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Genistein attenuates retinal inflammation associated with diabetes by targeting of microglial activation.

Ibrahim AS, El-Shishtawy MM, Peña A, Liou GI - Mol. Vis. (2010)

Bottom Line: Genistein represses the release of TNF-α and significantly inhibits ERK and P38 phosphorylation in activated microglial cells by acting as a tyrosine kinase inhibitor.These findings show genistein to be effective in dampening diabetes-induced retinal inflammation by interfering with inflammatory signaling (ERK and P38 MAPKs) that occurs in activated microglia.This beneficial effect of genistein may represent a new intervention therapy to modulate early pathological pathways long before the occurrence of vision loss among diabetics.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Medical College of Georgia, Augusta, GA 30912, USA.

ABSTRACT

Purpose: Diabetic retinopathy (DR) is associated with microglial activation and increased levels of inflammatory cytokines. Genistein, a tyrosine kinase inhibitor, has been shown to possess anti-inflammatory potential that so far untested in animal models of diabetes. The aims of this study are to evaluate the efficacy of genistein for alleviation of diabetes-induced retinal inflammation and also to gain insight into the molecular mechanisms involved therein by analyzing the effect of genistein on concomitant microglia activation in the diabetic retina and in isolated cells.

Methods: Streptozotocin (STZ)-induced diabetic Sprague Dawley rats were used. After diabetes was established for two weeks a single intravitreal injection of genistein or vehicle was performed. Forty-eight hours later, rats were killed, their retinal and vitreal samples were processed for Quantitative Real Time-PCR (qRT-PCR) and Enzyme-linked immunosorbent assay (ELISA) analyses, respectively. For the in vitro study, isolated microglial cells from retinas of newborn rats were used.

Results: mRNA as well as protein levels for tumor necrosis factor α (TNF-α), a robust marker of inflammation, were increased in the retina early in the course of diabetes. Moreover, diabetes resulted in elevation of ionized calcium binding adaptor molecule-1 (Iba1) mRNA, known to be upregulated in activated microglia. These effects of diabetes in retina were all reduced by intervention treatment with genistein. Using an in vitro bioassay, we demonstrated the release of TNF-α from microglia activated by glycated albumin, a risk factor for diabetic disorders. This inflammatory signal involves the activation of tyrosine kinase and its subsequent events, ERK and P38 MAPKs. Genistein represses the release of TNF-α and significantly inhibits ERK and P38 phosphorylation in activated microglial cells by acting as a tyrosine kinase inhibitor.

Conclusions: These findings show genistein to be effective in dampening diabetes-induced retinal inflammation by interfering with inflammatory signaling (ERK and P38 MAPKs) that occurs in activated microglia. This beneficial effect of genistein may represent a new intervention therapy to modulate early pathological pathways long before the occurrence of vision loss among diabetics.

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Accumulation of glycated albumin in diabetic retina and its inflammatory potential for microglia cells. A: Glycated albumin expression in normal, 2-weeks diabetic rats, and 12.5 ng of glycated albumin product (Sigma), analyzed by western blot using mouse monoclonal antibody A717. The mean ratio±SD of the intensity of glycated albumin versus actin was indicated below each group. The ratio of normal, non diabetic rats was taken as 1.0. B: Dose-dependent release of tumor necrosis factor α (TNF-α) in glycated albumin-treated microglial cells. Microglial cells were stimulated with 10, 50, 200, 500 µg/ml glycated albumin for 4 h. TNF-α levels assayed by enzyme-linked immunosorbent assay (ELISA) in culture supernatant were compared with corresponding dosage of non-glycated albumin-treated cells and are expressed as means±SD for three independent experiments.
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f2: Accumulation of glycated albumin in diabetic retina and its inflammatory potential for microglia cells. A: Glycated albumin expression in normal, 2-weeks diabetic rats, and 12.5 ng of glycated albumin product (Sigma), analyzed by western blot using mouse monoclonal antibody A717. The mean ratio±SD of the intensity of glycated albumin versus actin was indicated below each group. The ratio of normal, non diabetic rats was taken as 1.0. B: Dose-dependent release of tumor necrosis factor α (TNF-α) in glycated albumin-treated microglial cells. Microglial cells were stimulated with 10, 50, 200, 500 µg/ml glycated albumin for 4 h. TNF-α levels assayed by enzyme-linked immunosorbent assay (ELISA) in culture supernatant were compared with corresponding dosage of non-glycated albumin-treated cells and are expressed as means±SD for three independent experiments.

Mentions: From aforementioned evidence that highlighted the anti-inflammatory potential of genistein by targeting of microglial activity in STZ-injected rats, a cultured retinal microglia model was developed. This cultured model is advantageous because it offers a more detailed characterization of genistein’s anti-inflammatory actions and also helps elucidate the molecular mechanism responsible for this effect. In this model, we determined the ability of genistein to affect TNF-α release from retinal microglia in response to glycated albumin, a risk factor for diabetic disorders. Glycated albumin was found to be increased in the retina of 2-weeks diabetic rats (Figure 2A) and has been shown to elicit the inflammatory response in isolated microglia cells. This inflammatory response was characterized by a dose-dependent TNF-α production after 4 h incubation with increasing amounts of glycated albumin (Figure 2B).


