| Background and purposeStrok is a common disease in the World and is the first leading cause of death in China. It is characteristered by high rate of incidence, high disability and high mortality. Every year, country and family pay many for treatment this disease. However, the biochemical events that occurs after stroke do not well understand up-to-date, as a result, there lack of effective drugs and measures targeted the therapy for stroke. Tissue-type plasminogen activator (t-PA) is the only therapy approved by the Food and Drug Administration (FDA) for the treatment of acute ischemic stroke, but the therapeutic time window is limited within4.5hours of the onset of ischemic stroke, Beyond4.5h after stroke onset, t-PA administration show increased risk of hemorrhagic conversion and cerebral edema in the infarcted area of brain. For various reasons, the overwhelming majority of patients did not arrive medical institutions after stroke onset, so, they do not benefit from this therapeutic strategy. And, it is well known that tPA has neurotoxicity and could lead to neuronal apoptosis. The therapy of stroke has become one of the most important aims for the global medical workers. At present, the apoptosis that occurs in the ischemic had arised more and more attentions and been thought one of the targeted theragy for the stroke.Puerarin is an isoflavones that extracted from the traditional Chinese medicine, Pueraria lobata (Willd) Ohwi, It has been pasted more than20years since study Puerarin in laboratory to clinical cardiovascular and cerebrovascular diseases treatment. But puerarin has a poor lipid-soluble and can not permeate through blood-brain barrier (BBB), this shortcoming limits its extensive use in clinical. Acetylpuerarin, a new isoflavone compound, whose structure was modified based on puerarin, has been investigated. Acetylpuerarin has higher solubility in lipid than puerarin and can permeate BBB easily. Several studies have revealed that acetylpuerarin has protective effects against brain ischemia-reperfusion injury by increasing the expression of bcl-2and Bax, inhibiting cell apoptosis. Acetylpuerarin has a broad prospects in the treatmet of ischemic stroke.In this study, we furture explore the effect of acetylpuerarin on the ischemia/reperfusion. OGD/R-induced hippocampal neurons injured model were used to study the vibility, apoptosis and apoptic relating factors and the protective effects of acetylpuerarin. We choose a better time point by statistical analysis for the lasted study. We hope, our founding may do some use in the research and development of acetylpuerarin.Our study includes the following two parts:1. Effect of acetylpuerarin on the cell viability and morphology of hippocampal neurons following oxygen-glucose deprivation/reperfusion in rats.2. Acetylpuerarin reduces apoptosis of hippocampal neurons following oxygen-glucose deprivation/reperfusion in rats. Part I Effect of acetylpuerarin on the cell viability and morphology of hippocampal neurons following oxygen-glucose deprivation/reperfusion in ratsObjectivHippocampal neurons were cultured and OGD/R model was established, the effect of acetylpuearin on the cell viability and morphology following oxygen-glucose deprivation/reperfusion were detected by MTT and inverted microscope. In this part, we choose a best main point for the future research.MethodsHippocampi were removed from the brains of embryonic day18and Cells were cultured in a medium. Half volume of the medium was changed per3days for each. The following experiments were performed after8days.The neurons were divided into five groups according to the concentration of acetylpuearin:1.Control group:The primary embryonic hippocampal neurons were incubated in NB with B27for8days, then culture medium was removed and changed into warmLy D-Hank’s balanced salt solution with glucose and placed in5%CO2incubator for180min. After that, the neurons were incubated in NB without B27at37℃for scheduled time.2. Oxygen-glucose deprivation and reperfusion (O/R) group:The D-Hank’s balanced salt solution without glucose was placed in OGD chamber(95%N2/5%CO2)for30min and the oxygen was less than1%. The neuron were then rinsed with OGD buffer4times and transferred to an OGD chamber(95%N2/5%CO2)for3hours. After OGD treatment, the neurons were incubated in NB without B27for scheduled time.3. Low concentration of acetylpuearin for OGD/R:as soon as the OGD were taken and replaced in NB without B27, acetylpuerarin was added to medium to0.1μM and incubated for scheduled time. Other treating processes were the same as that of the OGD/R groups. 