Yonsei Med J.  2008 Jun;49(3):486-495. 10.3349/ymj.2008.49.3.486.

Protective Effect of Ultra Low Molecular Weight Heparin on Glutamate-Induced Apoptosis in Cortical Cells

• Yu TG
• Zhang QZ
• Zhang ZG
• Wang WW
• Ji SL
• Du GH

• KMID: 724266
• DOI: http://doi.org/10.3349/ymj.2008.49.3.486

Abstract

PURPOSE
To investigate the effect of ultra low molecular weight heparin (ULMWH) on glutamate induced apoptosis in rat cortical cells and to explore the possible mechanisms. MATERIALS AND METHODS: Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was first analyzed with Hoechst 33258 and then confirmed by DNA fragmentation. The concentration of free intracellular calcium ([Ca2+](i)) was determined with fura-2/AM fluorometry. The expression of Bcl-2 family protein and caspase-3 were evaluated with Western blot. RESULTS: Typical apoptotic morphological change in rat cortical cells treated with 100micromol/L glutamate for 24h was detected by Hoechst 33258 staining, which was then confirmed by the DNA ladder of agarose gel electrophoresis. The apoptotic rate of the glutamate treated cells was up to 33.21%, and 24 h of treatment with glutamate increased [Ca2+](i), down-regulated Bcl-2 expression, up-regulated Bax expression, and increased caspase-3 activation in rat cortical cells. Our research demonstrated that ULMWH pretreatment can prevent the glutamate- induced apoptosis, attenuate the increase of [Ca2+](i) not only in medium containing Ca2+ but also in Ca2+-free medium, up-regulate the expression of Bcl-2, down-regulate the expression of Bax, and decrease caspase-3 activation. CONCLUSION: ULMWH has neuroprotective capacity to antagonize glutamate-induced apoptosis in cortical cells, through decrease of Ca2+ release and modulation of apoptotic processes.

Keyword

Ultra low molecular weight heparin; glutamate; apoptosis; Ca2+; caspase-3; Bcl-2; Bax

MeSH Terms

Animals
Apoptosis/*drug effects
Blotting, Western
Calcium/metabolism
Caspase 3/metabolism
Cell Survival/drug effects
Cells, Cultured
Cerebral Cortex/cytology
DNA Fragmentation/drug effects
Glutamic Acid/*pharmacology
Heparin, Low-Molecular-Weight/*pharmacology
Proto-Oncogene Proteins c-bcl-2/metabolism
Rats
Rats, Wistar
bcl-2-Associated X Protein/metabolism

Figure

• 

  Fig. 1 Fluorescence photomicrographs of cortical cells with Hoechst 33258 staining. Assayed cells were from four treatment groups. (A) Control cells; (B) Treatment with 1 mg/L ULMWH; (C) Treatment with 100 µmol/L glutamate; (D) Treatment with 100 µmol/L glutamate and 1 mg/L ULMWH. Examples of cells that would be considered apoptotic by this assay are labeled with arrows.

• 

  Fig. 2 Effect of ULMWH on glutamate-induced DNA fragmentation (DNA ladder) in cortical cells. (A) Control cells; (B) Size mark; (C) Treatment with 1 mg/L ULMWH; (D) Treatment with 100 µmol/L glutamate and 1 mg/L ULMWH; (E) Treatment with 100 µmol/L glutamate.

• 

  Fig. 3 Quantitative apoptotic cortical cells evaluated by Hoechst 33258 staining. Multiple slides from the four treatment groups were viewed: Control cells (Con); Treatment with 1 mg/L ULMWH (U);Treatment with 100 µmol/L glutamate (Glu); Treatment with 100 µmol/L glutamate and 1 mg/L ULMWH (U1). At least 200 cells were viewed for each count. Data were expressed as mean ± SD (n=6). Significance was determined by Student t-test. *p < 0.01 vs control. †p < 0.01 vs glutamate.

• 

  Fig. 4 Effect of ULMWH on glutamate-induced decrease in cell viability. Cell viability was measured by MTT assay. Control cells (Con); Treatment with 100 µmol/L glutamate (Glu); Treatment with 1 mg/L ULMWH (U); Treatment with 100 µmol/L glutamate and 1 mg/L ULMWH (U1). Data were expressed as mean ± SD (n = 6). Significance was determined by Student t-test. *p < 0.01 vs control. †p < 0.01 vs glutamate.

• 

  Fig. 5 Effect of ULMWH on Bcl-2 and Bax protein levels in cortical cells treated with glutamate. (A) Western blot of proteins isolated from cortical cells after glutamate treatment. Control cells (Con); Treatment with 100 µmol/L glutamate (Glu); Treatment with 1 mg/LULMWH (U); Treatment with 100 µmol/L glutamate and 1 mg/L ULMWH (U1). (B) Quantitative analysis of ULMWH on the expression of Bcl-2and Bax (the relative abundance of the immunostaining) determined by Image Quant programmer. The levels of Bcl-2and Bax are expressed as % of the control. Data were expressed as mean ± SD (n = 3). Significance was determined by Student t-test. *p < 0.01 vs control. †p < 0.01 vs glutamate.

• 

  Fig. 6 Effect of ULMWH on the increase of [Ca2+]i in cortical cells induced by 100 µmol/L glutamate application. Control cells (Con); Treatment with 1mg/L ULMWH (U); Treatment with 100 µmol/L glutamate (Glu); Treatment with 100 µmol/L glutamate and 1 mg/L ULMWH (U1). The [Ca2+]i was measured by using fura-2/AM, based ratiometric fluorescence assay. Data were expressed as mean ± SD (n = 6). Significance was determined by Student t-test. *p < 0.01 vs control. †p < 0.05 vs glutamate.

• 

  Fig. 7 Effect of ULMWH on Ca2+ release induced by glutamate. When measuring the [Ca2+]i, the medium containing Ca2+ was changed into Ca2+-free medium. The [Ca2+]i was measured by using fura-2/AM, based ratiometric fluorescence assay. Control cells (Con); 100 µmol/L glutamate (Glu); Treatment with 100 µmol/L glutamate and 1 mg/L ULMWH (U1). Data were expressed as mean ± SD (n = 6). Significance was determined by Student t-test. *p < 0.05 vs control. †p < 0.01 vs glutamate.

• 

  Fig. 8 Effcet of ULMWH on glutamate-induced caspase-3 activation. (A) Western blot of proteins isolated from cortical cells after glutamate treatment. Control cells (Con); Treatment with 100 µmol/L glutamate (Glu); Treatment with 1 mg/LULMWH (U); Treatment with 100 µmol/L glutamate and 1 mg/L ULMWH (U1). (B) Quantitative analysis of ULMWH on the expression of caspase-3 (the relative abundance of the immunostaining) determined by Image Quant programmer. The levels of caspase-3 are expressed as % of the control. Data were expressed as mean ± SD (n = 6). Significance was determined by Student t-test. *p < 0.01 vs control. †p < 0.01 vs glutamate.

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