Effects And Underlying Mechanisms Of Novel Multi-functional Mercapoto-tacrine Derivative St09on Vascular Dementia In Rats

Part Ⅰ Synthesis and Biological Evaluation of Multifunctional Mercapoto-tacrine Derivative-ST09Aim:Incidences increasing with age, neurodegenerative diseases, such as vascular dementia (VaD), are becoming a severe threat to the whole world. Tacrine, the classic acetyleholinesterase inhibitor (AChEI) for the treatment of Alzheimer’s disease (AD), is still widely used as a pharmacophore to provide novel multi-target-directed ligands (MTDLs) endowed with additional biological properties beyond the simple AChE inhibition. In our previous studies, a series of novel multifunctional MTDLs have been synthesized, which cooperating thiol groups with tacrine in a single molecular. Therefore, ST09was view as the most promising candidate to investigate its neuropharmacological properties and to explore its possibility for the treatment of VaD.Methods:General chemical synthesis strategies, spectral anaylsis, cholinesterase inhibition assay, neuroprotectiDn, GOT/GPT assay, ESR spectroscopy, HE staining.Results:The yield of ST09remained at50-60%, after optimization of its preparation condition. Its structure was confirmed by spectral analysis. Pharmacokinetic results in vitro showed that as a prodrug, ST09was converted to deacetylated form ST10immediately, incubated in rat whole blood (the conversion rate reach96.72%within30min). ST09and ST10exhibit potent AChE inhibition with IC50value comparable with tacrine in vitro. Additionally, it exhibited neuroprotection on PC12cell line against H2O2-induced oxidative injury at1μM,3μM,10μM,30μM. In vivo, unlike tacrine, ST09did not affect the levels of GOT and GPT. Furthermore, in HE staining of liver, ST09was also demonstrated little hepatoxicity. The peak intensity of ESR signal reduced markedly in the persence of200μM ST10compared with the vehicle group (1%DMSO, v/v) and tacrine group (200μM).Conclusion:ST09and its active metabolite ST10exhibit good effects in AChE inhibition and neuroprotection with little hepatoxicity. Part Ⅱ Mercapoto-tacrine Derivative-ST09improves cognitive function deficits of vascular dementia ratsAim:VaD, a heterogeneous group of brain disorders in which cognitive impairment is attributable to cerebrovascular pathologies, is responsible for at least20-30%of cases of dementia, being second only to AD. Permanent, bilateral occlusion of common carotid arteries (2-VO) in rats has been established as a procedure to investigate the effects of chronic cerebral hypoperfusion on cognitive dysfunction and neurodegenetive processes.2-VO model has generated a large amount of data, which have been well recognized in the field of VaD research. We investigated the behavioral and the histopatho logical changes in cortex and hippocampus of VaD rats, examined the effects of ST09on2-VO-induced learning and memory impairment.Methods:SD rats were randomly divided into six groups:sham group, model group, Don group, ST09low-dose group, ST09medium-dose group and ST09high-dose group. The rats were anesthetized and bilateral common carotid arteries were ligated. And the rats of sham group received the same surgical operation without carotid artery ligation. One week after2-VO operation, sham group and model group were administrated with saline (1ml/kg, i.p.), Don group was administrated with0.3mg/kg donepezil, and the later three groups were administrated with ST09(1.73mg,3.46mg and6.92mg/kg) respectively, intraperitoneally daily. The2-VO rats were used as VaD model. The capabilities of spatial learning and memory of each group were examined by the conventional Morris water maze (MWM) task. And the histopathological changes of cortex and hippocampus in each group were examined by haematoxylin-eosin (HE) staining.Results:2-VO model of SD rats was used as an animal model of VaD in vivo. To evaluate whether the cognitive function of the VaD rats was improved after administration of ST09, we performed the MWM test to evaluate the abilities of spatial learning acquisition and memory retention. The escape latency of model group was markedly longer than the sham group (P<0.01), suggesting that2-VO successfully induced learing deficit. Daily administation of donepezil and ST09(3.46mg and6.92mg/kg, i.p.) significantly attenuated the2-VO-induced impairment in spatial acquisiton performance [F(3,32)=5.714, P=0.003]. After treatment with donepezil (0.3mg/kg) or ST09(3.46mg/kg), the escape latency was significantly decreased from day2to day4. On the probe test, the Don group and medium-dose ST09group showed significant preference (time and journey spent) for the target quadrant compared with model group rats (P<0.05). In sham rats, the pyramidal neurons in the CA1area of hippocampus were morphological intact, with full nuclei and clear nucleoli. But in the model group, some neurons died7weeks after2-VO, which represented by the increasing of inflammatory infiltration and vascular proliferation. The neuronal damages such as pyknosis of nuclei and vacuolation of neuropil were observed in the pyramidal neurons of hippocampal area of2-VO rats.Conclusion:These results show that ST09could rescue the behavioral and the histopathological changes in cortex and hippocampus of VaD rats. Part Ⅲ The mechanisms of mercapoto-tacrine derivative-ST09on alleviating brain damage and improving cognitive deficits in VaD ratsAim:Our previous studies have indicated that ST09could protect PC12cells against H2O2-induced oxidative injury and significantly alleviate the cognitive deficits and neuronal impairment induced by VaD in Morris water maze task. Furthermore, we investigated the possible multifunctional effects on VaD and the related mechanisms.Methods:The anti-oxidation effects of ST09were investigated in vitro using spectrophotometry assay to analysis the level of MDA and protein-bound sulfhydryl goup and the activity of SOD. The anti-apoptosis effect of ST09was evaluated using Western Blotting. The in vivo positron emission tomo graphy was used to investigate the effect of ST09on brain glucose metabolism.Results:2-VO induced a considarable increase in hippocampal AChE activity, compared with sham rats. Treatment with ST09significantly inhibited the rise of AChE activity. The BuChE activity in brain regions did not show significant changes. MDA levels in model group increased by93.19%in hippocampus and by81.37%in cortex, when compared with those in sham group. SOD activities in model group decreased from168.70±5.51U/mg protein to151.83±4.95U/mg protein in hippocampus and from450.07±11.17U/mg protein to343.15±18.59U/mg protein in cortex. Nevertheless, ST09significantly attenuated the alterations in both MDA levels and SOD activities induced by2-VO in these two brain regions. The levels of protein-bound sulfhydryl group in model group was decreased by16.37%in hippocampus and17.47%in cortex, campared with those in sham-operated group. ST09markedly attenuated the reduction of sulfhydryl group by2-VO in the two brain regions.2-VO operation induced significant increase of the expression of Bax and activated caspase-3in the two brain regions. The ratio of Bcl-2/Bax was decreased markedly compared with the levels obtained in sham group. However, administration with ST09(3.46mg/kg) significantly inhibited the elevation of active caspase-3from0.706±0.054to0.475±0.042(P<0.01) and attenuated the reduction of Bcl-2/Bax ratio from0.632±0.111to1.697±0.131(P<0.05) in cortex. In the hippocampus, the expressions of these three apotosis-related proteins were also regulated with the treatment of ST09in the same manner. Functional imaging of18F-FDG PET has been documented as a hypo metabolic pattern typical in VaD. Likewise, relative hypo metabolism in the ischemic brain of model group was remarkable marked in cerebral cortex and hippocampus. As we expected, the treatment of ST09greatly improved the glucose metabolism in the both brain regions.Conclusion:These results demonstrate that ST09induce multiple protective mechanisms against chronic cerebral hypoperfusion injury, including AChE inhibition, anti-apoptosis, anti-oxidation and improvement of glucose metabolism. We suggest that ST09may develop as a promising agent for the treatment of VaD.