Experimental Study Of Tetramethylpyrazine Diphenylmethyl Piperazidine On Vascular Dementia Rats

Dementia is an acquired lasting disturbance of intelligence syndromes due to cerebral vascular disease. Vascular dementia is the common type of senile dementia only secondly to Alzheimer disease. There is not an affirmative method to treat vascular dementia because of the complex etiology. The therapy of vascular dementia is to ameliorate the circulation of brain and oxygen supply of cerebral cell in order to prevent the new thrombus and infarct occurrence. At present, the common medicines to treat vascular dementia are the medicines to ameliorate the circulation of brain and the activator of cerebral energy and the accelerator of cholinergic system, ect. There aren't breakthrough break progresses on the vascular dementia pathological treatment. We have not satisfactory prevention and cure effects. Recovery after nervous system injury is a multi-link, milti-level system adjustment process, so that it is a significant subject in the field of the basic and clinical medicine.Tetramethylpyrazine (TMP), is the major efficient component of the Chuanxiong, which is used in China as a new kind of calcium antagonist and an antioxidant for the treatment of cardiovascular diseases and myocardial and cerebral ischemic diseases because of their low toxicity and their effectiveness. However, pharmacokinetics studies have shown that TMP presents low bioavailability and is metabolized quickly in vivo with short half-life of T1/2. Therefore, it is necessary to develop a new generation of the cerebrocardiac vascular drugs from molecular modification of TMP.Flunarizine is a second-generation piperazine calcium channel blocker, which could pass through the blood-brain barrier, and prevent the injury of neurons induced by calcium overload. It is an important drug for cardio-cerebrovascular diseases.The compound used in this study, tetramethylpyrazine diphenylmethyl piperazidine (TMPDP), a novel TMP derivate, was synthesized through replacing the methyl group of TMP molecules with the pharmacophores of Flunarizine. The piperazine ring acts as a linker in the molecular structure and is considered as a functional group of the drug activity. We supposed TMPDP have stronger effects on treatment of cardio-cerebrovascular diseases than TMP.In the first part of this study, we used KCL, L-Glu, NaN3 respectively to induce cytotoxicity in SH-SY5Y cells by different mechanism and observed the protective effect of TMPDP on cell damage. In the second part, vascular dementia rats' model was established by repeated bilateral carotid arteries occlusion to observe the therapeutic effects of TMPDP on vascular dementia and consequently to elucidate its mechanism.1. Effect of TMPDP on the viability of SH-SY5Y cells injured byThe cell viability was detected by MTT assay. The results indicated that SH-SY5Y cells viability decreased significantly after exposure to NMDA (P<0.01), post-treatment of cells with TMPDP effectively attenuated the KCL effect on cell viability (P<0.01) in a dose-dependent manner.2. Effect of TMPDP on the viability of SH-SY5Y cells injured by L-GluThe cell viability was detected by MTT assay. The results indicated that SH-SY5Y cells viability decreased significantly after exposure to L-Glu (3000μmol·L-1), post-treatment of cells with TMPDP effectively attenuated the effect L-Glu on cell viability (P<0.01) in a dose-dependent manner.3. Effect of TMPDP on the viability of SH-SY5Y cells injured by NaN3, The cell viability was detected by MTT assay. The results indicated that SH-SY5Y cells viability decreased significantly after exposure to NaN3 (64mmol·L-1), post-treatment of cells with TMPDP effectively attenuated the effect NaN3on cell viability (P<0.01) in a dose-dependent manner.4. Effect of TMPDP on motor performance in VD ratsVascular dementia rats made by repeated bilateral common carotid arteries occlusion. Prehensile traction test was performed to examine motor performance. The motor performance of rats was declined at 30 days after operation, and could be improved significantly by TMPDP treatment.5. Effect of TMPDP on the spatial learning and memory capability in VD rats Morris Water Maze was used to measure the spatial learning and memory capability of VD rats. In place navigation test, the escape latency of model group was longer than sham group obviously (p<0.01) and was improved by TMPDP (p<0.01); In spatial probe test, the sham-operated rats spent more time in the quadrant that had located at the hidden platform than rats in model group (P<0.01). After treatment with TMPDP at the dose of 80 mg/kg, the swimming time of rats was significantly prolonged (P<0.01).6. Effect of TMPDP on the content of Glu in the brain of VD ratsThe brain homogenate of the rats were collected to determine the content of Glu by kit. Results showed that the Glu content in the brain of rats in model group was higher than in the sham group (P<0.01).20 and 80mg/kg TMPDP treatment decreased the content of Glu in the brain significantly.7. Effect of TMPDP on the content of MDA, GSH and activity of SOD in the brain of VD ratsThe brain homogenate of the rats collected to determine the content of MDA, GSH and activity of SOD by kit. Compared with the sham-operated group, the content of MDA increased and the content of GSH and the activity of SOD decreased significantly in the brain of the rats in model group (P<0.01). TMPDP inhibited the increase of the formation of MDA and the decrease of GSH level and SOD activity in the brain of rats with VD.8. Effect of TMPDP on the content of NO and activity of NOS in the brain of VD ratsThe brain homogenate of the rats was collected to determine the content of NO and the activity of tNOS by kit. Results showed that the content of NO and activity of tNOS had no significant difference among all experimental groups.9. Effect of TMPDP on the swelling degree of mitochondria in the brain of VD ratsThe brain mitochondria were collected to detect the absorbance at 520nm which expressed the swelling degree of mitochondria. Compared with the sham-operated group, the mitochondria swelling degree of model group was increased (P<0.01), while TMPDP could decrease the mitochondrial swelling. The result indicated that TMPDP increased the function of brain mitochondria of VD rats (P<0.05).