Salvianolic acid A protects rats against cerebral ischemic injury by regulation Wnt/glycogen synthase-kinase-3β/β-catenin signaling pathway

Abstract

Objective To investigate the protective effect of salvianolic acid A (SAA) on permanent focal cerebral ischemia in rats and its possible mechanisms. Methods Fifty-four adult male Sprague-Dawley rats were randomly divided into a sham operation group, a cerebral ischemia group, and a SAA group (n=18 in each group). A model of permanent middle cerebral artery occlusion was induced by the intraluminal suture method.At 0 h and 6 h after modeling, the rats of the SAA groups were intraperitoneally injected SAA (3 mg/kg). The other groups were injected equal volume of saline. At 24 h after modeling, the neurological deficit scores were performed. 2, 3, 5-Triphenyl tetrazolium chloride (TTC) staining was used to detect cerebral infarction volume. TUNEL staining was used to detect cell apoptosis. Both immunohistochemical staining and Western blotting were used to detect the expressions of Wnt3a, β-catenin, and phosphor-glycogen synthase-kinase-3β (p-GSK-3β) in the ischemic cortex. Results The neurological deficit scores showed that no neurological deficits were observed in the sham operation group (score 0). The neurological deficit score in the SAA group (median and interquartile range) was significantly lower than that in the cerebral ischemia group (3 [2-3] vs. 4 [3-5]; Z=-2.679, P=0.007). No infarcts were observed in the sham operation group. The infarct volume in the SAA group was reduced significantly compared with the cerebral ischemia group (79.038±10.665 mm3vs. 212.702±8.029 mm3;t=24.525, P<0.001). Very few positive cells were observed in the sham operation group. The numbers of TUNEL-positive cells in the SAA group and the cerebral ischemia group were 29.667±1.366/HP and 63.333±0.894/HP, respectively. The former was significantly less than the latter (t=14.115, P<0.001). Immunohistochemical staining showed that the number of Wnt3a positive cells in the sham operation group, the cerebral ischemia group, and the SAA group were 35.500±2.572/HP, 18.056±3.765/HP, and 29.000±2.376/HP, respectively. There were significant differences among the 3 groups (F=115.972, P<0.001), and those in the SAA group were significantly more than the cerebral ischemia group (P<0.01). The numbers of p-GSK-3β positive cells in the sham operation group, the model group, and the SAA group were 7.944±2.127/HP, 37.444±3.434/HP, and 11.222±1.734/HP, respectively. There were significant differences among the three groups (F=730.580, P<0.001), and those in the SAA group were significantly less than the cerebral ischemia group (P<0.01). The numbers of β-catenin positive cells in the sham operation group, the cerebral ischemia group, and the SAA group were 26.722±26.722/HP, 16.556±1.854/HP, and 21.333±1.940/HP, respectively. There were also significant differences among the 3 groups (F<33.385, P<0.01), and those in the SAA group were significantly more than the cerebral ischemia group (P<0.01). Western blot analysis showed that Wnt3a expression levels in the sham operated group, the cerebral ischemia group, and the SAA group were 1.000±0.190, 0.800±0.185, and 1.198±0.262, respectively. There were significant differences among 3 groups (F=9.621, P<0.001), and those in the SAA group were significantly higher than the cerebral ischemia group (P<0.01). The p-GSK-3β expression levels in the sham operation group, the cerebral ischemia group, and the SAA group were 0.650±0.150, 1.290±0.250, and 1.190±0.250, respectively. There were also significant differences among the 3 groups (F=19.668, P<0.001), and those in the SAA group were significantly higher than the cerebral ischemia group (P<0.01). The β-catenin expression levels in the sham operation group, the cerebral ischemia group, and the SAA group were 1.200±0.210, 0.500±0.120, and 1.100±0.220, respectively. There were significant differences among the 3 groups (F=33.385, P<0.001), and those in the SAA group were significantly higher than the cerebral ischemia group (P<0.01). Conclusions SAA has certain protective effect on permanent cerebral ischemia injury in rats. Its mechanism may be associated with the up-regulation of the expression of Wnt3a and β-catenin and the down-regulation of the expression of p-GSK-3β. Key words: Brain Ischemia; Apoptosis; Caffeic Acids; Neuroprotective Agents; Wnt Proteins; Glycogen Synthase Kinase 3; βCatenin; Signal Transduction; Rats; Salvianolic Acid A