Protective Effects Of Tetramethylpyrazine Piperazine Derivates On SH-SY5Y Cells Damaged By NMDA And On Rats With Vascular Dementia Induced By Repeated Bilateral Common Carotid Arteries Occlusion

Vascular dementia (VD) is a degenerative cerebrovascular disease that leads to a progressive decline in memory and cognitive function. It occurs when the blood supply carrying oxygen and nutrients to the brain is interrupted by a blocked or diseased vascular system. VD can be caused in several different ways. Most commonly, there is a blockage of small blood vessels somewhere in the vast system of arteries that feeds the brain and enters through the base of the skull. Neurotoxicity of excitatory amino acid, free radical damage and apoptosis of neurons are regarded as the most important molecular biology mechanism of vascular dementia.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, CXC137, a tetramethylpyrazine piperazine 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 CXC137 have stronger effects on treatment of cardio-cerebrovascular diseases.In the first part of this study, we used NMDA to simulate excitatory amino acid toxicity in SH-SY5Y cells. In the second part, vascular dementia rats were induced by repeated bilateral carotid arteries occlusion.The aim of this study is to observe the protective effect of CXC137 on the apoptosis of SH-SY5Y cells induced by NMDA and on rats with vascular dementia, consequently to elucidate its mechanism.1. Effect of CXC137 on the viability of SH-SY5Y cells injured by NMDAThe cell viability was detected by MTT assay. The results indicated that SH-SY5Y cells viability decreased significantly after exposure to NMDA (P<0.01), Pretreatment and post-treatment of cells with CXC137 effectively attenuated the NMDA effect on cell viability (P＜0.01) in a dose-dependent manner.2. Effect of CXC137 on leaking rate of LDH from SH-SY5Y cells injured by NMDAThe supernate and lysate of SH-SY5Y cells collected to determine the activity of LDH by commercial kit. Treatment of cells with 200μmol/L NMDA for 0.5h caused significant increase of LDH release (an indicator of membrane integrity) (P<0.01). Pre-treated or post-treat of the cells with various concentration of CXC137 significantly prevented the LDH release induced by NMDA in a concentration-dependent manner (P＜0.01).3. Effect of CXC137 on the apoptosis in SH-SY5Y cells injured by NMDAThe DNA content and percentage of apoptosis were determined by flow cytometry analysis using Annexin V-FITC/PI double-stain system. An increase of apoptotic cells was observed in the NMDA-treated group with a lower number of living cells. CXC137 administration led to a reproducible decrease in the rate of early apoptosis and necrosis/late apoptosis in cells exposed to NMDA (P＜0.01).4. Effect of CXC137 on activity and expression of Caspase-3 in SH-SY5Y cells injured by NMDAThe lysate of SH-SY5Y cells was collected to determine the activity of Caspase-3 by kit and the expression of Caspase-3 by Western Blot assay. In cells exposed to NMDA, the activity and expression of caspase-3 was enhanced. However, NMDA-exposed cells treated with CXC137 at concentrations of 20,40 and 80μM, revealed an increase of both the activity and the expression of Caspase-3 (P＜0.05 or P＜0.01).5. Effect of CXC137 on [Ca2+]i in SH-SY5Y cells injured by NMDAThe fluorescent intensities of Fura 3-AM-loaded suspended cells were measured at 37℃with emission at 490 nm and excitation at 526 nm by Multilabel Counter. The results showed that addition of 20,40 and 80μmol/L CXC137 reduced [Ca2+]i in response to NMDA stimulation (P＜0.01).6. Effect of CXC137 on the content of ATP in SH-SY5Y cells injured by NMDAThe lysate of SH-SY5Y cells was collected to determine the content of ATP by kit. The results showed that 20,40 and 80μmol/L CXC137 could increase the content of ATP in cells in response to NMDA stimulation (P＜0.01).7. The free radical-scavenging activity of CXC137 by DPPH* assayThe antioxidative potential of CXC137 was studied against DPPH*. The absorbance of the DPPH* solution including CXC137/TMP/VitE was detected at 516nm.50mg/ml CXC137 demonstrated the %RSA of 40% radical scavenging activity, lower than TMP and VE.8. Effect of CXC137 on the content of MDA, GSH and activity of SOD in SH-SY5Y cells injured by NMDAThe lysate of SH-SY5Y cells injured by NMDA was collected to determine the content of MDA, GSH and activity of SOD by kit. There were significant increase of MDA content and decrease of GSH content and SOD activity in cells exposed to NMDA (P＜0.01).40 and 80μmol/L CXC137 significantly inhibited the generation of MDA(P＜.01) and enhanced the GSH content and SOD activity (P＜0.01).9. Effect of CXC137 on the content of NO and activity of iNOS/tNOS in SH-SY5Y cells injured by NMDAThe lysate of SH-SY5Y cells injured by NMDA was collected to determine the content of NO and activity of iNOS/tNOS by kit. The content of NO was increased in cells and reached to the peak at 24h after NMDA addition.40μmol/L CXC137 and flunarizine reduced the content of NO in response to NMDA. The activities of iNOS/tNOS were increased and reached to the peak at 12h after NMDA addition. 40μmol/L CXC137 and flunarizine decreased the activity of iNOS/tNOS in cells cultured with NMDA.10. Effect of CXC137 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 CXC137 treatment.11. Effect of CXC137 on the spatial learning and memory capability in VD ratsMorris 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 CXC137 (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 CXC137 at the dose of 80 mg/kg, the swimming time of rats was significantly prolonged (P＜0.01).12. Effect of CXC137 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 CXC137 treatment decreased the content of Glu in the brain significantly. 13. Effect of CXC137 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). CXC137 inhibited the increase of the formation of MDA and the decrease of GSH level and SOD activity in the brain of rats with VD.14. Effect of CXC137 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.15. Effect of CXC137 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 CXC137 could decrease the mitochondrial swelling. The result indicated that CXC137 increased the function of brain mitochondria of VD rats (P＜0.05).