Objective:

2

To investigate protective effects of hirudin on oxidative stress and apoptosis of spinal dorsal root ganglion cells in high-glucose rats at the cellular and molecular level.

Methods:

2

Dorsal root ganglion neurons (DRGn) were harvested from embryonic day in 15 SD rats

purified and identificated after primary culture. They were divided into the normal control group

high-glucose (HG) group

positive control (alpha-lipoic acid

ALA) group

low-dose hirudin group (H1)

medium-dose hirudin group (H2) and high-dose hirudin group (H3). The control group was cultured by neuron specific culture medium

while the HG group was cultured by neuron specific culture medium and 20 mmol/L glucose (HG medium). The hirudin groups were cultured by HG medium+0.25 IU/mL hirudin (H1)

HG medium+0.5 IU/mL hirudin (H2) and HG medium+1 IU/mL hirudin (H3). The ALA group was cultured by HG medium+100 μmol/L ALA. 3-(4

5-dimethylthiazol-2-yl)-2

5-diphenylt etrazolium bromide (MTT) assay was used to explore the optimum concentration and intervention time. Flow cytometry assay was used to detect the level of reactive oxygen series (ROS). Western blot and quantificational realtime polymerase chain reaction (qRT-PCR) were used to detect the expression of protein and mRNA of nuclear factor erythroid 2-related factor 2 (Nrf-2)

hemeoxygence-1 (HO-1)

nuclear factor-κB (NF-κB) and Caspase-3. TUNEL assay was used to test the apoptosis rate of different groups.

Results:

2

After 24 h of culture

the cell activity of hirudin and ALA groups were higher than that of HG group

and there was a statistical difference between the H1 group and HG group (

P

<

0.05). In hirudin groups

the apoptosis rate of cells

the expression of activated Caspase-3 protein and Caspase-3 mRNA were lower than those of HG group (

P

<

0.01)

higher than those of ALA group (

P

<

0.01 or

P

<

0.05). The ROS level of hirudin groups was higher than that of ALA group (

P

<

0.01)

lower than that of HG group (

P

<

0.01 or

P

<

0.05). The expression of NF-κB (P65) protein in H3 group were lower than those of HG group (

P

<

0.05). The expression of Nrf-2 protein in hirudin groups was higher than that of HG group (

P

<

0.01)

lower than that of ALA group (

P

<

0.01 or

P

<

0.05). The expression of HO-1 protein in hirudin groups was lower than that of ALA group (

P

<

0.01 or

P

<

0.05)

higher than that of HG group (

P

<

0.01 or

P

<

0.05).

Conclusions:

2

The activity of DRGn cells can be promoted by hirudin under HG conditions. The effects of hirudin on the inhibition of HG on DRGn cells damage mainly include scavenging ROS

up-regulating Nrf-2/ HO-1 pathway

inhibiting activation of NF-κB pathway

down-regulating the expression of and Caspase-3 and reducing DRGn cell apoptosis.