| Background:Alzheimer’s disease (Alzheimer’s disease, AD) has a high incidence rate in the elderly, which is one of the most harmful degenerative diseases in central nervous system and it is also the most common form of senile dementia. Due to the aggravation of population aging, this disease already ranks among the elderly cause of death in the first few. At present, the pathogenesis of the the disease has not been fully elucidated so that there is no satisfactory therapy for AD. Qingkailing (QKL) is one of the famous traditional Chinese medicines which have significant clinical effect on cerebral illness of TCM, the acute cerebral ischemic stroke. A series of pharmaceutical and pharmacodynamic studies have been conducted that identified more than60compounds in QKL and found3compounds, i.e.baicalin, jasminoidin, and cholic acid as the most active ones. Previous research has suggested that extracting baicalin, geniposide and acid as a combination of refined QKL(RQKL) can replace QKL in the treatment of stroke patients. Furthermore, based on the "toxic brain collateral damage" theory, RQKL also showed better therapeutic efficacy in the clinical treatment of post-stroke dementia. By the virtue of the common risk factors and overlapped pathologic change shared by AD and vascular dementia, and with the deeper understanding of AD pathogenesis, researchers found that "toxic brain collateral damage" is also the core of the development of AD. Hence, we proposed that on the basis of unblock collaterals and subcollaterals, the potential mechanism of RQKL, would increase the curative effect of AD.Objective:This study applied a comprehensive method combined with behavioristics, histopathology, neuroimaging and gene expression to assess the effectiveness and mechanism of refined Qingkailing when based on "JieDuTongLuo " treatment, then to do a further exploration about the "toxic damaged brain collaterals" theory and the significance of "JieDuTongLuo" treatment in the AD field of Chinese medicine research in. Furthermore, the research may provide a new train of thought for finding effective Chinese medical ways in AD treatment.Methods:To model dementia rats by injecting ibotenic acid (IBO) into the basal forebrain of male rats (weights between250g and300g), then devided modeling rats into contral group, model group and medicine group. Medicine group would be given gavage 3ml/kg each day with drug solution from the third day after operation,and the drug solution contained baicalin1.25mg/ml,geniposide6.25mg/ml, bile acids1.75mg/ml, while the contral group and IBO model group would be given gavage with normal saline after modeling. Within gavage for one month, rats would accept Morris water maze behavioral experiments in order to test whether the dementia model was successed and whether the RQKL could improve spatial learning and memory of Alzheimer’s disease model rats by hidden platform experiments and reverse assays. To assess the improvement of energy metabolism of brain glucose in AD model rats with the usage of RQKL, rats would receive neuroimaging of FDG-PET test in the second day after Morris water maze behavioral experiments ended. And then, researchers picked three rats randomly from each group for a histological examination in order to abserve structural changes in hippocampal neurons after taking RQKL, while rest of rats would be tested within DNA microchips and western blot methods. The research might assess effects of AD Chinese medical treatment from molecular level and it also provided an import ant theoretical reference and basis for improving compatibility of modern Chinese medicines.Result:RQKL treatment significantly attenuated IBO-induced abnormalities in cognition, brain functional images, and brain histological morphology. Morris water maze test results as follows:in the hidden platform test, the escape latency time was dependent on both the time effect (F4,108=18.178, P＜0.001) and the group effect (F2,27=41.426, P＜0.001); the control and durg groups escaped significantly faster than the IBO-model group (both P<0.001).A similar result was observed for the swim distance (F4,108=14.393and P＜0.001for the time effect; F2.27=8.784and P<0.001for the group difference; and P＜0.001and P＜0.05for the comparisons of the control group and the durg group to the IBO-model group). Swim speed was not significantly different between the groups. In the reverse hidden platform test, the time effect and differences between the groups were both significant factors in the escape latency time (F2,54=12.607, P＜0.001; F2,27=23.013, P＜0.001); compared with the IBO-model group, the escape latency times in the control group and the durg group were significantly shorter (both P<0.001). A similar result was observed for the swim distance (F2,54=12.886and P＜0.001for the time effect; F2,27=12.268and P<0.001for the group difference; and P＜0.001for the comparisons of the control group and the durg group to the IBO-model group). No significant differences in the swim speed were observed between groups.FDG-PET examination showed that compared with the control group, significant decreases in FDG-PET signals were observed in the IBO-model group for numerous brain regions. The affected regions included the anterior nucleus, the hippocampus, the midbrain, the intragyral nuclei and the primary cortex on both sides, the olfactory bulb and the hypothalamus on the left side, and the nucleus accumbens and the primary barrel cortex on the right side (P<0.05). Compared with the IBO-model group, in the durg group, the decrease in the left olfactory bulb and the left anterior nucleus was significantly attenuated; additionally,a significant increase in the FDG-PET signal was observed in the left hippocampus (P<0.05).Light microscope to observe the change of the structure of the hippocampus results showed in the control group, neurons in the hippocampal CA1region showed an orderly arrangement with no apparent abnormalities in cellular morphology. In the IBO-model group, neuron arrangement was disrupted with severe lesions in the nucleus and cytoplasm such as karyolysis and eosinophilia, and a neuronal cell loss was noted. In the durg group, these abnormalities were ameliorated.DNA microarray analysis showed that compared with the IBO-model group, in the durg group, the expression of19genes was significantly changed (P<0.05or P<0.01). Of these19genes,11are related to neuroprotection and neurogenesis and the other8are related to anti-oxidation, protein degradation, cholesterol metabolism, angiogenesis, stress response, and apoptosis. Gene expression was increased in all cases except for the gene related to apoptosis.TRH, EGF, CRBP1, CBR1, and ACE were selected for verification by western blot because previous studies have shown that they are more important in preventing AD in comparison to the others in the19genes significantly regulated by RQKL.The results showed that the protein expression levels of these5genes were significantly increased in the durg group compared with the IBO-model group (P<0.05or P<0.01), which was consistent with the results of the DNA microarray. Conclusion:RQKL can ameliorate the IBO-induced dementia in rats and may show significant effects in the treatment of AD. The therapeutic mechanism may be related to RQKL’s modulation of a number of processes, mainly through promotion of neuroprotection and neurogenesis, with additional promotion of anti-oxidation, protein degradation, etc. |
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