Potential neuroprotection of protodioscin against cerebral ischemia-reperfusion injury in rats through intervening inflammation and apoptosis

Abstract

The aim of the current research is to investigate the cerebral-protection of protodioscin on a transient cerebral ischemia-reperfusion (I/R) model and to explore its possible underlying mechanisms. The rats were preconditioned with protodioscin at the doses of 25 and 50mgkg(-1) prior to surgery. Then the animals were subjected to right middle cerebral artery occlusion (MCAO) using an intraluminal method by inserting a thread (90min surgery). After the blood flow was restored in 24h via withdrawing the thread, some representative indicators for the cerebral injury were evaluated by various methods including TTC-staining, TUNEL, immunohistochemistry, and Western blotting. As compared with the operated rats without drug intervening, treatment with protodioscin apparently lowered the death rate and improved motor coordination abilities through reducing the deficit scores and cerebral infarct volume. What's more, an apparent decrease in neuron apoptosis detected in hippocampus CA1 and cortex of the ipsilateral hemisphere might attribute to alleviate the increase in Caspase-3 and Bax/Bcl-2 ratio. Meanwhile, concentrations of several main pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in the serum were also significantly suppressed. Finally, the NF-κB and IκBa protein expressions in the cytoplasm of right injured brain were remarkably up-regulated, while NF-κB in nucleus was down-regulated. Therefore, these observed findings demonstrated that protodioscin appeared to reveal potential neuroprotection against the I/R injury due to its anti-inflammatory and anti-apoptosis properties. This therapeutic effect was probably mediated by the inactivation of NF-κB signal pathways.

Keywords: Bax/Bcl-2 ratio; Caspase-3; Middle cerebral artery occlusion; NF-κB; Pro-inflammatory cytokines; Protodioscin.

Figure 1:

The chemical structure of protodioscin with numbers marked on carbon atoms in aglycone which are consistent with the expression of the ¹H-NMR and ¹³C-NMR in section 3.1.

Figure 2:

The distribution of major blood vessels in the right cerebral hemisphere of a rat. MCA: Middle Cerebral Artery; ICA: Internal Carotid Artery; ECA: External Carotid Artery; CCA: Common Carotid Artery

Figure 3:

The flow diagram of the procedure used for extraction of protodioscin from the rhizome of Dioscorea zingiberensis C.H. Wright

Figure 4:

The flow diagram of isolating pure protodioscin from the extracts with a series of chromatographic technologies. Phase I: The extracts were separated on silica gel column (particle size in 100~160 mesh), and eluted with the solvent system composed of dichloromethane-methanol-Water (the lower layer of this system) at various ratios under a gradient mode. Phase II: The fraction after elution at the ratio of 8:3:1 in previous step was further separated on silica gel column (particle size in 200~300 mesh), and used the solvent system at the same ratio of 8:3:1 again. Phase III: The obtained Frac. A after phase II was loaded on semi-preparative HPLC DAC-HB50 C18 (450 mm×50 mm, 10 μm) and successively separated under the mobile phrase of acetonitrile-water. The ratios in this graph were all the ratios of volumes.

Figure 5:

The chromatograph of purifying protodioscin from Frac. A obtained after tedious steps. The mobile phrase: acetonitrile-water; Flow rate: 50 mL min⁻¹; Conditions of ELSD: the drift tube temperature was set 55, and the gas flow rate was 3 L min⁻¹.

Figure 6:

Therapeutic effect of protodioscin on neurological deficit scores and cerebral infarct volume. The rats were exposed to transient ischemia for 90 min exerted by MCAO and sacrificed 24 h after reperfusion. The treatments with drugs were kept for seven days. The brain was sliced into coronal sections and stained with TTC solution. (a): Representative TTC-stained photos; (b): Neurological deficit scores; (c): Cerebral infarct volume. The obtained values were presented as mean ± SD (n=12 each group). #p versus SG group and *p versus the I/R group, respectively. SG, sham group without occluding the blood flow and drug treatment; I/R, model group without drug treatment after MCAO operation; NG, nimodipine-treated group after MCAO; LG, protodioscin (low dose)-treated group after MCAO; HG, protodioscin (high dose)-treated group after MCAO.

