J Korean Neurosurg Soc.  2016 May;59(3):259-268. 10.3340/jkns.2016.59.3.259.

Sildenafil Ameliorates Advanced Glycation End Products-Induced Mitochondrial Dysfunction in HT-22 Hippocampal Neuronal Cells

• Sung SK
• Woo JS
• Kim YH
• Son DW
• Lee SW
• Song GS

• KMID: 2192103
• DOI: http://doi.org/10.3340/jkns.2016.59.3.259

Abstract

OBJECTIVE
Accumulation of advanced glycation end-products (AGE) and mitochondrial glycation is importantly implicated in the pathological changes of the brain associated with diabetic complications, Alzheimer disease, and aging. The present study was undertaken to determine whether sildenafil, a type 5 phosphodiesterase type (PDE-5) inhibitor, has beneficial effect on neuronal cells challenged with AGE-induced oxidative stress to preserve their mitochondrial functional integrity.
METHODS
HT-22 hippocampal neuronal cells were exposed to AGE and changes in the mitochondrial functional parameters were determined. Pretreatment of cells with sildenafil effectively ameliorated these AGE-induced deterioration of mitochondrial functional integrity.
RESULTS
AGE-treated cells lost their mitochondrial functional integrity which was estimated by their MTT reduction ability and intracellular ATP concentration. These cells exhibited stimulated generation of reactive oxygen species (ROS), disruption of mitochondrial membrane potential, induction of mitochondrial permeability transition, and release of the cytochrome C, activation of the caspase-3 accompanied by apoptosis. Western blot analyses and qRT-PCR demonstrated that sildenafil increased the expression level of the heme oxygenase-1 (HO-1). CoPP and bilirubin, an inducer of HO-1 and a metabolic product of HO-1, respectively, provided a similar protective effects. On the contrary, the HO-1 inhibitor ZnPP IX blocked the effect of sildenafil. Transfection with HO-1 siRNA significantly reduced the protective effect of sildenafil on the loss of MTT reduction ability and MPT induction in AGE-treated cells.
CONCLUSION
Taken together, our results suggested that sildenafil provides beneficial effect to protect the HT-22 hippocampal neuronal cells against AGE-induced deterioration of mitochondrial integrity, and upregulation of HO-1 is involved in the underlying mechanism.

Keyword

Sildenafil; Glycation end products, advanced; Mitochondria; Hippocampus

MeSH Terms

Adenosine Triphosphate
Aging
Alzheimer Disease
Apoptosis
Bilirubin
Blotting, Western
Brain
Caspase 3
Cytochromes c
Diabetes Complications
Glycosylation End Products, Advanced
Heme Oxygenase-1
Hippocampus
Membrane Potential, Mitochondrial
Mitochondria
Neurons*
Oxidative Stress
Permeability
Reactive Oxygen Species
RNA, Small Interfering
Transfection
Up-Regulation
Sildenafil Citrate
Adenosine Triphosphate
Bilirubin
Caspase 3
Cytochromes c
Glycosylation End Products, Advanced
Heme Oxygenase-1
RNA, Small Interfering
Reactive Oxygen Species

Figure

• 

  Fig. 1 Effect of sildenafil on MTT reduction ability in AGE-treated cells. A : Cells were incubated for indicated time periods in control media with BSA (open circle), in the presence of AGE alone (200 µg/mL, solid circle), or AGE with anti-RAGE antibody (1 µg/mL, solid square). B : MTT reduction was measured after 48 hr incubation in media containing combinations of AGE, Vehicle (Veh), sildenafil citrate (SC, 20 µM), Rp-8-Br-cGMP (RBcGMP, 20 µM). Data were represented as the mean±SEM of 5 experiments. *p<0.01 vs. control, †p<0.01 vs. Veh, ‡p<0.01 vs. SC.

• 

  Fig. 2 Effect of sildenafil on cellular ATP content in AGE-treated cells. A : Cells were incubated for indicated time periods in control media with BSA (open circle), in the presence of AGE alone (200 µg/mL, closed circle), or AGE with anti-RAGE antibody (1 µg/mL, solid square). B : Cellular ATP was measured after 48 hr incubation in media containing combinations of AGE, vehicle (Veh), sildenafil citrate (SC, 20 µM), Rp-8-Br-cGMP (RBcGMP, 20 µM). Data were represented as the mean±SEM of 6 experiments. *p<0.01 vs. control, †p<0.01 vs. Veh, ‡p<0.01 vs. SC.

• 

  Fig. 3 Effect of sildenafil on ROS generation in AGE-treated cells. Flow cytometric analysis of DCFH-DA-loaded cells. A : Control. B : AGE-treated. C : ROS formation was measured by flow cytometric analysis of DCF fluorescence after 48 hr incubation in media containing combinations of AGE, vehicle (Veh), sildenafil citrate (SC, 20 µM), Rp-8-Br-cGMP (RBcGMP, 20 µM). Data were represented as the mean±SEM of 5 experiments. *p<0.01 vs. control, †p<0.01 vs. Veh, ‡p<0.01 vs. SC.