Genistein attenuates retinal inflammation associated with diabetes by targeting of microglial activation.

Ibrahim AS, El-Shishtawy MM, Peña A, Liou GI - Mol. Vis. (2010)

Accumulation of glycated albumin in diabetic retina and its inflammatory potential for microglia cells. A: Glycated albumin expression in normal, 2-weeks diabetic rats, and 12.5 ng of glycated albumin product (Sigma), analyzed by western blot using mouse monoclonal antibody A717. The mean ratio±SD of the intensity of glycated albumin versus actin was indicated below each group. The ratio of normal, non diabetic rats was taken as 1.0. B: Dose-dependent release of tumor necrosis factor α (TNF-α) in glycated albumin-treated microglial cells. Microglial cells were stimulated with 10, 50, 200, 500 µg/ml glycated albumin for 4 h. TNF-α levels assayed by enzyme-linked immunosorbent assay (ELISA) in culture supernatant were compared with corresponding dosage of non-glycated albumin-treated cells and are expressed as means±SD for three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2965567&req=5

f2: Accumulation of glycated albumin in diabetic retina and its inflammatory potential for microglia cells. A: Glycated albumin expression in normal, 2-weeks diabetic rats, and 12.5 ng of glycated albumin product (Sigma), analyzed by western blot using mouse monoclonal antibody A717. The mean ratio±SD of the intensity of glycated albumin versus actin was indicated below each group. The ratio of normal, non diabetic rats was taken as 1.0. B: Dose-dependent release of tumor necrosis factor α (TNF-α) in glycated albumin-treated microglial cells. Microglial cells were stimulated with 10, 50, 200, 500 µg/ml glycated albumin for 4 h. TNF-α levels assayed by enzyme-linked immunosorbent assay (ELISA) in culture supernatant were compared with corresponding dosage of non-glycated albumin-treated cells and are expressed as means±SD for three independent experiments.
Mentions: From aforementioned evidence that highlighted the anti-inflammatory potential of genistein by targeting of microglial activity in STZ-injected rats, a cultured retinal microglia model was developed. This cultured model is advantageous because it offers a more detailed characterization of genistein’s anti-inflammatory actions and also helps elucidate the molecular mechanism responsible for this effect. In this model, we determined the ability of genistein to affect TNF-α release from retinal microglia in response to glycated albumin, a risk factor for diabetic disorders. Glycated albumin was found to be increased in the retina of 2-weeks diabetic rats (Figure 2A) and has been shown to elicit the inflammatory response in isolated microglia cells. This inflammatory response was characterized by a dose-dependent TNF-α production after 4 h incubation with increasing amounts of glycated albumin (Figure 2B).

Bottom Line: Genistein represses the release of TNF-α and significantly inhibits ERK and P38 phosphorylation in activated microglial cells by acting as a tyrosine kinase inhibitor.These findings show genistein to be effective in dampening diabetes-induced retinal inflammation by interfering with inflammatory signaling (ERK and P38 MAPKs) that occurs in activated microglia.This beneficial effect of genistein may represent a new intervention therapy to modulate early pathological pathways long before the occurrence of vision loss among diabetics.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Medical College of Georgia, Augusta, GA 30912, USA.

ABSTRACT

Purpose: Diabetic retinopathy (DR) is associated with microglial activation and increased levels of inflammatory cytokines. Genistein, a tyrosine kinase inhibitor, has been shown to possess anti-inflammatory potential that so far untested in animal models of diabetes. The aims of this study are to evaluate the efficacy of genistein for alleviation of diabetes-induced retinal inflammation and also to gain insight into the molecular mechanisms involved therein by analyzing the effect of genistein on concomitant microglia activation in the diabetic retina and in isolated cells.

Methods: Streptozotocin (STZ)-induced diabetic Sprague Dawley rats were used. After diabetes was established for two weeks a single intravitreal injection of genistein or vehicle was performed. Forty-eight hours later, rats were killed, their retinal and vitreal samples were processed for Quantitative Real Time-PCR (qRT-PCR) and Enzyme-linked immunosorbent assay (ELISA) analyses, respectively. For the in vitro study, isolated microglial cells from retinas of newborn rats were used.

Results: mRNA as well as protein levels for tumor necrosis factor α (TNF-α), a robust marker of inflammation, were increased in the retina early in the course of diabetes. Moreover, diabetes resulted in elevation of ionized calcium binding adaptor molecule-1 (Iba1) mRNA, known to be upregulated in activated microglia. These effects of diabetes in retina were all reduced by intervention treatment with genistein. Using an in vitro bioassay, we demonstrated the release of TNF-α from microglia activated by glycated albumin, a risk factor for diabetic disorders. This inflammatory signal involves the activation of tyrosine kinase and its subsequent events, ERK and P38 MAPKs. Genistein represses the release of TNF-α and significantly inhibits ERK and P38 phosphorylation in activated microglial cells by acting as a tyrosine kinase inhibitor.

Conclusions: These findings show genistein to be effective in dampening diabetes-induced retinal inflammation by interfering with inflammatory signaling (ERK and P38 MAPKs) that occurs in activated microglia. This beneficial effect of genistein may represent a new intervention therapy to modulate early pathological pathways long before the occurrence of vision loss among diabetics.

Show MeSH
Related in: MedlinePlus