J Korean Med Sci.  2009 Jan;24(Suppl 1):S204-S209. 10.3346/jkms.2009.24.S1.S204.

The Protective Effects of Green Tea Extract against L-arginine Toxicity to Cultured Human Mesangial Cells

• Shin BC
• Ryu HH
• Chung JH
• Lee BR
• Kim HL

• KMID: 1778163
• DOI: http://doi.org/10.3346/jkms.2009.24.S1.S204

Abstract

The aim of this study was to investigate whether green tea extract (GTE) has the protective effects on excess L-arginine induced toxicity in human mesangial cell. Human mesangial cells treated with L-arginine were cultured on Dulbecco's modified eagle medium in the presence and absence of inducible nitric oxide synthase (iNOS) inhibitor and GTE. The cell proliferation was determined by 3 (4,5-dimethylthiazol- 2-yl)-2, 5-diphengltetrqzolium bromide, a tetrazole assay. The iNOS mRNA and its protein expression were detected by reverse transcription polymerase chain reaction and Western blot, respectively. The concentration of nitric oxide (NO) was measured by NO enzyme-linced immuno sorbent assay kit. L-arginine significantly inhibited the proliferation of human mesangial cells, and induced the secretion of NO to the media. NO production by L-arginine was significantly suppressed by GTE and iNOS inhibitor (p<0.01). The expression level of iNOS mRNA and its protein that was significantly increased by L-arginine was decreased by iNOS inhibitor but not by GTE. GTE protected the mesangial cells from the NO-mediated cytotoxicity by scavenging the NO rather than by iNOS gene expression. Therefore, we conclude that GTE has some protective effect for renal cells against oxidative injury possibly by polyphenols contained in GTE.

Keyword

Green Tea Extract; Arginine; Nitric Oxide; Polyphenols

MeSH Terms

Antioxidants/metabolism
Arginine/metabolism/pharmacology/*toxicity
Cell Line
Cell Proliferation
Cell Survival
Flavonoids/metabolism
Glomerular Mesangium/cytology/metabolism
Humans
Mesangial Cells/*cytology/metabolism
Nitric Oxide/chemistry/metabolism
Nitric Oxide Synthase Type II/metabolism
Phenols/metabolism
RNA, Messenger/metabolism
Reverse Transcriptase Polymerase Chain Reaction
Tea

Figure

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  Fig. 1 Toxicity of L-arginine on the proliferation of human mesangial cells. Cell viabilities were measured by MTT assay. Indicated amount of L-arginine were added to mesangial cell for 48 hr (n=10 wells. Values expressed as mean±SD. *p<0.05 and †p<0.01 as compared to control group. OD, Optical density.

• 

  Fig. 2 Effect of iNOS inhibitor (L-NAME) on L-arginine induced human mesangial cells toxicity (96-well microplates). Cell viabilities were measured by MTT assay. Mesangial cells were treated with indicated amounts of L-arginine in the presence and absence of iNOS inhibitor (L-NAME, 3 mM/L, 4 mM/L, and 5 mM/L) for 48 hr (n=9 wells). Values expressed as mean±SD. *p<0.05 as compared to L-arginine group. OD, Optical density.

• 

  Fig. 3 Effect of green tea extract on proliferation of human mesangial cells (96-well microplates). Cell viabilities were measured by MTT assay. Mesangial cells were treated with indicated amounts of L-arginine in the presence and absence of GTE (10 µL, 20 µL, and 30 µL) for 48 hr (n=9 wells). Values expressed as mean±SD. *p<0.05 and †p<0.01 as compared to L-arginine group. OD, Optical density.

• 

  Fig. 4 The expression of iNOS mRNA and its protein in human mesangial cells by RT-PCR and Western blot. (A) Mesangial cell pretreated with L-arginine were cultured with iNOS inhibitor (L-NAME) or GTE and were analyzed by RT-PCR for iNOS mRNA. M, molecular marker; C, control; A1, L-arginine 25 µM/L; A2, L-arginine 50 µM/L; A3, L-arginine 100 µM/L; I, iNOS inhibitor 5 mM; G1, GTE 10 µL; G2, GTE 20 µL; G3, GTE 30 µL. (B) Mesangial cell pretreated with L-arginine (50 µM/L) were cultured with iNOS inhibitor (L-NAME, 5 mM) or GTE (30 µL) and were analyzed by Western blot. C, control; A, L-arginine 50 µM/L; A+I, L-arginine 50 µM/L+iNOS inhibitor 5 mM; A+G, L-arginine 50 µM/L+GTE 30 µL.

• 

  Fig. 5 Concentration of NO (nitric oxide). Mesangial cell pretreated with L-arginine (50 µM/L) were cultured with iNOS inhibitor (L-NAME, 5 mM) or GTE (30 µL) and were analyzed by NO ELISA kit. *p<0.01 as compared to control group; †p<0.01 as compared to L-arginine group.

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