HMGB-1/RAGE signaling inhibition by dioscin attenuates hippocampal neuron damage induced by oxygen-glucose deprivation/reperfusion

Abstract

Cerebral ischemia is one of the most common clinical diseases characterized by high morbidity and mortality. Neurocyte apoptosis and a cascade of inflammatory signals following cerebral ischemia-reperfusion injury (IRI) may contribute to secondary brain damage, resulting in severe neurological damage. It has been reported that dioscin, a natural steroid saponin, exerts anti-inflammatory properties against different diseases. The present study aimed to investigate the role of dioscin in oxygen-glucose deprivation/reperfusion (OGD/R) induction in hippocampal cells in vitro and in vivo. For the in vitro study, hippocampal cells were collected from rat embryos of gestational age of E18. The oxygen-glucose deprivation model in primary hippocampal neurons was used to mimic cerebral IRI in vitro. To select the optimum dioscin concentration and acting time, cell viability was evaluated by a Cell Counting Kit-8 (CCK-8) assay. Neurons subjected to OGD/R were treated with dioscin and the inflammatory cytokines, high mobility group box chromosomal protein 1 (HMGB-1)/receptor for advanced glycation end products (RAGE) signaling molecules and apoptosis-associated genes were determined. The intracellular reactive oxygen species (ROS) generation was detected. Furthermore, the effects of dioscin on the antioxidant defense mechanisms were evaluated by measuring the activity of glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and the glutathione (GSH)/glutathione disulphide (GSSG) ratio. In addition, OGD/R-induced cells were transfected with pcDNA3.1-HMGB-1 and treated with dioscin, and the neuronal cell apoptosis rate was determined using a terminal deoxynucleotidyl transferase-mediated 2-deoxyuridine 5-triphosphate-biotin nick-end labeling (TUNEL) assay. The mRNA and protein expression levels of the inflammatory factors were measured using real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. For the in vivo investigation, the oxidation and anti-oxidation system in rat hippocampal tissue was evaluated by detecting the expression of the aforementioned oxidative stress-associated proteins, 3-NT as well as 8-oxo-deoxyguanosine (8-OHdG). In the hippocampal region, the apoptotic rate was determined using a TUNEL assay. The results demonstrated that dioscin at a dose of 400 ng/ml significantly reversed the increase in the expression levels of the inflammatory factors and attenuated those of apoptotic cytokines induced by OGD/R. Additionally, dioscin notably reversed the OGD/R-mediated activation of the HMGB-1/RAGE signaling pathway in vitro and in vivo. Cell treatment with dioscin significantly attenuated ROS production and increased the activity of antioxidant enzymes. Additionally, increasing the expression of HMGB-1 inhibited the protective effects of dioscin on cell apoptosis in the OGD/R-induced neurons. Furthermore, HMGB-1 overexpression reversed the antiapoptotic and anti-inflammatory effects of dioscin on neurons. The results of the present study indicated that dioscin exerted anti-inflammatory, antiapoptotic and antioxidant effects via the HMGB-1/RAGE signaling pathway. These results suggest a novel perspective of the protective effects of dioscin as a prospective remedial factor for IRI.

Keywords: apoptosis; dioscin; high mobility group box-1 protein; inflammation; oxygen-glucose deprivation.

Figure 1

Dioscin affects cell viability of hippocampal neurons. Neurons were treated with different concentrations (100, 200, 400 and 800 ng/ml) of dioscin with or without OGD/R for 24 h. Values are presented as the mean ± standard deviation (SD). ^*P<0.05 and ^**P<0.01 compared with the OGD/R-free condition of the control group. ^#P<0.05 compared with OGD/R-free condition of the dioscin-treated group (400 ng/ml). ^&P<0.05 compared with the OGD/R condition of the dioscin-treated group (400 ng/ml). OGD/R, oxygen-glucose deprivation/reperfusion.

