Ameliorating the effect of astragaloside IV on learning and memory deficit after chronic cerebral hypoperfusion in rats

Abstract

Astragaloside IV (AS-IV) has been reported to have a prominent antioxidant effect and was proposed as a promising agent for the prevention of neurodegenerative disorders accompanied by cognitive impairment. The present study investigated the ameliorating effect of AS-IV on learning and memory deficits induced by chronic cerebral hypoperfusion in rats. Rats were treated with two doses of AS-IV (10 and 20 mg/kg, i.p.) daily for 28 days starting from the 5th week after permanent bilateral common carotid artery occlusion. AS-IV treatment (at dose of 20 mg/kg) significantly improved the spatial learning and memory deficits assessed using the Morris water maze test in rats with chronic cerebral hypoperfusion. AS-IV significantly attenuated neuronal apoptosis as well as the levels of superoxide dismutase and lipid peroxidation markers, including malondialdehyde and 4-hydroxy-2-nonenal, in the hippocampus. AS-IV also significantly reduced 8-hydroxy-2'-deoxyguanosine expression, a maker of oxidative DNA damage, while significantly inhibited the astrocyte and microglia activation in the hippocampus. The results indicate that AS-IV has therapeutic potential for the prevention of dementia caused by cerebral hypoperfusion and suggest that the ameliorating effect of AS-IV on learning and memory deficits might be the result of suppressing neuronal apoptosis and oxidative damage in the hippocampus.

Figure 1

Chemical structure of astragaloside IV.

Figure 2

Effect of astragaloside IV (AS-IV) on the acquisition training trials. The escape latency of total trials for 3 days was significantly different between the control, pBCAO, AS-IV(10), and AS-IV(20) groups (A). AS-IV showed significantly shorter escape latency on the 3rd day, particularly at a dose of 20 mg/kg, compared to the pBCAO group (B). Data are presented as mean ± SEM (n = 12 in each group; †, p < 0.05; ††, p < 0.01; †††, p < 0.001 between the control and the pBCAO groups; *, p < 0.05 between the pBCAO and AS-IV groups).

Figure 3

Effects of astragaloside IV (AS-IV) on the retention test. Computerized grid design which used in the retention test (A). Discrete zones are labeled with letters, zone A representing the platform site. Representative swimming tracts in study groups (B). AS-IV on the swimming time spent in discrete zones (C). AS-IV significantly prolonged the swimming time spent in zone B and significantly shortened it in zones F and G at a dose of 20 mg/kg. AS-IV significantly increased the number of target heading on platform site (D) and memory score (F) in the retention test at 20 mg/kg administration. The time for 1st target heading was not different (E). Data are presented as mean ± SEM (n = 12 in each group; †, p < 0.05; †††, p < 0.001 between the control and the pBCAO groups; *, p < 0.05; **, p < 0.01 between the pBCAO and AS-IV groups).

Figure 4

Effects of astragaloside IV (AS-IV) on neuronal apoptosis in the hippocampus. Representative photographs show the CA1 of the hippocampus stained with cresyl violet, TUNEL, Bax and caspase-3 expression (A). Scale bar is 50 µm, applicable to all sections. AS-IV, at both 10 and 20 mg/kg dose, significantly attenuated the neuronal loss and thinned thickness of the CA1 (B,C). AS-IV also significantly reduced TUNEL-positive cells (D) and significantly attenuated the upregulation of Bax (E) and caspase-3 (F) in the CA1 of the hippocampus at both 10 and 20 mg/kg treatment. Representative western blots illustrating differences in the bands of Bax and caspase-3 (G). The upregulation of Bax and caspase-3 protein expression was significantly attenuated by AS-IV at 20 mg/kg dose (H). Data are presented as mean ± SEM (n = 6 in each group; †††, p < 0.001 between the control and the pBCAO groups; *, p < 0.05; **, p < 0.01 between the pBCAO and AS-IV groups).

Figure 5

Effects of astragaloside IV (AS-IV) on the levels of SOD and MDA in brain tissue. AS-IV significantly reduced SOD levels in the cerebral cortex and the hippocampus at a dose of 20 mg/kg (A). AS-IV significantly attenuated the upregulation of MDA levels in the hippocampus at 20 mg/kg treatment (B). Data are presented as mean ± SEM (n = 6 in each group; ††, p < 0.01; †††, p < 0.001 between the control and the pBCAO groups; *, p < 0.05 between the pBCAO and AS-IV groups).

Figure 6

Effects of astragaloside IV (AS-IV) on 4-HNE and 8-OHdG expressions in hippocampal tissue. Representative photographs show the CA1 and DG regions of the hippocampus immuno-stained against 4-HNE and 8-OHdG (A). Scale bar is 100 µm, applicable to all sections. AS-IV at 20 mg/kg significantly attenuated the upregulation of 4-HNE expression in the CA1 of the hippocampus (B). AS-IV 10 and 20 mg/kg treatment significantly attenuated the upregulation of 8-OHdG expression in the CA1 and DG regions of the hippocampus (C). AS-IV treatment at 20 mg/kg significantly attenuated the upregulation of 4-HNE and 8-OHdG protein levels as determined by ELISA method (D,E). Data are presented as mean ± SEM (n = 6 in each group; †††, p < 0.001 between the control and the pBCAO groups; *, p < 0.05; **, p < 0.01 between the pBCAO and AS-IV groups).

Figure 7

Effects of astragaloside IV (AS-IV) on GFAP and Iba1 expressions in hippocampal tissue. Representative western blots illustrating differences in the bands of GFAP and Iba1 (A). GFAP expression was significantly attenuated by AS-IV 20 mg/kg treatment, while Iba1 expression was significantly attenuated at both 10 and 20 mg/kg AS-IV treatment (B). Representative photographs show the CA1 and DG regions of the hippocampus immuno-stained against GFAP and Iba1 (C). Scale bar is 100 µm, applicable to all sections. AS-IV significantly attenuated the upregulation of GFAP expression in the CA1 and DG regions of the hippocampus (D) and significantly reduced the % area of the GFAP-labeled astrocytes in the DG region of the hippocampus (E) at both 10 and 20 mg/kg treatment. AS-IV at 20 mg/kg significantly attenuated the upregulation of Iba1 expression in the DG region of the hippocampus (F) and significantly reduced the % area of the Iba1-labeled microglia in the DG region of the hippocampus (G). Data are presented as mean ± SEM (n = 6 in each group; ††, p < 0.01; †††, p < 0.001 between the control and the pBCAO groups; *, p < 0.05; **, p < 0.01; ***, p < 0.001 between the pBCAO and AS-IV groups).