compared with the 30 mM glucose-treated group. Values are expressed as means±S.E. of three independent experiments.

• 

  FIG. 3 Effects of EGCG on the protein expression of TGF-β1. TGF-β1 and GAPDH expression was assessed by Western blotting with anti-TGF-β1 mAb or anti-GAPDH mAb, respectively. Results are shown in the top panels and quantified by densitometry (bottom panels). *p<0.005 compared with the control group, †p<0.001, ‡p<0.0005, compared with the 30 mM glucose-treated group. Values are expressed as means±S.E. of three independent experiments.

• 

  FIG. 4 Effects of EGCG on the phosphorylation of PKC-α/βII. Cells (2×106) were incubated in DMEM-serum-free media for 24 hours. Then the cells were treated with EGCG for 2 hours before adding glucose (30 mM). Phosphorylation of PKC-α/βII was assessed by Western blotting with anti-PKC-α mAb or anti-phospho-PKC-α/βII mAb, respectively. *p<0.005 compared with the control group. Results are shown in the top panels and quantified by densitometry (bottom panels). *p<0.001 compared with the control group. †p<0.001 compared with the 30 mM glucose-treated group. Values are expressed as means±S.E. of three independent experiments.

• 

  FIG. 5 EGCG decreased the binding activities of NF-κB increased by 30 mM glucose in GECs. Cells (2×106) were incubated in DMEM-serum-free media for 24 hours. Then the cells were treated with EGCG for 2 hours before adding glucose (30 mM). Lane 1, 5 mM glucose medium nuclear extract; lane 2, 30 mM glucose-treated nuclear extract; lane 3, EGCG 100 µM alone treated nuclear extract; lanes 4-6, EGCG 1-100 µM pretreated for 2 hours before treatment with 30 mM glucose nuclear extract; lane 7, 30 mM mannan-treated nuclear extract; lane 8, nuclear extract with competing unlabeled NF-κB probe.

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