Figure 2

Dioscin downregulates the protein expression levels of IL-1, IL-6 and TNF-α. (A) Representative western blot analysis results showing IL-1, IL-6 and TNF-α expression at 24 h following dioscin treatment. (B) Densitometric quantification of the IL-1/β-actin, (C) IL-6/β-actin and (D) TNF-α/β-actin ratio at 24 h following dioscin treatment. (E) Relative mRNA expression of IL-1, IL-6 and TNF-α at 24 h following dioscin treatment. Values are expressed as the mean ± standard error of the mean (SEM). ^**P<0.01 vs. the control group. ^&P<0.05 and ^&&P<0.01 vs. the OGD/R group. IL-1, interleukin 1; TNF-α, tumor necrosis factor α; OGD/R, oxygen-glucose deprivation/reperfusion.

Figure 3

Dioscin treatment inhibits the expression of apoptosis-associated proteins. (A) Representative western blot analysis results indicating the protein expression of cleaved caspase-3, Bax and Bcl-2 at 24 h following dioscin treatment. Densitometric quantification of the (B) cleaved caspase-3/β-actin and (C) Bax/Bcl-2 ratio at 24 h following dioscin treatment. (D) Relative mRNA expression of cleaved caspase-3, Bax and Bcl-2 at 24 h following dioscin treatment. Values are expressed as the mean ± standard error of the mean (SEM). ^**P<0.01 vs. the control group. ^&P<0.05 and ^&&P<0.01 vs. the OGD/R group. OGD/R, oxygen-glucose deprivation/reperfusion.

Figure 4

Dioscin inhibits the OGD/R-mediated activation of the HMGB-1/RAGE pathway. (A) Western blot analysis results showing HMGB-1 and RAGE protein expression at 24 h following dioscin treatment. Densitometric quantification of the (B) HMGB-1/β-actin and (C) RAGE/β-actin ratio at 24 h following dioscin treatment. (D) Relative mRNA expression levels of HMGB-1 and RAGE at 24 h following dioscin treatment. Values are expressed as the mean ± standard error of the mean (SEM). ^*P<0.05 and ^**P<0.01 vs. the control group. ^&P<0.05 and ^&&P<0.01 vs. the OGD/R group. OGD/R, oxygen-glucose deprivation/reperfusion; HMGB-1, high mobility group box chromosomal protein 1; RAGE, receptor for advanced glycation end products.

Figure 5

Dioscin remarkably decreases ROS production and promotes the antioxidant defense system. Dioscin inhibited ROS generation in OGD/R-subjected neurons. (A) DCHF-DA fluorescence intensity was quantified by flow cytometry. Dioscin improved the enzymatic antioxidant defense mechanisms in OGD/R-subjected neurons. (B) GPx, (C) CAT and (D) SOD activities were evaluated in cultured primary hippocampal cells. Dioscin improved the non-enzymatic antioxidant defense mechanisms in OGD/R-subjected neurons. (E) GSH/GSSG ratio was evaluated in primary hippocampal cells. Values are expressed as the mean ± standard error of the mean (SEM). ^*P<0.05 and ^**P<0.01 vs. the control group. ^&P<0.05 and ^&&P<0.01 vs. the OGD/R group. ROS, reactive oxygen species; OGD/R, oxygen-glucose deprivation/reperfusion; DCHF-DA, 2',7'-dichlorofluorescein diacetate; GPx, glutathione peroxidase; CAT, catalase; SOD, superoxide dismutase; GSH/GSSG, glutathione/glutathione disulphide.

Figure 6

Effect of HMGB-1 overexpression on the apoptosis rate of primary hippocampal neurons following dioscin treatment. The transfection effect was confirmed by (A) PCR and (B) western blot analysis. ^##P<0.01 vs. the control vector group. (C) Bar chart of TUNEL-positive cell ratios in different groups. Values are presented as means ± standard deviation (SD). ^**P<0.01 vs. the OGD/R group. (D) TUNEL-stained (green) cells indicate apoptosis-positive cells, DAPI stain (blue) indicates nucleated cells, and the ‘merge’ column shows cells stained with TUNEL and DAPI. Magnification, x200. White boxes represented magnified view of the typical apoptosis cells. Magnification, x400. ^&&P<0.01 vs. the OGD/R + dioscin group. HMGB-1, high mobility group box chromosomal protein 1; TUNEL, terminal deoxynucleotidyl transferase-mediated (dUTP) nick-end labeling; DAPI, 4',6-diamidino-2-phenylindole; OGD/R, oxygen-glucose deprivation/reperfusion.

