| Background and ObjectiveNow, life expectancy has been risen, the speed of the acceleration of the agingis increasing, the changes in diet after the improvement of living standards isobvious, the lack of physical exercise is remarkable, all these factors promotes theincidence and the prevalence rate of ischemic cerebrovascular disease to rise year byyear. It is seriously harmful and even life-threatening to people who suffer from thecerebrovascular disease. So except for exploring the mechanism of stroke, how tofind the strategy to reduce the incidence of the disease and cure it is also a criticaltask now. The cerebral ischemic lesion is divided into the ischemic core and theischemic penumbra, the damage on the neurons of the ischemic penumbra isreversible, so it is helpful to repair the impaired neurons from improving theirplasticity. The dynamic changes of the level of cytoskeletal proteins has a closerelation to the neurons’ survival and functional states, furthermore, the cytoskeletalprotein can accelerate the process of the neurons’ growth and increase their plasticity,so it’s very significative to explore how to increase the ability of cytoskeletal proteinon neuron’s remairment, which could improve patient’s outcome.Sonic hedgehog (Shh) signaling pathway is one of the main hedgehogsignaling pathways. Recent studies show that Shh signaling pathway is closely related to the remodeling of the cytoskeletal protein. The activation of Shh signalingpathway can not only increase the expression level of cytoskeletal protein, butimprove their stability. The microtubule (MTs) is one of the main components in thecytoskeleton, α-tubulin is one of the important components which consist MTs. Inthe nervous system, the MTs is not only the marker protein during the neurocytes’growth, but is the pivotal protein in the process of neurons’ repairment on theirstructure and functions after the neurons were damaged. MAP-2is also one of themain cytoskeletal components, which acts as the symbolic microtubule-associatedprotein, it plays an important role in neurons’ regeneration, apoptosis and plasticity.However, the studies about Shh signaling pathway in the models of stroke is mainlyfocused on vascular regeneration and oxidative stress, few research of Shh signalingpathway on cytoskeletal protein is reported.Shh signaling pathway mainly consists of Shh protein, Ptch protein, Smoprotein and Gli protein. Gli-1protein is the major downstream target gene in the Shhsignaling pathway. When the Shh is lack, the level of Gli-1decreases, when the Shhis activated, the level of Gli-1increases, so Gli-1protein is usually used as themarker of activated Shh signaling pathway. Purmorphamine is the specific agoniston Shh signaling pathway. It could combine with Smo protein and change Smoprotein’s conformation, which has the ability to activate the downstream protein ofShh signaling pathway. In this study, we use the acute cerebral artery occlusionmodel which was made by middle cerebral artery occlusion to observe the level ofα-tubulin and MAP-2in the ischemic brain after the activation of Shh signalingpathway by purmorphamine, then try to explore the effect on the cytoskeleton ofischemic brain in rats after the Shh signaling pathway is activated, which may offer anew clue for the cure of stroke.Materials and methodsIn this study, we chose96adult male healthy clean Sprague Dawley (SD) rats,the weight of the rats was controled about220±30g, then the models of acutemiddle cerebral artery occlusion was made according to Longa’s report. The96SDrats were divided randomly into three groups: sham operation group, model groupand treatment group. Every group was divided into four subgroups:1d,3d,7d,14d. Every subgroup has8rats. Purmorphamine powder was completely dissolved intothe dimethyl formamide (DMF) solution before we used it. The treatment group wasgiven purmorphamine0.69mg/Kg by intraperitoneal injection after the operation,while the sham operation group and the model group were given corresponding doesof pure DMF solution (0.69mg/Kg) also by intraperitoneal injection. We observedthe rats’ neurological deficit and evaluate their behavior when they completely cometo life after the operation and then all rats were executed at the given time point, thebrain tissue was taken out through the methods according to the demand of the study.In this study, we detected the levels of α-tubulin in the brain tissue byimmunohistochemistry and the expression of Gli-1and MAP-2was detected byRT-PCR.Data analysisWe use SPSS21.0statistical software to process the data. All the results areexpressed by x±s. ANOVA are used between groups comparison. As to pairwisecomparisons, we use LSD test (test level: α=0.05).Results1.The expression of α-tubulin by immunohistochemistryThe positive expression of α-tubulin is mainly in the neuronal cytoplasm andnuclei, it is also expressed in gliocyte and axon. In the sham operation group, it has astrong positive reaction of α-tubulin at every time point. In the model group, it hasan obvious decrease at the1d and3d time point, but is presents a stronger positivereaction of α-tubulin as time went by and tend to a stable level at the7d and14d timepoint. Compare the treatment group with the model group, the expression ofα-tubulin is more in the treatment group than the model group at every time point,the difference is statistically significant.2.The expression of MAP-2by RT-PCRThe expression of MAP-2is mainly in the soma and dendrite of the neurons.Compared with the sham operation group, the model group and the treatment grouphave a poor level expression of MAP-2(P<0.05). The expression of MAP-2in themodel group is decreased right after the stroke, particularly at the1d and3d timepoint which has a poorer expression of MAP-2, then it shows an increased expression of MAP-2as the time went by and tend to a stable level at the7d and14dtime point. In the treatment group, the expression of MAP-2has a big differencebetween model group at every time point (p<0.05).3.The expression of Gli-1by RT-PCRThe Shh signaling pathway was not activated in the brain of the shamoperation group, so it has a very poor level of Gli-1. Compared with the shamoperation group, the model group and the treatment group show an increasedexpression of Gli-1, and the difference is statistically significant (p<0.05). Theexpression of Gli-1of the model group and the treatment group shows the highestexpression level at the1d time point, then began to decrease, which still presents ahigher level than the sham operation group. Compare the treatment group with themodel group at every time point, the expression of Gli-1in the treatment group has ahigher level, and the difference is statistically significant (p<0.05).Conclusions1.Purmorphamine can effectively increase the expression level of Gli-1;2.The Shh pathway activated by purmorphamine in cerebral ischemia couldincrease the expression of α-tubulin and MAP-2, which maight be one of themechanism to reduce the stroke damage after ischemia. |
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