4. Middle concentration of acetylpuearin for OGD/R:as soon as the OGD were taken and replaced in NB without B27, acetylpuerarin was added to medium to0.4μM and incubated for scheduled time. Other treating processes were the same as that of the OGD/R groups.5. High concentration of acetylpuearin for OGD/R:as soon as the OGD were taken and replaced in NB without B27, acetylpuerarin was added to medium to1.6μM and incubated for scheduled time. Other treating processes were the same as that of the OGD/R groups.OGR/R-12h,24h and36h, we use inverted microscope, MTT and DAPI to observed the morphology, viability and apoptosis of neurons. Through statistical analysis, we choose OGD/R-24h as a main point for the future research;The effect of acetylpuerarin on hippocampal neuron apoptosis induced by OGD/R-24h were determined with TUNEL (terminal-deoxynucleotidyl transferase mediated nick end labeling) staining.Results1. With inverted microscope, we observed, that the neurons refraction were decreased, projections were shorten, or even disappear, part of the cell had dissolved, scatterd nucleus.0.1μM,0.4μM and1.6μM acetylpuerarin can significantly improve the morphological injury after the OGD/R, acetylpuerarin show a neuron protection against OGD/R injury.2. OGD/R-12h,24h and36h decreased the vibility of neurons. Acetylpuerarin could improve neuronal viability, the value of high concentration of acetylpuerarin treatment increasing neuron survival rates was more obvious than those of middle and low concentrations of acetylpuerarin treatments.3. OGD/R treatment resulted in a time-dependent increase in numbers of apoptotic cells with a peak time at24h. Acetylpuerarin could decrease neuron apoptosis in high and middle concentration treatment except for OGD/R-12h, The low concentration of acetylpuerarin has no significant changes for all three scheduled time. 4. Acetylpuerarin with the concentration of0.1μM,0.4μM and1.6μM μM could inhibit the apoptosis of the injured neurons induced by OGD/R-24h significantly. The influence of acetylpuerarin on hippocampal neuronal cell death with OGD/R was determined with TUNEL staining. Results showed that acetylpuerarin decreased the apoptotic index.Conclusion1. OGD/R could damage the cultured hippocampal neurons at different points in vitro, decrease the cell viability and cause neurons apoptosis, the peak time of apoptosis is OGD/R-24h.2. Acetylpuerarin can alleviate neuronal damage, enhance the vitality of neurons and maintain the structural integrity of neurons.3. Acetylpuerarin in the concentration of0.1μM,0.4μM and1.6μM could reduce the number of apoptosis positive cells, show a protective effect on the ischemic-reperfusion injury. Part Ⅱ Acetylpuerarin reduces hippocampal neuron apoptosis following oxygen-glucose deprivation/reperfusion in ratsObjectiveUsing the point from the first part, the effect of acetylpuerarin on the apoptosis related factors were detection from the change of molecular level, provide effective theory basis for the clinical treatment of cerebral ischemia/reperfusion injury.MethodHippocampal neurons cultured and model established as same as the part Ⅰ. The point of experiment were OGD/R-6h,24h. The groups were oxygen-glucose deprivation and reperfusion (O/R) group, Low concentration of acetylpuearin for OGD/R, middle concentration of acetylpuearin for OGD/R, high concentration of acetylpuearin for OGD/R, respectively.The activities of caspase-8and caspase-3of hippocampal neurons were detected by fluorescence spectrophotometry; The effect of acetylpuerarin on OGD/R-24-h-induced Fas-L, FADD, TNF-αand Hsp70were determined by western blot on cultured hippocampal neuron in vitro.It were used the methods of RT-PCR to investigate the level of HIF-lamRNA, NF-κB mRNA and P53mRNA and the influence of acetylpuerarin on hippocampal neuronal cell death with OGD/R-24h.Results1.0.1μM,0.4μM and1.6μM acetylpuerarin could inhibit the activities of caspase-8and caspase-3with OGD/R. The examination with fluorescence spectrophotometry method showed that acetylpuerarin treatment led to an decrease in the expression of enzymatic activity of caspase-8and caspase-3.2. The expression of Fas-L, FADD and TNF-awere examined through western blotting and the results showed0.1μM,0.4μM and1.6μMacetylpuerarin could decrease the Fas-L, FADD and TNF-α after OGD/R was added to the cultured.3. After OGD/R-24h, the level of Hsp70increased, whereas Hsp70significantly increased when neurons cultured with0.1μM,0.4μM and1.6μM acetylpuerarin. 4.0.1μM,0.4μM and1.6μM acetylpuerarin could inhibited the expression of HIF-1α mRNA and P53mRNA24h after OGD/R was added to the cultured cell. Through the reverse transcriptase polymerase chain reaction, we find the area and the mean gray value of HIF-1α mRNA, NF-κBmRNA and P53mRNA all decrease.ConclusionOGD/R could injuried hippocampal neurons at different point, increasing the viability of caspase-3and8, the level of Fas-L, FADD and TNF-aprotein were increasing, decreasing the level of Hsp70, increasing the expressing of HIF-la mRNA, NF-κB mRNA and P53mRNA. Acetylpuerarin had protective effects against injury induced by OGD/R in hippocampal neurons via attenuating the level of Fas-L, FADD and TNF-α, and down-regulating the activities of caspase-8and caspase-3, as well as the level of HIF-1α mRNA, NF-κBmRNA and P53mRNA, and enhanced the expression of the Hsp70. These results show that the protection of acetylpuerarin is related its anti-apoptosis and effect some apoptosis related factors. |
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