Puerarin Alleviates Vascular Cognitive Impairment in Vascular Dementia Rats

Abstract

Cerebral ischemia triggers vascular dementia (VD), which is characterized by memory loss, cognitive deficits, and vascular injury in the brain. Puerarin (Pur) represents the major isoflavone glycoside of Radix Puerariae, with verified neuroprotective activity and cardiovascular protective effects. However, whether Pur ameliorates cognitive impairment and vascular injury in rats with permanent occlusion of bilateral common carotid arteries (BCCAO) remains unknown. This work aimed to assess Pur's effects on BCCAO-induced VD and to dissect the underlying mechanisms, especially examining the function of transient receptor potential melastatin-related 2 (TRPM2) in alleviating cognitive deficits and vascular injuries. Rats with BCCAO developed VD. Pur (50, 100, and 150 mg/kg) dose-dependently attenuated the pathological changes, increased synaptic structural plasticity in the dorsal CA1 hippocampal region and decreased oxidative stress, which eventually reduced cognitive impairment and vascular injury in BCCAO rats. Notably, Pur-improved neuronal cell loss, synaptic structural plasticity, and endothelial vasorelaxation function might be mediated by the reactive oxygen species (ROS)-dependent TRPM2/NMDAR pathway, evidenced by decreased levels of ROS, malondialdehyde (MDA), Bax, Bax/Bcl2, and TRPM2, and increased levels of superoxide dismutase (SOD), Bcl2, and NR2A. In conclusion, Pur has therapeutic potential for VD, alleviating neuronal cell apoptosis and vascular injury, which may be related to the ROS-dependent TRPM2/NMDAR pathway.

Keywords: NR2A; ROS; TRPM2; cognitive impairment (dementia); puerarin; vascular dementia.

FIGURE 1

Effects of puerarin (Pur) on occlusion of bilateral common carotid arteries (BCCAO)-induced spatial memory impairment. (A) Timeline of surgery (BCCAO), drug administration and behavioral assays. (B) Results of the Morris water maze (MWM) (days 28 following BCCAO) in the sham and BACCO groups. (C) Escape latency (starting site to the hidden platform) at days 24–28 post-BCCAO). (D) Escape latency at days 59–63, during Pur treatment. (E) Numbers of crossings in the target quadrant within 60 s in the probe trial on day 63. (F) Percentages of time spent in the target quadrant within 60 s in the probe trial (no platform) on day 63. (G) Swimming speed (path length by escape latency) in each group at days 59–63. Values were mean ± SEM (n = 6). *P < 0.05, **P < 0.01 vs. Sham; ^#P < 0.05, ^##P < 0.01 vs. BCCAO; ^$$P < 0.01 vs. BCCAO + Pur (150 mg/kg/day).

FIGURE 2

Effects of Pur on middle brain artery relaxation. (A) Pur’s effect on acetylcholine-associated endothelium-dependent vasorelaxation in BCCAO rats. (B) Pur’s effect on sodium nitroprusside-associated endothelium-independent vasorelaxation in BCCAO rats. Values were mean ± SEM (n = 6). **P < 0.01 vs. Sham; ^#P < 0.05, ^##P < 0.001 vs. BCCAO group; ^$P < 0.05 vs. BCCAO + Pur (150 mg/kg/day).

FIGURE 3

Pur’s effects on morphological alterations and dendrites in the hippocampus of BCCAO rats (H&E staining and Golgi-Cox staining). (A) HE staining in groups, Representative micrographs are shown at ×200. (B) Histological score. (C) Golgi staining in groups. (D) Percentage of mushroom spines. (E) Number of spines. Values were mean ± SEM (n = 6). **P < 0.01 vs. Sham; ^#P < 0.05, ^##P < 0.001 vs. BCCAO group.

FIGURE 4

Puerarin’s effects on apoptosis in dorsal CA1 region of hippocampal neurons in BCCAO rats (TUNEL staining). (A) Tunel staining to identify positive apoptotic cells (Red arrows), representative micrographs are shown at ×40. (B) The statistics of TUNEL positive cells. Values were mean ± SEM (n = 6). **P < 0.01 vs. Sham; ^##P < 0.01 vs. BCCAO; ^$P < 0.05 vs. BCCAO + Pur (150 mg/kg/day); ^&&P < 0.01 vs. BCCAO + NGF (20 mg/kg/day).

FIGURE 5

Expression and co-localization of Bax and Bcl2 in the rat dorsal CA1 region of hippocampus. (A) Protein expression of Bax in the hippocampal dorsal CA1 region of rats. (B) Protein expression of Bcl2 in the hippocampal dorsal CA1 region of rats. (C) Bax/Bcl2 ratios in the hippocampal dorsal CA1 region of rats. (D) Pur’s effects on Bax and Bcl2 co-localization in the hippocampal dorsal CA1 region of rats, assessed by immunofluorescent double staining. Representative micrographs are shown at ×200. Results were mean ± SEM (n = 6). *P < 0.05, **P < 0.01 vs. Sham; ^##P < 0.01 vs. BCCAO; ^$P < 0.05, ^$$P < 0.01 vs. BCCAO + Pur (150 mg/kg/day); ^&P < 0.05, ^&&P < 0.01 vs. BCCAO + NGF (20 mg/kg/day).

FIGURE 6

Effect of Pur on (A) ROS, (B) MDA, and (C) SOD levels in experimental rats. Results were mean ± SEM (n = 6). **P < 0.01 vs. Sham; ^##P < 0.01 vs. BCCAO; ^$P < 0.05 vs. BCCAO + Pur (150 mg/kg/day).

FIGURE 7

Expression and co-localization of NR2A and transient receptor potential melastatin-related 2 (TRPM2) in the rat hippocampus. (A) Protein amounts of TRPM2 in the hippocampal dorsal CA1 region of rats. (B) Protein amounts of NR2A in the hippocampal dorsal CA1 region of rats. (C) Effects of Pur on NR2A and TRPM2 co-localization in the hippocampal dorsal CA1 region, examined by immunofluorescence double staining. Representative micrographs are shown at ×200. Results were mean ± SEM (n = 6). **P < 0.01 vs. Sham; ^##P < 0.01 vs. BCCAO; ^$$P < 0.01 vs. BCCAO + Pur (150 mg/kg/day).