Quercetin protects rat dorsal root ganglion neurons against high glucose-induced injury in vitro through Nrf-2/HO-1 activation and NF-κB inhibition

Abstract

Aim: To examine the effects of quercetin, a natural antioxidant, on high glucose (HG)-induced apoptosis of cultured dorsal root ganglion (DRG) neurons of rats.

Methods: DRG neurons exposed to HG (45 mmol/L) for 24 h were employed as an in vitro model of diabetic neuropathy. Cell viability, reactive oxygen species (ROS) level and apoptosis were determined. The expression of NF-кB, IкBα, phosphorylated IкBα and Nrf2 was examined using RT PCR and Western blot assay. The expression of hemeoxygenase-1 (HO-1), IL-6, TNF-α, iNOS, COX-2, and caspase-3 were also examined.

Results: HG treatment markedly increased DRG neuron apoptosis via increasing intracellular ROS level and activating the NF-κB signaling pathway. Co-treatment with quercetin (2.5, 5, and 10 mmol/L) dose-dependently decreased HG-induced caspase-3 activation and apoptosis. Quercetin could directly scavenge ROS and significantly increased the expression of Nrf-2 and HO-1 in DRG neurons. Quercetin also dose-dependently inhibited the NF-κB signaling pathway and suppressed the expression of iNOS, COX-2, and proinflammatory cytokines IL-6 and TNF-α.

Conclusion: Quercetin protects rat DRG neurons against HG-induced injury in vitro through Nrf-2/HO-1 activation and NF-κB inhibition, thus may be beneficial for the treatment of diabetic neuropathy.

Figure 1

Double fluorescent labeling of MAP-2 and DAPI. MAP-2-labeled DRG neurons, DAPI-labeled nuclei of all cells in culture. (Scale bar=75 μm).

Figure 2

Cell viability data from MTT assays. HG had a negative effect on cell viability, which was expressed as the percentage of cytoprotection compared to the control group (set at 100%) (^cP<0.01 vs CON). Treatment with quercetin resulted in a partial reversal of the HG effect (^fP<0.01 vs HG).

Figure 3

Intracellular ROS measurements with a DCFH-DA assay. Results from 10 000 events were analyzed in each sample and corrected for autofluorescence from unlabeled cells. (A) Representative flow cytometric images of hydrogen peroxides. (B) Quantitative analysis of ROS. Data are the mean±SEM from three separate experiments. There was a marked increase in ROS levels in the HG group compared to the control group. Quercetin treatment attenuated this HG effect dose-dependently. ^cP<0.01 vs CON. ^fP<0.01 vs HG.

Figure 4

TUNEL assay demonstrating effects of HG and quercetin on apoptosis of DRG neurons. (A) Apoptotic nuclei labeled with green fluorescence were visualized by fluorescence microscopy (100×) (a, CON; b, HG; c, HG+2.5 mmol/L quercetin (Q2.5); d, HG+5 mmol/L quercetin (Q5); e, HG+10 mmol/L quercetin (Q10). (B) Images of apoptotic cells (×400). TUNEL labeling is revealed by black-brown DAB staining; negative cells were counterstained blue-purple by hematoxylin (image key same as in A). (C) Quantitative analysis of percentages of apoptotic cells. Data are the mean±SEM from three independent experiments. The percentage of apoptotic cells was increased in HG-exposed cells. The presence of quercetin dose-dependently reduced the frequency of apoptotic DRG neurons under the HG condition (^cP<0.01 vs CON. ^fP<0.01 vs HG).

Figure 5

Relative levels of Nrf-2 and HO-1 in different groups of cells as determined by Western blot assays and RT-PCR. (A) Representative Western blots. (B) Quantitative analysis of Nrf-2 and HO-1 protein. Protein expression levels of Nrf-2 and HO-1 were significantly decreased in the HG group compared to the normal glucose exposure control group. Quercetin attenuated the HG-induced reductions in Nrf-2 and HO-1. (C) Relative mRNA transcript levels of Nrf-2 and HO-1. Quercetin normalized the mRNA levels of Nrf-2 and HO-1. Data are the mean±SEM from three independent experiments. ^bP<0.05, ^cP<0.01 vs CON. ^eP<0.05, ^fP<0.01 vs HG.

Figure 6

Relative levels of NF-кB, IкBα, and p-IкBα as determined by RT-PCR and Western blot assays. (A) Representative images of Western blots. (B) Quantitative analysis of NF-кB, IкBα, and p-IкBα protein levels. Protein expression levels of NF-кB, IкBα, and p-IкBα were significantly increased in the HG group compared to the normal glucose exposure control group. Quercetin attenuated the effects of HG on the expressions of NF-кB, IкBα, and p-IкBα. (C) Relative mRNA transcript levels of NF-кB. Quercetin normalized the mRNA levels of NF-кB. Data are the mean±SEM from three independent experiments. ^bP<0.05, ^cP<0.01 vs CON. ^eP<0.05, ^fP<0.01 vs HG.

Figure 7

Relative levels of IL-6 and TNF-α expression as determined by Western blot assays and RT-PCR. (A) Representative images of Western blots. (B) Quantitative analysis of IL-6 and TNF-α protein. Protein expression levels of IL-6 and TNF-α were significantly increased in the HG group compared to the control group. Quercetin attenuated the effect of HG on the expressions of IL-6 and TNF-α. (C) Relative mRNA transcript levels of IL-6 and TNF-α. Treatment with quercetin normalized the mRNA levels of IL-6 and TNF-α. Data are the mean±SEM from three independent experiments. ^bP<0.05, ^cP<0.01 vs CON. ^eP<0.05, ^fP<0.01 vs HG.

Figure 8

Relative expressions of iNOS and COX-2 as determined by Western blot and RT-PCR experiments. (A) Representative Western blots. (B) Quantitative analysis of iNOS and COX-2 protein. iNOS and COX-2 expression were increased in the HG group compared to the control group. Quercetin attenuated the effects of HG on the expression levels of iNOS and COX-2. (C) Relative mRNA transcript levels of iNOS and COX-2. Quercetin normalized the mRNA levels of iNOS and COX-2. Data are the mean±SEM from three independent experiments. ^bP<0.05, ^cP<0.01 vs CON. ^eP<0.05, ^fP<0.01 vs HG.

Figure 9

Caspase-3 levels as determined Western blot assays and RT-PCR. (A) Representative images. (B) Quantitative analysis of the cleaved caspase-3. Relative levels of cleaved caspase-3 were increased markedly in HG-treated cells compared to normal glucose conditions. Addition of quercetin reduced caspase-3 cleavage induced by HG in a dose-dependent manner. Intact caspase-3 levels were similar across all five groups. (C) Relative mRNA transcript levels of caspase-3. Quercetin normalized the mRNA levels of caspase-3, but not in parallel with caspase-3 protein, which indicates that quercetin decreases the apoptosis of DRG neurons mainly by inhibiting the cleavage of caspase-3. Quantitative data are expressed as the mean±SEM from three independent experiments. ^bP<0.05, ^cP<0.01 vs CON. ^eP<0.05, ^fP<0.01 vs HG.