N-acetylcysteine-mediated antioxidation prevents hyperglycemia-induced apoptosis and collagen synthesis in rat mesangial cells.
OBJECTIVE
High-glucose (HG)-induced mesangial apoptosis and fibrogenesis possibly involves reactive oxygen species (ROS) formation and activated mitochondrial stress. We investigated the therapeutic effect of the antioxidant N-acetylcysteine (NAC) on cellular apoptosis and matrix accumulation in HG-treated rat mesangial cells (RMCs).


METHODS
RMCs were cultured in media containing 5 (control) or 35 mM (HG) glucose. Cellular apoptosis was assayed by TdT-mediated dUTP nick-end labeling staining. Collagen and transforming growth factor-1 gene expression were measured by reverse transcriptase-polymerase chain reaction or Northern blotting. Mitochondrial capacity and intracellular ROS generation was assayed by fluorescence microscopy and flow cytometry, respectively. Cellular ATP production and malondialdehyde (MDA) formation were determined by a luciferin-luciferase reaction and high-performance liquid chromatography, respectively. Cytochrome c release, caspase activation and poly(ADP)ribose polymerase cleavage were assayed by Western blotting.


RESULTS
HG-treated RMCs displayed enhanced cellular apoptosis (65%) and collagen gene expression (1.8-fold increase); these reactions could be significantly suppressed by 1 mM NAC (p < 0.05). Intracellular ROS generation, production of ATP and MDA, and caspase-3, -8 and -9 activities were significantly increased in HG-treated RMCs, and were effectively attenuated by addition of NAC.


CONCLUSIONS
It is concluded that NAC prevents HG-induced mesangial apoptosis and fibrogenesis pathways by the reduction of oxidative stress.
Hung
Kuan-Yu
KY
Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC.
Liu
Shin-Yun
SY
Kao
Shu-Huei
SH
Huang
Jenq-Wen
JW
Chiang
Chih-Kang
CK
Tsai
Tun-Jun
TJ
D000111:Acetylcysteine / Q000494:pharmacology* / Q000627:therapeutic use; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis*; D045304:Cytochromes c / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005109:Extracellular Matrix / Q000187:drug effects; D016166:Free Radical Scavengers / Q000494:pharmacology* / Q000627:therapeutic use; D015870:Gene Expression / Q000187:drug effects; D006943:Hyperglycemia / Q000188:drug therapy*; D008315:Malondialdehyde / Q000378:metabolism; D050527:Mesangial Cells / Q000187:drug effects* / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000096:biosynthesis
D016166:Free Radical Scavengers; D017382:Reactive Oxygen Species; D053773:Transforming Growth Factor beta1; D008315:Malondialdehyde; D000255:Adenosine Triphosphate; D003094:Collagen; D045304:Cytochromes c; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases; D000111:Acetylcysteine