• 

  Fig. 4 Effect of AGE and sildenafil on mitochondrial membrane potential. Flow cytometric analysis of DIOC6(3)-loaded cell. A : Control. B : AGE-treated. C : Flow cytometric analysis was performed after 48 hr incubation in media containing combinations of AGE, vehicle (Veh), sildenafil citrate (SC, 20 µM), Rp-8-Br-cGMP (RBcGMP, 20 µM). Data were represented as the mean±SEM of 6 experiments. *p<0.01 vs. control, †p<0.01 vs. Veh, ‡p<0.01 vs. SC.

• 

  Fig. 5 Effect of sildenafil on mitochondrial permeability transition in AGE-treated cells. A and B : Confocal microscopic images showing cells with intact mitochondria and cells with mitochondrial permeability transition. Intact mitochondria accumulate TMR and are discriminated as punctuated bright red spots, whereas mitochondria undergoing MPT get deprived of TMR, became permeable to calcein, and as a consequence, loose their visible contour. Arrows indicate cells undergoing MPT. C : Confocal microscopic analysis was performed after 48 hr incubation in media containing combinations of AGE, vehicle (Veh), sildenafil citrate (SC, 20 µM), Rp-8-Br-cGMP (RBcGMP, 20 µM). Data were represented as the mean±SEM of 6 experiments. *p<0.01 vs. control, †p<0.01 vs. Veh, ‡p<0.01 vs. SC.

• 

  Fig. 6 Effect of sildenafil on cytochrome C release in AGE-treated cells. A : Western blot analysis was performed for cytosolic released cytochrome C after incubation for indicated time periods in control media with BSA, in the presence of AGE (200 µg/mL, closed circle), or AGE with sildenafil (SC, 20 µM). B : Western blot analysis was performed after 48 hr incubation in media containing combinations of AGE, vehicle (Veh), sildenafil citrate (SC, 20 µM), Rp-8-Br-cGMP (RBcGMP, 20 µM). Data were represented as the mean±SEM of 6 experiments. *p<0.01 vs. control, †p<0.01 vs. Veh, ‡p<0.01 vs. SC.

• 

  Fig. 7 Effect of sildenafil on apoptosis in AGE-treated cells. A : Representative micrographs of Hoechst-33258-stained cells. Arrows indicate apoptotic cells. B : Apoptosis was analyzed by staining with Hoechst-33258 after 48 hr incubation in media containing combinations of AGE, vehicle (Veh), sildenafil citrate (SC, 20 µM), Rp-8-Br-cGMP (RBcGMP, 20 µM). Data were represented as the mean±SEM of 6 experiments. *p<0.01 vs. control, †p<0.01 vs. Veh, ‡p<0.01 vs. SC.

• 

  Fig. 8 Increased HO-1 expression by sildenafil. Cells were incubated for 48 hr in the presence of each 200 µg/mL of control BSA or AGE with vehicle (DMSO, Veh), 20 µM sildenafil citrate (SC) and 20 µM cobalt protophorphyrin (CoPP). A : HO-1 mRNA was determined by qRT-PCR. B and C : HO-1 protein was determined by Western blot analysis. Data were represented as the mean±SEM of 4 experiments. *p<0.01 vs. Veh.

• 

  Fig. 9 Effects of HO-1 related agents on AGE-induced changes in MTT reduction and MPT induction. Cells were incubated in the presence of each 200 µg/mL of control BSA or AGE-BSA (AGE) for 24 hr with or without each 20 µM of sildenafil citrate (SC), cobalt protophorphyrin (CoPP), bilirubin (Bili), and zinc protoporphyrin IX (ZnPP). A : MTT reduction was determined by colorimetric analysis of formazan. B : Confocal microscopic analysis was performed in cells double-stained with TMRM and calcein/AM. Data were represented as the mean±SEM of 4 experiments. *p<0.01 vs. control, †p<0.01 vs. AGE+SC.

• 

  Fig. 10 A : Effect of siRNA transfection on HO-1 protein expression. Cells were transfected with 30 picomoles of HO-1 siRNA or scrambled siRNA (siRNA control) and incubated for 24 hr with vehicle (Veh) or each 20 µM of sildenafil citrate (SC) and cobalt protophorphyrin (CoPP). HO-1 protein was determined by Western blot analysis of the cell extracts. B : MTT reduction was determined by colorimetric analysis. C : Confocal microscopic analysis was performed in cells double-stained with TMRM and calcein/AM. Data were represented as the mean±SEM of 4 experiments. *p<0.01 vs. BSA control, †p<0.01 vs. AGE alone, ‡p<0.01 vs. siRNA control.

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