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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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Related in: MedlinePlus

As a tyrosine kinase inhibitor, genistein mitigates tumor necrosis factor α (TNF-α) release in stimulated retinal microglia cells. A: Genistein’s dose-dependent inhibition of TNF-α release from activated microglia. Cells were treated with (500 μg/ml) glycated albumin for 4 h in the presence of indicated concentrations of genistein. TNF-α released was analyzed by enzyme-linked immunosorbent assay (ELISA). Values represent the means percentage±SD of TNF-α release compared with that of glycated albumin-treated in presence of vehicle for three experiments. B: Genistein also had no effect on cell viability, as determined by trypan blue exclusion test. C: Time-dependent, glycated albumin-induced tyrosine phosphorylation in microglial cells. Cells were treated with (500 μg/ml) glycated albumin in the presence or absence of 100 µM genistein for the indicated time. Phosphorylated tyrosine was determined by Western analysis. Intensities of phosphorylated tyrosine for each time points were compared with the control (time 0). Data shown is the mean±SD of three experiments. * p<0.001 compared with 0 time; $ p<0.001 compared with non-genistein treated, 1 h; # p<0.001 compared with non-genistein treated, 4 h.
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f3: As a tyrosine kinase inhibitor, genistein mitigates tumor necrosis factor α (TNF-α) release in stimulated retinal microglia cells. A: Genistein’s dose-dependent inhibition of TNF-α release from activated microglia. Cells were treated with (500 μg/ml) glycated albumin for 4 h in the presence of indicated concentrations of genistein. TNF-α released was analyzed by enzyme-linked immunosorbent assay (ELISA). Values represent the means percentage±SD of TNF-α release compared with that of glycated albumin-treated in presence of vehicle for three experiments. B: Genistein also had no effect on cell viability, as determined by trypan blue exclusion test. C: Time-dependent, glycated albumin-induced tyrosine phosphorylation in microglial cells. Cells were treated with (500 μg/ml) glycated albumin in the presence or absence of 100 µM genistein for the indicated time. Phosphorylated tyrosine was determined by Western analysis. Intensities of phosphorylated tyrosine for each time points were compared with the control (time 0). Data shown is the mean±SD of three experiments. * p<0.001 compared with 0 time; $ p<0.001 compared with non-genistein treated, 1 h; # p<0.001 compared with non-genistein treated, 4 h.

Mentions: Of note, the increment of TNF-α level is significantly measureable at 4 h after glycated albumin treatment and continued for 24 h (Ibrahim et al. submitted). Moreover, this early time point was chosen in this study to minimize the positive feed-back effect of TNF-α [19]. The concentration of glycated albumin (500 µg/ml) chosen in our study is close to what has been used previously by several investigators to study other glycated albumin-mediated responses such as stimulation of glomerular endothelial and mesangial cell expression of collagen type IV [20] and represents those found in clinical specimens. In non diabetic individuals, ~1% of serum albumin is in the glycated form, which is equivalent to concentrations of 300–400 µg/ml of glycated albumin. The concentration of glycated albumin is increased one-and-a-half- to threefold in diabetic subjects, according to recent glycemic status [21]. To assess the ability of genistein to reduce TNF-α release, microglial cells were pretreated with indicated concentrations of genistein for 1/2 h then stimulated with glycated albumin for 4 h. The supernatants were collected and assayed for TNF-α by ELISA. As shown in Figure 3A, genistein inhibited glycated albumin-mediated TNF-α release in a dose-dependent manner. To ensure that this effect was not caused by nonspecific cytotoxicity of genistein, we assessed cell viability in microglial cells 4 h after an exposure to genistein, using the trypan blue exclusion test. As shown in Figure 3B, genistein did not affect cell viability (88%–94% vital cells), which indicates that the decrease in TNF-α release was indeed consecutive to genistein’s anti-inflammatory effect but not to cell death.


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

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

As a tyrosine kinase inhibitor, genistein mitigates tumor necrosis factor α (TNF-α) release in stimulated retinal microglia cells. A: Genistein’s dose-dependent inhibition of TNF-α release from activated microglia. Cells were treated with (500 μg/ml) glycated albumin for 4 h in the presence of indicated concentrations of genistein. TNF-α released was analyzed by enzyme-linked immunosorbent assay (ELISA). Values represent the means percentage±SD of TNF-α release compared with that of glycated albumin-treated in presence of vehicle for three experiments. B: Genistein also had no effect on cell viability, as determined by trypan blue exclusion test. C: Time-dependent, glycated albumin-induced tyrosine phosphorylation in microglial cells. Cells were treated with (500 μg/ml) glycated albumin in the presence or absence of 100 µM genistein for the indicated time. Phosphorylated tyrosine was determined by Western analysis. Intensities of phosphorylated tyrosine for each time points were compared with the control (time 0). Data shown is the mean±SD of three experiments. * p<0.001 compared with 0 time; $ p<0.001 compared with non-genistein treated, 1 h; # p<0.001 compared with non-genistein treated, 4 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: As a tyrosine kinase inhibitor, genistein mitigates tumor necrosis factor α (TNF-α) release in stimulated retinal microglia cells. A: Genistein’s dose-dependent inhibition of TNF-α release from activated microglia. Cells were treated with (500 μg/ml) glycated albumin for 4 h in the presence of indicated concentrations of genistein. TNF-α released was analyzed by enzyme-linked immunosorbent assay (ELISA). Values represent the means percentage±SD of TNF-α release compared with that of glycated albumin-treated in presence of vehicle for three experiments. B: Genistein also had no effect on cell viability, as determined by trypan blue exclusion test. C: Time-dependent, glycated albumin-induced tyrosine phosphorylation in microglial cells. Cells were treated with (500 μg/ml) glycated albumin in the presence or absence of 100 µM genistein for the indicated time. Phosphorylated tyrosine was determined by Western analysis. Intensities of phosphorylated tyrosine for each time points were compared with the control (time 0). Data shown is the mean±SD of three experiments. * p<0.001 compared with 0 time; $ p<0.001 compared with non-genistein treated, 1 h; # p<0.001 compared with non-genistein treated, 4 h.
Mentions: Of note, the increment of TNF-α level is significantly measureable at 4 h after glycated albumin treatment and continued for 24 h (Ibrahim et al. submitted). Moreover, this early time point was chosen in this study to minimize the positive feed-back effect of TNF-α [19]. The concentration of glycated albumin (500 µg/ml) chosen in our study is close to what has been used previously by several investigators to study other glycated albumin-mediated responses such as stimulation of glomerular endothelial and mesangial cell expression of collagen type IV [20] and represents those found in clinical specimens. In non diabetic individuals, ~1% of serum albumin is in the glycated form, which is equivalent to concentrations of 300–400 µg/ml of glycated albumin. The concentration of glycated albumin is increased one-and-a-half- to threefold in diabetic subjects, according to recent glycemic status [21]. To assess the ability of genistein to reduce TNF-α release, microglial cells were pretreated with indicated concentrations of genistein for 1/2 h then stimulated with glycated albumin for 4 h. The supernatants were collected and assayed for TNF-α by ELISA. As shown in Figure 3A, genistein inhibited glycated albumin-mediated TNF-α release in a dose-dependent manner. To ensure that this effect was not caused by nonspecific cytotoxicity of genistein, we assessed cell viability in microglial cells 4 h after an exposure to genistein, using the trypan blue exclusion test. As shown in Figure 3B, genistein did not affect cell viability (88%–94% vital cells), which indicates that the decrease in TNF-α release was indeed consecutive to genistein’s anti-inflammatory effect but not to cell death.

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