Figure 7

Inhibiting the activity of the HMGB-1/RAGE pathway reversed the effect of dioscin on the expression of proinflammatory cytokines, ROS generation and antioxidant stress. (A) Western blot analysis results showing IL-1, IL-6 and TNF-α protein expression. β-actin served as an internal control. (B) Bar chart showing the IL-1/β-actin, IL-6/β-actin and TNF-α/β-actin ratio from the western blot analysis results in different groups. (C) Bar chart showing IL-1/β-actin, IL-6/β-actin and TNF-α/β-actin ratio from RT-qPCR results in different groups. (D) DCHF-DA fluorescence intensity in different groups using flow cytometry. (E) GPx, (F) CAT and (G) SOD activity and (H) GSH/GSSG ratio in different groups. Values are presented as the means ± standard deviation (SD). ^*P<0.05 and ^**P<0.01 vs. the OGD/R group. ^&P<0.05 and ^&&P<0.01 vs. the OGD/R + dioscin group. HMGB-1, high mobility group box chromosomal protein 1; RAGE, receptor for advanced glycation end products; ROS, reactive oxygen species; IL-1, interleukin 1; TNF-α, tumor necrosis factor α; DCHF-DA, 2',7'-dichlorofluorescein diacetate; GPx, glutathione peroxidase; CAT, catalase; SOD, superoxide dismutase; GSH/GSSG, glutathione/glutathione disulphide; OGD/R, oxygen-glucose deprivation/reperfusion.

Figure 8

Dioscin remarkably alleviates oxidative stress and apoptosis of hippocampal neurons in the MCAO model via the HMGB-1/RAGE pathway. (A) The effect of dioscin and HMGB-1 on the expression of the 3-NT protein in rat hippocampal neurons. (B) DCHF-DA fluorescence intensity was quantified by flow cytometry. Dioscin improved the enzymatic antioxidant defenses in rat hippocampal neurons in the MCAO group via the HMGB-1 pathway. (C) GPx, (D) CAT and (E) SOD activities were evaluated in rat hippocampal neurons. Dioscin improved the non-enzymatic antioxidant defenses in rat hippocampal neurons of the MCAO group. This effect was not mediated via the HMGB-1 pathway. (F) The GSH/GSSG ratio was evaluated in rat hippocampal neurons of the MCAO group. (G) The effect of dioscin on the aggregation of the 8-OHdG protein in rat hippocampal neurons. 8-OHdG (green) was immunolabeled in rat hippocampal neurons. Magnification, x400. (H) Treatment with dioscin decreased the number of TUNEL-positive apoptotic cells in rat hippocampal tissue. Magnification, x200. Rat brain tissue sections were stained with TUNEL (green) and DAPI (blue). The bar chart shows the quantification of the TUNEL-positive cells (%). Values are expressed as the mean ± standard error of the mean (SEM). ^**P<0.01 vs. the sham group. ^&P<0.05 and ^&&P<0.01 vs. the MCAO group. ^#P<0.05 and ^##P<0.01 vs. the MCAO + dioscin group. MCAO; middle cerebral artery occlusion; HMGB-1, high mobility group box chromosomal protein 1; RAGE, receptor for advanced glycation end products; DCHF-DA, 2',7'-dichlorofluorescein diacetate; GPx, glutathione peroxidase; CAT, catalase; SOD, superoxide dismutase; GSH/GSSG, glutathione/glutathione disulphide; 8-OHdG, 8-hydroxy-2'-deoxyguanosine; TUNEL, terminal deoxynucleotidyl transferase-mediated (dUTP) nick-end labeling; DAPI, 4',6-diamidino-2-phenylindole.