Figure 7:

Therapeutic effect of protodioscin on neurons apoptosis measured by TUNEL staining. The rats were exposed to transient ischemia for 90 min exerted by MCAO and sacrificed 24 h after reperfusion. The treatments with drugs were kept for seven days. The positive TUNEL cells on the photomicrographs were stained with tawny colour and indicated the apoptotic neurons. a and c: The representative images of TUNEL assay in hippocampus CA1 and cortex, respectively. b and d: The quantitative analysis of the positive cells in hippocampus CA1 and the cortex, respectively. The obtained values were presented as mean ± SD (n=5 each group). #p versus SG group and *p versus the I/R group, respectively. Photomicrographs are magnified by 200 times, and scale bar equals 100 μm. SG, sham group without occluding the blood flow and drug treatment; I/R, model group without drug treatment after MCAO operation; NG, nimodipine-treated group after MCAO; LG, protodioscin (low dose)-treated group after MCAO; HG, protodioscin (high dose)-treated group after MCAO.

Figure 8:

Therapeutic effect of protodioscin on Caspase-3 activity evaluated by immunohistochemistry. The rats were exposed to transient ischemia for 90 min exerted by MCAO and sacrificed 24 h after reperfusion. The treatments with drugs were kept for seven days. a and c: The representative pictures of positive Caspase-3 cells in hippocampus CA1 and the cortex, respectively. b and d: The quantitative analysis of the positive cells in hippocampus CA1 and the cortex, respectively. The obtained values were presented as mean ± SD (n=5 each group). #p versus SG group and *p versus the I/R group, respectively. Photomicrographs are magnified by 200 times, and scale bar equals 100 μm. SG, sham group without occluding the blood flow and drug treatment; I/R, model group without drug treatment after MCAO operation; NG, nimodipine-treated group after MCAO; LG, protodioscin (low dose)-treated group after MCAO; HG, protodioscin (high dose)-treated group after MCAO.

Figure 9:

Effect of protodioscin and Nimodipine on expression Bcl-2 and Bax protein in the ipsilateral ischemic hemisphere. The rats were exposed to transient ischemia for 90 min exerted by MCAO and sacrificed 24 h after reperfusion. The treatments with drugs were kept for seven days. (a) Representative results of Western blotting. (b) Quantitative analysis. The protein levels were normalized against the Actin, which served as loading control. Values are expressed in relative optical density and represented as means ± SD (n=3 per group). #p<0.05 versus the Sham group, *p<0.05 versus the I/R group, respectively. SG, sham group without occluding the blood flow and drug treatment; I/R, model group without drug treatment after MCAO operation; NG, nimodipine-treated group after MCAO; LG, protodioscin (low dose)-treated group after MCAO; HG, protodioscin (high dose)-treated group after MCAO.

Figure 10:

Therapeutic effect of protodioscin on pro-inflammatory cytokines determined by ELISA. The rats were exposed to transient ischemia for 90 min exerted by MCAO and sacrificed 24 h after reperfusion. The treatments with drugs were kept for seven days. The units were presented as ng L⁻¹. The obtained values were presented as mean ± SD (n=12 each group). #p versus SG group and *p versus the I/R group, respectively. SG, sham group without occluding the blood flow and drug treatment; I/R, model group without drug treatment after MCAO operation; NG, nimodipine-treated group after MCAO; LG, protodioscin (low dose)-treated group after MCAO; HG, protodioscin (high dose)-treated group after MCAO.

Figure 11:

Therapeutic effect of protodioscin on NF-κB and IκBα protein expression in cytoplasm and nucleus by western blotting analysis. The rats were exposed to transient ischemia for 90 min exerted by MCAO and sacrificed at 24 h after reperfusion. The treatments with drugs were kept for seven days. a: The typical western blotting of NF-κB and IκBα in cytoplasm; b: The typical western blotting of NF-κB in nucleus; c and d: The quantitative analysis. The protein expression levels were normalized to the level of H3, a loading control in this research. The obtained values were presented as mean ± SD (n=3 each group). #p versus SG group and *p versus the I/R group, respectively. SG, sham group without occluding the blood flow and drug treatment; I/R, model group without drug treatment after MCAO operation; NG, nimodipine-treated group after MCAO; LG, protodioscin (low dose)-treated group after MCAO; HG, protodioscin (high dose)-treated group after MCAO.