| Our team have done a lot of work to declare the effects of FUZHI Capsule on preventing and treatment of Vascular Dementia(VD). It is know that FUZHI Capsule is efficient on VD by means of protecting the cholinergic system, making regulation of the hippocampus cholinergic system; reducing the damage to the nervous system which caused by ischemic inflammation; improving the recovery progression of cerebrovascular hemoperfusion, lowing the degree of platelets aggregation and activating the anti-oxidization system in the body to eliminate free radicals, etc. In recent years, the research found that potassium channels particularly the delayed rectifier potassium channel is intimately linked with cell apoptosis, our research will pay attention to the relationship between mitochondrial apoptotic pathways and IK current to verify the effects of FUZHI Capsule and its effective component Emodin on VD.ObjectiveIn order to further study the possible relationship between apoptotic mechanism and potassium channels particularly the delayed rectifier potassium channel which based on mitochondria-dependent pathway of the neron loss in Vascular Dementia by FUZHI capsule, we focus on the apoptosis-related genes Bcl-2, Bax and the key enzyme responsible for diseases related to cell apoptosis-Caspase-3. The gene expression were detected by real-tim PCR and the protein expression were detected by ELISA and western-blot. Emodin is the effective components of the Radix Polygonum Multiflorum Preparata, which is the sovereign drug of FUZHI Capsule. we investigated the effects of emodin on the voltage-gated potassium channels especially on delayed rectifier potassium current(IK) in cultured human neuroblastoma cells (SH-SY5Y) cells by using whole-cell clamp patch, cytotoxity test were detected by MTT.MethodsIn Vivo:According to random number table,40 SD rats were divided randomly into normal control group and Sham group with 20 in each group, After building successful VD model by bilateral carotid artery occlusion (2-VO) method, the other 60 SD rats were randomly divided into VD model group, Aricept treatment group, and FUZHI Capsule treatment group with 20 in each group. After continuous infusion medicine for 4 weeks, groups of animals were detected the corresponding indicators. Cleaved Caspase 3 expression in serum was detected by enzyme linked immunosorbent assay (ELISA), Bcl-2 and Bax mRNA in the brain were detected by real-time PCR; Bcl-2, Bax, Caspase-3 and Cleaved Caspase-3 protein expression in brain hippocampus were detected by Western blot.In Vitro:fter cytotoxic detection by MTT, SH-SY5Y cell lines were randomly divided into normal control group, OGD model group, OGD+emodin (X mol/L) group, using whole cell patch clamp technique record model group and normal control group respectively emodin to observe the changes of Ik and draw the I-V curve.Results(1) Compared to the Control Group, the anti-apoptotic factor Bcl-2 were intensely low expressed in the VD model group while the pro-apoptotic factor Bax were increased, and FUZHI Capsule has a resistance to apoptosis by changing the Bcl-2/Bax ratio which can decrease the level of Bax and increase the level of Bcl-2 in the VD model group.(2) Compared to the Control Group, the apoptisis related protein Caspase-3 and Cleaved Caspase-3 were up regulated in the VD model group, and FUZHI Capsule can protect the neuron from VD by down regulated the level of Caspase-3 and Cleaved Caspase-3 in hippocampus of VD rats.(3) Compared to the Control Group, SH-SY5Y cells appear a significant reduction in the cell survival rate after Oxygen Glucose Deprivation, this phenomenon can be reversed after intervention by emodin, it proved that emodin has a protective effect in SH-SY5Y cells growth and dynamic state from hypoxia and glucose deprivation.(4) Compared to the Control Group, Oxygen Glucose Deprivation inhibited the current amplitude of IK. Emodin could eliminate the inhibition by OGD, the IK peak density increase to (5.947+0.7415) pA/pF (P< 0.05) from (4.474+0.4182) pA/pF when the voltage of+50 mV.(5) Emodin can rise voltage-current curve (I-V) of IK which was inhibited by OGD, and the I-V curve gradually increased with the further depolarization.Conclusion(1) The apoptosis related experiments show that FUZHI Capsule can inhibit the expression of apoptosis gene Bax, down-regulate Caspase 3 which is the key enzyme responsible for apoptosis, and raised apoptosis inhibiting gene expression Bcl-2, that is based on the classical mitochondrial-depended apoptotic pathway, FUZHI Capsule could activate antiapoptotic mechanisms in order to achieve the neuronal protection of vascular dementia.(2) Emodin which is the effective components of FUZHI Capsule has the obvious active effect on voltage-gated potassium channels IK peak and I-V curve in SH-SY5Y cells. The data suggested FUZHI capsule attributes preventive and therapeutic effects on VD through the activation of IK and mitochondrial-depended apoptotic pathway.
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