| Objective: To date, dementia has become one of the top threats to the public worldwide. Vascular dementia(VD) is the second most common cause of dementia in the elderly after Alzheimer's disease. Clinical studies have demonstrated that Cerebralcare Granule can improve cognitive function in patients with VD. However, the related acting mechanism paid far too little attention to the regulation of autophagy and its signal transduction pathways in nerve cells. Permanent cerebral hypoperfusion, which mimics the vascular dementia model, was induced by occlusion of the bilateral common carotid arteries. The study was to investigate the protective effects of Cerebralcare Granule on cognitive function in rats with VD and futher to explore whether it was related to the autophagy mediated by JNK /Bcl-2 pathway in nerve cells.Methods: 3 months male Sprague Dawley rats weighing 250-330 g were used throughout the study. The permanent occlusion of bilateral common carotid arteries(2-VO) was used to establish the vascular dementia(VD) model. Rats were randomly divided into four groups: Sham+Saline group?2VO+Saline group ? 2VO+CG0.4 group ? 2VO+CG0.8 group, and Cerebralcare Granule was intragastrically administrated to 2VO+CG0.4 group and 2VO+CG0.8 group after operation for 28 d, while equal volume physiological saline was used as control to Sham+Saline group and 2VO+Saline group. The behavioral testing was conducted at day 29-34, including the place navigation test and the spatial probe test, to evaluate the spatial cognitive function of four groups. After the Morris water maze test, nissl staining was used to observe the pathological changes in the hippocampus. MDA content and SOD activity were measured by the reagent kits. Western blot were applied to detect JNK?p-JNK?p-bcl-2?Beclin 1?LC3-?/? and p62 expressions in hippocampus tissue. And immunohistochemistry was used to observe the positive cell number of Beclin 1 and p62 in hippocampal CA1 region.Results: 1 The Morris water maze test results 1.1 The place navigation test: Four groups showed a progressive decline in the escape latency with training. The first day, the escape latency of four groups had no statistical difference(P>0.05). From the second day on, 2VO+Saline group exhibited significant prolonged escape latency compared with the Sham+Saline group(P>0.01). 2VO+CG0.4 group and 2VO+CG0.8 group showed shorter escape latency compared with the 2VO+Saline group(the third day P<0.05,others P<0.01). 1.2 The spatial probe test: Compared with the Sham+Saline group, the 2VO+Saline group spent significantly less time in the target quadrant(P<0.01). 2VO+CG0.4 group and 2VO+CG0.8 group evidently increased the time spent in the target quadrant compared with the 2VO+Saline group(P<0.05), and the latter was more significant(P<0.05). 2 Nissl staining observation Nissl staining demonstrated that the pyramidal neurons had complete neuronal structure and they were tightly ranked in order in hippocampal CA1 region of Sham+Saline group. The nucleus and nucleolus were clear. Compared with the Sham+Saline group, the 2VO+Saline group showed loss of pyramidal neurons and loose arrangement. Part of neurons manifested neuronal shrinkage. The morphological structure of pyramidal neurons had improved in hippocampal CA1 region of the 2VO+CG0.4 group and 2VO+CG0.8 group. 3 MDA content and SOD activity Compared with the Sham+Saline group, MDA content increased significantly in hippocampus of 2VO+Saline group(P<0.01). 2VO+CG0.4group and 2VO+CG0.8 group showed less compared with 2VO+Saline group(P<0.01), and the latter was more significant(P<0.01). Compared with the Sham+Saline group, SOD activity decreased significantly in hippocampus of 2VO+Saline group(P<0.01). 2VO+CG0.4 group and 2VO+CG0.8 group represented higher compared with 2VO+Saline group(P<0.01), and the latter was more significant(P<0.01). 4 Western blot results 4.1 The expression of JNK?p-JNK ?p-bcl-2? Beclin 1?LC3?/ and p62 ?in hippocampus of VD rats: Compared with the Sham+Saline group, the expression of p-JNK ?p-bcl-2?Beclin 1 and the ratio of LC3?/?were up-regulated in 2VO+Saline group, but the expression of p62 was significantly decreased with statistical significance(P <0.01). There was no significant difference in the expression of JNK among them. 4.2 Effect of Cerebralcare Granule on the expression of JNK?p-JNK ?p-bcl-2? Beclin 1?LC3?/ and p62 ? in hippocampus of VD rats: Compared with the 2VO+Saline group, the expression of JNK showed no marked difference, and the expression of p-JNK ?p-bcl-2?Beclin 1 and the ratio of LC3?/ ?were declined in 2VO+CG0.4 group and 2VO+CG0.8 group, but the expression of p62 was significantly increased, and the latter was more significant(P<0.01). 5 Immunohistochemistry observation 5.1 Beclin1 positive cells count: The Beclin1 immunoreactive material was brown, localized in cytoplasm. Compared with the Sham+Saline group, positive cells count was significantly increased in hippocampal CA1 region of 2VO+Saline group(P<0.01), 2VO+CG0.4 group and 2VO+CG0.8 group represented lower compared with 2VO+Saline group(P<0.01). 5.2 p62 positive cells count: The p62 positive cells, like Beclin1, was brown, localized in cytoplasm. Compared with the Sham+Saline group, p62 positive cells count was fewer in hippocampal CA1 region of 2VO+Saline group(P<0.01), 2VO+CG0.4 group and 2VO+CG0.8 group showed more compared with 2VO+Saline group(P<0.05).Conclusions: In conclusion, the results demonstrated that Cerebralcare Granule has anti-oxidant activity against hippocampus oxidative damage and allexiate the cognitive deficits induced by chronic cerebral circulation insufficiency in rats. Excessive autophagy is a contributing factor to vascular dementia. Cerebralcare Granule could attenuate spatial learning and memory deficit and pathological damage in rat hippocampus via modulating JNK/Bcl-2 signaling pathway which will inhibit autophagic neuron death. |
PDF Full Text Request